PROTECTION OF THE PROTEOLYTIC ACTIVITY OF CRUDE PAPAIN
AND CHEMICAL MODIFICATION OF PAPAIN BY TETRATHIONATE
by
Guillermo Eleazar Arteaga Mac Kinney
Biochemical Engineer Manager in Food Processing, The Institute of Technology and Higher Studies of Monterrey, Mexico, 1984
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
in
THE FACULTY OF GRADUATE STUDIES (The Department of Food Science)
We accept this thesis as conforming to the required standard
THE UNIVERSITY OF BRITISH COLUMBIA JULY 1988
©Guillermo E. Arteaga Mac Kinney , 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.
Department of f-poci ^ciewcg
The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 ABSTRACT
In the first chapter, sodium tetrathionate (TT), a sulfhydryl blocking agent, is assessed for its ability to protect the proteolytic activity (PA) of papaya latex during air, sun or vacuum drying, and of crude papain during storage.
X3 By means of Taguchi's L2? (3 ) fractional factorial design,
it was found that the addition of 1% TT significantly increased the retention of PA of papaya latex when it was air dried at a temperature of 55°C. This protection of PA was found to be 23% higher than the one given by the addition of 1% sodium metabisulfite, the compound commonly used in the commercial processing of papaya latex. When drying was carried out either under 27 inches vacuum at 50°C or in the sun, the protective effect of TT on the PA was not significantly different from that of metabisulfite.
The PA of crude papain during storage at room temperature was also protected by TT. A loss of 20% of the original PA occurred
over a period of 13 wk when crude papain contained 1% TT, compared to a loss of 45% when the crude enzyme preparation
contained 1% metabisulfite.
In the same chapter five different oxidants for synthesis of
TT from thiosulfate are compared, namely: iodine, hydrogen
peroxide, ferric chloride, cupric sulfate and sodium vanadate.
The results indicated that hydrogen peroxide or sodium vanadate
were not only effective in the oxidation but also much less expensive than iodine, which is the most popular oxidant for the synthesis of TT.
The results obtained in this chapter warrant the use of TT in the commercial production of commercial papain to prevent the destruction of the enzymes during harvesting, storage,
transportation and processing.
In the second chapter, chemical modification of pure papain by
TT is discussed. Optimization techniques were applied for
improving the precision of two methods used in this study:
circular dichroism (CD) and proteolytic activity determination.
Simplex optimization significantly improved repeatability and
signal to noise ratio of the CD scan of papain. A new
optimization approach, which was a combination of a central
composite rotatable design and simplex optimization, was
successfully applied to achieve maximum precision for the
proteolytic activity assay of papain using casein as a substrate.
This approach may also be applied to other analytical methods to
improve the reliability of the experimental data.
Influential factors in the inactivation of PA of papain by
using TT and reactivation of the inactivated papain by cysteine
were carried out using two Taguchi's Lie (21S) fractional
factorial designs. The results indicated that when inactivation
was carried out at pH 6.8, with a reaction time of 5 min at
22<>C, and a molar ratio of TT to papain of 10, the inactivation
reaction was highly reversible upon addition of 20 mM cysteine.
-iii- Although some interactions of the factors were significant, 70% reactivation was achieved in most cases.
Analysis of UV absorbance, near-UV and far-UV CD spectra
indicated that there were no major changes in the spectra in papain upon the chemical modification of the enzyme with TT.
Secondary structure computed from far-UV CD spectra also demonstrated no significant changes upon this modification.
Sulfhydryl data and pH-fluorescence profiles of the modified
papain support the hypothesis that reversible blocking by TT
results from binding with the single reactive cysteine residue
present in papain. Quenching of the intrinsic fluorescence of
papain when the modification was carried out using high molar
ratios of TT to papain was suggestive of modification of
tryptophan residues in the enzyme during the oxidation reaction
with TT.
Precipitation or insolubilization of pure papain, and of the
proteins of papaya latex and commercial papain was observed upon
the chemical modification with TT under certain conditions.
Addition of fl-mercaptoethanol and TT at levels of 100 mM and 50
mM, respectively, precipitated 90% of pure papain.
Solubility studies together with electrophoretic analysis of
the precipitated papain suggested formation of insoluble
aggregates due to the insoluble aggregation as a result of
inter-molecular disulfide bonds formation.
TT was found to be a competitive inhibitor of both reversible
and irreversible inhibition of the enzyme action, when
-iv- carbobenzoxyglycine p-nitrophenyl ester was used as a substrate.
The second order inactivation constant in the absence of substrate was computed to be 16,919 M^sec"1, indicating that the reaction had a high rate.
-v- TABLE OP CONTENTS Page
ABSTRACT ii
TABLE OF CONTENTS vi
LIST OF TABLES xii
LIST OF FIGURES XV
LIST OF APPENDICES xxi
ACKNOWLEDGEMENTS XXi i
GENERAL INTRODUCTION 1
CHAPTER 1. Protection of the proteolytic activity of papaya latex and crude papain by tetrathionate
LITERATURE REVIEW 4 A. Definitions 4 B. Production of papaya latex 5 1. Agronomic factors 5 2. Papaya latex harvesting 6 3. Yields of papaya latex 7 C. Production of crude papain and commercial papain 8 1. Drying 8 2. Refining 10 3. Grades and prices of crude and commercial papain 11 D. Losses of proteolytic activity of papaya latex due to drying and during storage of crude and commercial papain 13 E. Process to improve the stability of crude and commercial papain 15 1. Improvement of tapping and collecting procedures 16 2. The Boudart process 16 3. Addition of reducing agents 17 F. Sodium tetrathionate as a stabilizing agent of sulfhydryl proteases 18 1. Mechanism of reaction 18 2. Reversible inactivation 19 G. Chemical properties of tetrathionate 21 1. Some properties of tetrathionate 22 2. Structure of tetrathionate... 23 3. Uses 23 4. Toxicity 25
-vi- Page
H. Preparation of tetrathionate 27 1. Iodine oxidation 29 2. Oxidation with other compounds 31 (a) Oxidation with hydrogen peroxide 31 (b) Oxidation with metal salts 32 (c) Oxidation with vanadate 32 3. Other methods of synthesis of tetrathionate 33
MATERIALS AND METHODS 35 A. Materials 35 B. Rehydration of crude papain 35 C. Drying characteristics of papaya latex 36 D. Determination of influential factors on the losses of proteolytic activity due to drying of papaya latex..36 E. Effect of different types of drying and additives on the losses of proteolytic activity of papaya latex..40 P. Losses of the proteolytic activity of crude papain during storage 41 G. Proteolytic activity assay 41 H. Preparation of sodium tetrathionate 43 1. Iodine oxidation 43 2. Hydrogen peroxide oxidation 43 3. Ferric oxidation 44 4. Cupric oxidation 45 5. Vanadate oxidation 46 I. Determination of purity and yield 46 1. Iodate-iodine titration 46 2. Alkaline cyanolysis 49 3. Melting point determination 50 4. Calculation of purity and yield 50 J. Cost evaluation 51 K. Inactivation/activation efficiency of the synthesized tetrathionates 51
RESULTS AND DISCUSSION 53 A. Drying Rates of papaya latex 53 B. Determination of influential factors on the losses of proteolytic activity due to drying of papaya latex 57 C. Effect of different types of drying and additives on the loss of proteolytic activity of papaya latex 62 D. Effect of additives on loss of the proteolytic activity of crude papain during storage 64 E. Comparison of the different methods to synthesize tetrathionate 66
-vii- Page
F. Cost evaluation of the different methods of tetrathionate synthesis 68 G. Melting point determination 74 H. Inactivation/activation efficiency of the synthesized tetrathionates 96
CONCLUSION 98
CHAPTER 2. Chemical modification of papain by tetrathionate
LITERATURE REVIEW 100 A. Chemical modification of proteins 100 B. Chemical modification of food related proteins 102 C. Modification of sulfhydryl groups in proteins 103 1, Oxidation 104 (a) Modification by aromatic disulfides 106 (b) Modification by tetrathionate 107 I. Tetrathionate as a stabilizing agent for sulfhydryl proteases 108 II. Tetrathionate as a blocking agent of cysteine residues 108 III. Tetrathionate as a chemical modification agent of cysteine residues 109 (c) Modification by iodobenzoates and mercurials....112 2. Alkylation 113 D. Papain 114 1. Definition and isolation 114 2. Physicochemical properties and structure 115 3. Stability 118 4. Activity 119 E. Chemical modification of papain 119 1. Modification of Cys-25 120 2. Modification of other residues 121 F. Enzyme kinetics 125 1. Reactions rates 125 (a) Zero order reactions 125 (b) First order reactions 126 (c) Second order reactions 126 I. Type I 127 II. Type II 127 III. Type III 127 2. States of an enzymatic reaction 128 (a) The pre-steady state 128 (b) The steady state 130 (c) The nonlinear state 130
-viii- Page
3. Measurement of velocity of enzyme catalyzed reactions 130 (a) Effect of substrate concentration on the initial velocity 131 (b) Determination of Km and Vmax 133 G. Determination of initial velocities 134 H. Nitrophenyl esters as substrates for papain 136 1. Steady state kinetics for the papain catalyzed hydrolysis of carbobenzoxyglycine p-nitrophenyl ester 137 2. Pre-steady state kinetics for the papain catalyzed hydrolysis of carbobenzoxyglycine p-nitrophenyl ester 138 I. Enzyme inhibitors 140 1. Definition and classification .140 (a) Irreversible inhibitors 140 I. Kinetics of irreversible inhibitors 141 II. Irreversible inhibitors of sulfhydryl enzymes 144 (b) Reversible inhibition 144 I. Kinetics or reversible inhibition 145 II. Competitive inhibition 145 III. Uncompetitive inhibition 146 IV. Noncompetitive inhibition 146 V. Reversible inhibition of sulfhydryl enzymes 146
MATERIALS AND METHODS 149 A. Materials 149 B. Determination of protein concentration 149 C. Determination of proteolytic activity 150 1. Optimization of the conditions to measure proteolytic activity of papain 150 2. Proteolytic activity determination 151 D. Determination of the influencial factors for maximum inhibition and reactivation of the proteolytic activity of papain by tetrathionate 154 E. Preparation of tetrathionate modified papain 158 F. Circular Dichroism ....159 1. Optimization of the conditions for measuring the CD spectra of papain 160 2. Far-UV CD Spectra (190-240 nm) 161 3. Near-UV Spectra (250 -350 nm) 162 G. Secondary structure prediction 163 H. Fluorescence and differential absorption spectroscopy 163 I. Determination of total -SH groups of papain 165
-ix- Page
J. Insolubilization of papain with tetrathionate 166 1. Effect of concentration of tetrathionate and 0-mercaptoethanol on the precipitation of papain 166 2. Effect of pH and temperature on the precipitation of papain by tetrathionate 168 3. Resolubilization of the precipitated protein 168 4. Analysis of the precipitated protein 169 K. Determination of Vmax and Km for the papain-catalyzed hydrolysis of carbobenzoxyglycine p-nitrophenyl ester 171 L. Inhibition experiments 173 1. Reversible inhibition experiments 173 2. Irreversible inhibition experiments 174 (a) In the absence of substrate 174 (b) In the presence of substrate 175 M. Statistical analysis 176
RESULTS AND DISCUSSION 177 A. Optimization of the conditions to measure proteolytic activity of papain 177 1. Localization of optimum conditions 180 B. Determination of the influencial factors for maximum inhibition by tetrathionate and reactivation by cysteine of the proteolytic activity of papain 189 C. Optimization of the conditions for measuring the CD spectra of papain 201 D. CD of native and tetrathionate modified papain 208 E. Secondary structure of native and tetrathionate-modified papain 212 F. Fluorescence and UV-absorption of native and tetrathionate modified papain 214 G. Determination of -SH groups in native and tetrathionate modified papain 219 H. Insolubilization of papain with tetrathionate 221 I. Kinetics parameters for the papain-catalyzed hydrolysis of carbobenzoxyglycine p-nitrophenyl ester 236 J. Characterization of the inhibition effect of tetrathionate 244 1. Reversible inhibition 244 2. Irreversible inhibition 245 (a) In the presence of substrate 245 (b) In the absence of substrate 248
-x- Page
CONCLUSIONS 255
REFERENCES CITED 259
APPENDIX 275
-xi- LIST OF TABLES
Page
Table 1. Conditions reported for oven drying of
papaya latex 9
Table 2. Prices of crude, commercial and pure papain 12
Table 3. Standard electrode potential (E°) for some chemical reactions 28 Table 4. Chemical reactions in the synthesis of tetrathionate 30
Table 5. Factors and their assigned levels investigated for their possible influence on the losses of proteolytic activity of papaya latex 38
Table 6. Analysis of variance (Taguchi's L»» 3") for proteolytic activity values of papaya latex obtained from 27 drying experiments 58
Table 7. Mean retention of the proteolytic activity of the crude papain resulting from papaya latex treated with 1% tetrathionate or 1% metabisulfite and dried using different methods 63
Table 8. Yields and purity of tetrathionate synthesized by the different methods 67
Table 9. Commercial price of tetrathionate, metabisulfite and chemicals used to synthesize tetrathionate 70
Table 10. Inactivation and activation efficiency of tetrathionates synthesized using different methods 97
Table 11. Oxidation products of sulfhydryl groups 105
Table 12. Physicochemical properties of some cysteine proteases 116
Table 13. Michaelis-Menten parameters for the papain catalyzed hydrolysis of carbobenzoxyglycine p-nitrophenyl ester 139
Table 14. Upper and lower limits for the three factors used for optimization of the proteolytic activity determination of papain 151
-xii- Page
Table 15. Factors and their assigned levels investigated for their possible influence on the inhibition of the proteolytic activity of papain, and on the subsequent reactivation of the activity by cysteine 155
Table 16. Levels of the factors used in the RSM experiment of the precipitation of papain by tetrathionate 167
Table 17. Central compositive rotatable design matrix used for the optimization of the conditions to measure proteolytic activity of papain, and results for each experiment 178
Table 18. Analysis of variance of the second order model 179
Table 19. Analysis of variance for the modified second order model 181
Table 20. Analysis of variance (Taguchi's Lx« 2") for the inhibition of the proteolytic activity of papain by tetrathionate 191
Table 21. Analysis of variance (Taguchi's Li« 21*) for the reactivation by cysteine of the proteolytic activity of tetrathionate-inactivated papain 193
Table 22. Simplex-centroid optimization of the conditions for CD spectrophotometry of papain 203
Table 23. Values of mean residue ellipticity and corresponding coefficient of variation at three wavelengths for the different vertices of the simplex-centroid optimization 207
Table 24. Predicted secondary structure fractions of native and tetrathionate modified papain based on CD data, using two algorithms, and X-ray determine secondary fractions 213
Table 25. Sulfhydryl content of native and tetrathionate modified papain 220
-xiii- Page
Table 26. Experimental data for the two-factor, five-level response surface analysis of the effect of tetrathionate and 0-mercaptoethanol concentration on the precipitation of papain 223
Table 27. Analysis of variance for the second order model for the precipitation of papain by tetrathionate, obtained using backward multiple regression 224
Table 28. Effect of temperature and pH on the precipitation of papain by tetrathionate 226
Table 29. Effect of different reagents on the resolubilization of precipitated papain 228
Table 30. Proteolytic activity of native and insoluble papain 230
Table 31. Values of B and corresponding empirical reaction order with respect to time at different initial substrate concentrations for the reaction of papain-carbobenzoxyglycine p-nitrophenyl ester 240
Table 32. Results of curve fitting the kinetics data for the reaction of papain-carbobenzoxyglycine p-nitrophenyl ester at various initial substrate concentrations. Initial velocites (Vo) determined by the fixed time assay method are included for comparative purposes 242
Table 33. Kinetics parameters of the reaction of the papain-carbobenzoxyglycine p-nitrophenyl ester reaction (+ standard error), computed with the program of Oestreicher and Pinto (1983) using initial velocities estimated by fixed time assays or derived from experimentally determined curves. 243
Table 34. Second order rate constants for inactivation for some papain inhibitors and for tetrathionate with two different enzymes 254
-xiv- LIST OF FIGURES
PAGE
Figure 1. Scheme used in the Taguchi Li? fractional factorial experiment 39
Figure 2. The effect of three different drying temperatures on the drying rate of papaya latex at a drying load of 2,381 g/m* 54
Figure 3. The effect of drying load on the drying rate of papaya latex at a drying temperature of 80°C 55
Figure 4. The effect of the addition of 1% tetrathionate or metabisulfite on the drying rate of papaya latex at 550C and drying load of 2,381 g/m* 56
Figure 5. Effect curve for the interaction between drying temperature and treatment prior to drying on the proteolytic activity retained by crude papain. (Meanj-Confidence limits calculated at p<0.05) 59
Figure 6. Effect curve for the interaction between drying load and treatment prior to drying on the proteolytic activity retained by crude papain 61
Figure 7. Change in the proteolytic activity of crude papain with or without additives during storage at room temperature 65
Figure 8a. The cost of synthesis of tetrathionate for the different methods evaluated. Cost including that of the solvents 71
Figure 8b. The cost of synthesis of tetrathionate for the different methods evaluated. Cost without including that of the solvents 72
Figure 9. Distribution of the cost of synthesis of tetrathionate using the iodine and the hydrogen peroxide methods 73
Figure 10. Typical DSC thermogram of a commercial tetrathionate sample from ICN Pharmaceutical, Inc. Tetrathionate content (%) determined using the iodate-iodine reaction (Mean+S.D n=3)= 102.0 + 5% 76
-xv- PAGE
Figure 11. Typical DSC thermogram of tetrathionate synthesized using the iodine reaction. Tetrathionate content (%) determined using the iodate-iodine reaction (Mean+S.D n=3)= 98.31+6% 78
Figure 12. Typical DSC thermogram of tetrathionate synthesized using the peroxide method. Tetrathionate content (%) determined using the iodate-iodine reaction (Mean+S.D n=3)= 37.9 + 6% 80
Figure 13. Typical DSC thermogram of tetrathionate synthesized using the cupric method. Tetrathionate content (%) determined using the iodate-iodine reaction (Mean+S.D n=3)= 20.4 + 8% 82
Figure 14. Typical DSC thermogram of tetrathionate synthesized using the ferric method. Tetrathionate content (%) determined using the iodate-iodine reaction (Mean+S.D n=3)= 18.3 + 5% 84
Figure 15. Typical DSC thermogram of tetrathionate synthesized using the vanadate method with hydrochloric acid. Tetrathionate content (%) determined using the iodate-iodine reaction (Mean+S.D n=3)= 38.6 + 7% 86
Figure 16. Typical DSC thermogram of tetrathionate synthesized using the vanadate method with acetic acid. Tetrathionate content (%) determined using the iodate-iodine reaction (Mean + S.D n=3)= 35.0 + 7% 88
Figure 17. Typical DSC thermograms of a sample of pure thiosulfate from Fisher Scientific Comp 91
Figure 18. Typical DSC thermogram of a mixture of pure thiosulfate and tetrathionate at a 1:1 (w/w) ratio 93
Figure 19. Effect of addition of thiosulfate on the area of two endothermic peaks of tetrathionate 94
Figure 20. Effect of addition of thiosulfate on the melting point of tetrathionate 95
-xv i- PAGE Figure 21. Progress reaction curve for an ideal enzyme reaction 129
Figure 22. Typical graph of the initial velocity of an enzymatic reaction as a function of initial substrate concentration 132
Figure 23. Scheme used in the Taguchi Lit fractional factorial experiment 156
Figure 24. Plot of predicted values according to the modified second order model of the SD of the determination of the proteolytic activity of papain against the corresponding experimental ones 182
Figure 25. Plot of residuals for the modified second order model 183
Figure 26. Computational optimization used to obtain the best experimental conditions for proteolytic activity determination of papain. The response is the standard deviation calculated using the modified second order model 185
Figure 27. Response surface of standard deviation against incubation time and enzyme concentration, at a constant incubation temperature of 35<>C 186
Figure 28. Response surface of standard deviation against incubation time and temperature, at a constant papain concentration of 0.1 mg/ml 187
Figure 29. Response surface of standard deviation against incubation temperature and enzyme concentration, at a constant incubation time of 5 min 188
Figure 30. Effect of two levels of the molar ratio of tetrathionate to papain in the inactivation of papain 192
Figure 31. Effect curve for the interaction between the molar ratio of tetrathionate to papain and reaction time of inactivation reaction on the % reactivation of the proteolytic activity of papain 195
-xvii- PAGE
Figure 32. Effect curve for the interaction between the molar ratio of tetrathionate to papain and pH of the inactivation reaction on the % reactivation of the proteolytic activity of papain 197
Figure 33. Effect curve for the interaction between the cysteine concentration during the reactivation and pH of the inactivation on the % reactivation of the proteolytic activity of papain ....198
Figure 34. Effect curve for the interaction between the cysteine concentration during reactivation and temperature of inactivation on the % reactivation of the proteolytic activity of papain 199
Figure 35. Effect curve for the interaction between the cysteine concentration during reactivation and time of inactivation on the % reactivation of the proteolytic activity of papain 200
Figure 36. Effect curve for the interaction between the pH and temperature of inactivation on the % reactivation of the proteolytic activity of papain 202
Figure 37. Trace of the far-UV CD spectrum of papain measured under different conditions. (A) CD spectrum measured under the conditions of vertex 1. (B) CD spectrum measured under the optimum conditions (Vertex 15) 205
Figure 38. Far-UV CD spectra of native and TT-papain (molar ratio tetrathionate to papain during reaction = 200). Note: both proteins gave the same spectra 209
Figure 39. Near-UV CD spectra of native and TT-papain (molar ratio tetrathionate to papain during reaction = 200)... 211
Figure 40. Effect of pH on the relative fluorescence intensity at 22°C of native papain 215
-xviii- PAGE
Figure 41. Effect of pH on the relative fluorescence intensity at 22°C of TT-papain (molar ratio tetrathionate to papain during reaction = 200) 216
Figure 42. Effect of the molar ratio of tetrathionate to papain during the chemical modification on the relative fluorescence intensity at 352 nm (pH 6.8 and 22<>c) 218
Figure 43. Response curve for the effect of tetrathionate and 0-mercaptoethanol concentration on the precipitation of papain 222
Figure 44. Change of pH during the reaction between tetrathionate, 0-mercaptoethanol and papain. Final [tetrathionate] = [B-mercaptoethanolJ = 100 mM, final [papain]= 10 mg/ml 227
Figure 45. Electrophoretogram of native and insoluble papain 231
Figure 46. Electrophoretogram of soluble and insoluble papaya latex and commercial papain 233
Figure 47. Electrophoretogram under non-reduced conditions of soluble tetrathionate-raodidified pure papain and of tetrathionate-treated commercial papain and papaya latex 234
Figure 48. Papain-catalyzed hydrolysis of carbobenzoxyglycine p-nitrophenyl ester at pH 6.8 and 22<>C. The reaction mixture contained 0.02 M sodium phosphate buffer 1 mM EDTA, 0.35 mM cysteine, 6.7% (v/v) acetonitrile and 3.3 x 10"T papain. Initial substrate concentration as shown 237
Figure 49. Schematic diagram of the system used for the enzyme kinetics experiments 238
Figure 50. Idealized progress reaction curve (A«0o vs. time) for the papain-catalyzed hydrolysis of carbobenzoxyglycine p-nitrophenyl ester 239
Figure 51. Lineweaver-Burk plot for the papain activity at different concentrations of tetrathionate 246
-xix- PAGE
Figure 52. Inhibition effect of tetrathionate (TT) in the presence of three different initial substrate concentrations at a constant TT concentration (1.06 x 10-J M) 247
Figure 53. Effect of carbobenzoxyglycine p-nitrophenyl ester concentration on the second order rate constant of inactivation of tetrathionate on papain. (Mean±S.D., n=3). The regression line is shown 249
Figure 54. Change in the catalytic activity of papain as a result of the reaction with tetrathionate. Activity was measured with carbobenzoxyglycine p-nitrophenyl ester as the substrate 250
Figure 55. Effect of the molar ratio of tetrathionate to papain on the catalytic activity of papain at two reactions times. Activity was measured with carbobenzoxyglycine p-nitrophenyl ester as the substrate 251
Figure 56. Progress of inactivation of papain by different levels of tetrathionate (TT). The value of Y was calculated based on Eq.ll when the molar ratio of TT to papain was 1:1, for the other molar ratios Eq. 12 was used. Activity was measured with carbobenzoxyglycine p-nitrophenyl ester as the substrate 252
-xx- LIST OF APPENDIX
PAGE
Appendix 1. Listing of the IBM-BASIC version of the computer program used by Siegel et al. (1980) to determine secondary structure fractions from CD spectra 275
Appendix 2. Listing of the IBM-BASIC computer program which utilizes the simplex algorithm of Morgan and Deming (1974) used for computational optimization of the conditions to measure the proteolytic activity of papain 277
Appendix 3. Listing of the IBM-BASIC computer program used to generate the data (grid) for plotting the response surface of the effect of 0-mercaptoethanol and tetrathionate on the precipitation of papain 280
-xxi- ACKNOWLEDGEMENTS
I sincerely wish to thank Dr. S. Nakai for suggesting this project, for many helpful comments and suggestions, for answering my many questions, for patience, and - by being such an expert in his field- for serving as a role model...I hope that someday I may know so much !
I would also like to thank professors B. J. Skura, J.
Vanderstoep, L. E. Hart and Dr. E. Li-Chan for their interest and painstaking task of checking the technical aspects and overall structure of this thesis. In my view, their sharp remarks have considerably improved the quality of this report.
I am also in debt to Ms. A. Smith and Ms. S. Weintraub for kindly helping with the formidable task of proofreading this thesis.
Finally, I must thank my best friend, my wife Cecy, whose constant support and encouragement helped to make this thesis possible and helped me keep my sanity during the writing of this work .
Financial support from the Consejo Nacional de Ciencia y
Tecnologia (CONACYT) (Mexico) in the form of a Graduate
Scholarship is gratefully acknowledged.
-xxii- GENERAL INTRODUCTION
Food-related enzymes can be classified in two major groups: those enzymes which are added to food and those which occur naturally in foods (Beck and Scott, 1974).
Although many enzymes have been isolated, there are less than
20 industrial enzymes on a scale that have had significant impact in either the enzyme industry or the food industry. Most industrial enzymes are usually not highly purified and relatively cheap, with a cost per kg of < US$10.00 (Hepner and Male, 1983).
These industrial enzymes can be classified in three categories: proteolytic, amylolytic and other enzymes. The proteolytic enzymes account for over 50% of the market and amylolytic enzymes for 33% (Hepner and Male, 1983).
Industrial proteolytic enzymes are derived from a wide range of sources and may have widely different pH and temperature optima and specificities. They are also one of the few groups of industrial enzymes obtained from animal, plant and microbial sources (Cheetham, 1985).
Among the proteolytic enzymes, plant proteases are the most widely used in the food industry. These enzymes are sulfhydryl or thiol proteases such as papain, ficin and bromelain, with papain being most commonly used (Liener, 1974).
-1- Chill-proofing of beer using papain, a process patented in
1911- represents the earliest industrial application of commercial papain and still represents the single most significant market for this enzyme (Brocklehurst et al., 1981).
The second largest use of papain is in meat tenderization
(Liener, 1974). Commercial papain has also been used in biscuit products and for the preparation of protein hydrolyzates
(Brocklehurst et al., 1981). Many other uses of papain for food applications have been cited but most of them have not been commercialized.
Enzymes, unlike many other substances, are recognized and sold by their activity rather than their weight, so the stability of the enzyme during its isolation, purification, formulation and storage is of prime importance (Cheetham, 1985).
It is well documented that during the manufacturing process of commercial papain a significant loss of proteolytic activity (PA) occurs. In addition, commercial papain loses its PA during storage, especially when it is in solution (Liener, 1974).
As a sulfhydryl enzyme the major causes of PA losses in papain are irreversible oxidation of its essential cysteine residue and autolysis. In order to prevent losses of PA in the commercial processing of papaya latex the addition of reducing agents (e.g. sulfites) prior to drying is usually done.
-2- There were two objectives of the first part of this thesis research. The first objective was to evaluate a new method to stabilize the PA of papaya latex. This new method consisted of the addition of sodium tetrathionate to achieve controlled oxidation or blocking of the sulfhydryl groups of the proteases of papaya latex. Due to the fact that sodium tetrathionate is presently more expensive than sulfites, the second objective was to develop a chemical method to synthesize sodium tetrathionate at a cost that will make this new method of protection of the PA of papaya latex more economically attractive.
Since the reaction between tetrathionate and the enzymes in papaya latex was basically a chemical modification of the proteins, the second part of this study examined the effect of sodium tetrathionate on selected physicochemical properties of the pure enzyme papain. In addition, kinetics of the reaction between papain and tetrathionate are reported.
-3- CHAPTER 1. Protection of the proteolytic activity of papaya latex and crude papain by tetrathionate.
LITERATURE REVIEW
A. DEFINITIONS
The material termed "papain", in industrial usage, is not a pure form of the enzyme (E.C. 3.4.22.2), but a partially purified preparation of the latex of papaya (Caricia papaya).
Papaya latex contains, in addition to the pure enzyme papain
(E.C. 3.4.22.2), other cysteine proteases, cellulase, lysozyme, glutamine cyclotransferases and low molecular weight thiols
(Caygill, 1979). Surprisingly, pure papain is not the major component of papaya latex (Goodenough and Owen, 1987). The
individual enzyme papain (E.C. 3.4.22.2) is the most thoroughly characterized of the cysteine proteases. Its structure has been determined by X-ray diffraction studies and its physico-chemical characteristics and mechanism of action are still the subject of intensive study in many laboratories
(Brocklehurst et al., 1981).
In order to avoid confusion, the term "pure papain" will be used in this paper to describe the individual proteolytic enzyme (E.C. 3.4.22.2); "crude papain" will be used to indicate the dry papaya latex without further purification; and
"commercial papain" will be used to name the product resulting from partial purification of crude papain.
-4- B. PRODUCTION OF PAPAYA LATEX
The production of papaya latex has changed little in recent decades. The major cost is the establishment and maintenance of the plantation (Caygill, 1979).
The production and processing of papaya latex has been discussed in detail by Jones and Mercier (1974), Caygill (1979) and more recently by Poulter and Caygill (1985).
1. Agronomic Factors
Poulter and Caygill (1985) covered the main agronomic factors that influence a papaya plantation. An average diurnal temperature range of 31 to 33°C with a regular rainy season lasting from six to eight months, followed by dry weather appears to be ideal. A slightly acidic soil (pH 6.0 to 6.5) is preferable, and near the equator in Africa, altitudes of about
1000 m are considered favorable for papaya cultivation for latex
(Poulter and Caygill, 1985). There is little information about the best varieties for papain yield, although Lassoudiere
(1969) mentioned Red Panama, Floride, Richbourg and Ineac 329 as among the best varieties. These cultivars were reported to have latex yields several times that of Solo, the variety favored for fruit production in Hawaii (Poulter and Caygill,
1985).
-5- 2. Papaya latex harvesting
Latex is harvested by tapping unripe fruit about four months after setting and about six weeks before ripening. Tapping involves the cutting of longitudinal incisions on the surface of the green unripe fruit using a sharp blade made of stainless steel, such as a razor blade (Poulter and Caygill, 1985;
Brocklehurst et al., 1981).
The latex is found in the laciferous ducts only 1-2 mm below the surface. Cuts must not be deep, in order to avoid infections of the fruit and contamination of the latex with fruit juice
(CONAFRUT, 1973). The latex flows for only a few minutes and drips into cups (Brocklehurst et al., 1981), or into collection devices, resembling inverted umbrellas attached around the trunk of the papaya tree (Poulter and Caygill, 1985).
Additional latex coagulates on the fruit and is subsequently scraped off. Although Brocklehurst et al. (1981) mentioned that this coagulated latex was a second grade material, with a lower enzyme content, Madrigal et al. (1980) reported no difference in proteolytic activity between the coagulated and the first latex.
The fruit should be wiped with a cloth soaked with sulfites or a suitable fungicide to prevent infection of unprotected cuts
(Salunke and Desai, 1984).
Fruit may be tapped several times, at approximately 4-day intervals, until ripening. After the final tapping, the fruit
-6- should be removed from the tree (Poulter and Caygill- 1985).
3. Yields of papaya latex
The yields and quality of the latex depend on many factors including fruit sex (Madrigal et al., 1980), size of the fruit
(Castro, 1981), soil characteristics (Becker, 1958), temperature and season (Castro, 1981). Yields of fresh latex are frequently unreliable guides to yield of proteolytic activity (Madrigal et al., 1980). Fruits from the same tree give different enzyme concentrations in the latex (Madrigal et al., 1980).
Yields in carefully managed plantations can be as high as
1,200 Kg of latex/ha per year (Lassoudiere, 1969), though yields are often appreciably lower (Lewis and Woodward, 1948).
Krishnamurty et al. (1960) and Madrigal et al. (1980) reported fresh latex yields of 3-5 g/fruit/day (approximately
0.17% of the fruit weight). However, Castro (1981) reported a much lower production of 0.22-0.38 g/fruit/day under different climatic conditions. Kimmel and Smith (1957) indicated that the average annual harvest of dried latex was approximately 100 g per tree.
Shanmugavelu et al. (1976) reported that the yields of papaya latex were increased four fold over the controls with the use of the latex stimulant compound Ethephon.
-7- C. PRODUCTION OF CRUDE AND COMMERCIAL PAPAIN
1. Drying
Papaya latex contains approximately 15-20% total solids and must be dried without delay in order to increase its stability (Poulter and Caygill, 1985). The moisture content of crude papain (dry papaya latex) is usually about 5-10% (Ortiz et al., 1980; Vaidya et al., 1977).
The earliest commercial methods of drying papaya latex were sun drying or kiln drying. The latex was thinly spread on trays or on cotton cloths and dried by exposure to the sun or by open fire in an oven (Poulter and Caygill, 1985). Sun drying causes browning of the papaya latex resulting in a considerable loss of enzymatic activity. The final dry latex
(i.e. crude papain) is frequently malodorous, as a result of microbial growth (Salunke and Desai, 1984). Exposure to ultraviolet radiation in sunlight reduces proteolytic activity, and thus these methods are not recommended (Poulter and
Caygill, 1985; Jones and Mercier, 1974). Becker (1958) recommended a drying shed in which latex can be dried at 55°C by indirect heat. Cabinet dryers have been used to prepare crude papain as a white granulated powder of higher activity than that obtained by sun or kiln drying (Poulter and Caygill, 1985; Ortiz et al., 1980). A summary of the different conditions used for oven drying of papaya latex is shown in Table 1.
-8- Table 1. Conditions reported for oven drying of papaya latex.
Drying Conditions
Temperature Time Load Reference*- (°C) (hr) (g/m«) Additives
(1) 55 24 2,083 no
(2) 40-80 N.R.B N.R. no
(3) 55 3-4 944 0.5% PMBC
(4) 55 6 1,600 1.0% SMBD+ 0.2% EDTA
*Reference: (1) Becker (1958) (2) Castro (1981) (3) Krishnamurty et al. (1960)
(4) Ortiz et al. (1980)
^.R. = Not reported in the reference
^MB = Potassium metabisulfite
DSMB = Sodium metabisulfite
-9- Drying under vacuum (55<>c and 28 in vacuum) minimizes proteolytic activity losses as compared to sun or oven drying
(Krishnamurty et al., 1960).
The patented Boudart process (Boudart, 1968; 1970; 1972)
involves filtration and centrifugation of the freshly collected latex to remove smaller insoluble impurities. The latex is concentrated under vacuum at low temperature to 25% (w/v) solids or more. The liquid passes to a conventional low capacity spray drier. This process produces a fine white to off-white powder with high proteolytic activity (Jones and Mercier,
1974) .
2. Refining
Crude papain is usually exported and refined in the primary importing country (Brocklehurst et al., 1981). Refining consists of dissolving the soluble constituents, including the enzymes from the latex, and precipitating the enzymes with agents (e.g. ammonium sulfate) that do not inactivate them
(Caygill, 1979).
The main objective of refining is to produce a stable, readily soluble material, with a low microbial count and substantially free from the gummy substances and the offensive flavor found in crude papain (EDC, 1985).
In the United States imported crude papain is purified by numerous methods, which generally involve filtration, solvent or chemical precipitation and/or spray drying, to yield commercial
-10- purified enzyme preparations of various grades. These grades are usually standardized by adding lactose, sucrose, or starch as a diluent, to give commercial papain (Leung, 1980; Miles, 1985).
These purified preparations, often called "papain", are not pure papain. Pure papain is not commercially available on a large scale, and is used mainly for research purposes (Leung, 1980).
3. Grades and prices of crude and commercial papain
Until the mid 1950s when the trade was dominated by supplies from Sri Lanka, three grades of crude papain were known: (1) fine white powder form prepared by a specific process, (2) oven-dried white crumb, and (3) sun-dried dark crumb (Salunke and Desai,
1984). Since 1970, other grades of papain, resulting from new processing techniques, have arrived on the market. As a result, crude papain can be reclassified into three groups
(Salunke and Desai, 1984):
1. Crude papain: ranging from first grade white to second
grade brown.
2. Crude papain in flake or powder form sometimes referred to
as semirefined.
3. Spray-dried crude papain of higher activity, in powder
form.
Table 2 presents prices, obtained from different sources, of crude and commercial papain. Proteolytic activity determines the price of commercial papain. A direct comparison of the prices of commercial papain is sometimes difficult because of the different
-11- Table 2. Prices of crude, commercial and pure papain.
Type of Price papain Grade (USD/kg) Source*
Crude Brown No. 2 3. 60 - 5.20 (1)
Crude White No.2 5. 20 - 8.60 (1)
Crude Al 3. 00 - 8.50 (2)
Crude Type I 103 .40 (3)
Crude Type II 148 .50 (3)
Commercial Papain 16,00 24 .25 (4)
Commercial Papain 30,000 44 .09 (4)
Pure 2X Crystallized 115 .55* (3)
"•Sources: (1) Salunke and Desai (1984) (2) Becker (1958) (3) Sigma Chemical Co. Ltd (1988) (4) Miles Laboratories (1987)
BPrice for pure papain in USD/g
-12- methods used by enzyme marketing companies to measure the activity of the products. The price of the pure enzyme papain is also included in Table 2.
Prices of commercial papain exhibit a cyclical behavior, rising to a higher figure and falling, a full cycle taking about five years. There is also an overall tendency for exports and prices to rise slowly over time (Flynn, 1975). A more detailed discussion on the international trade of crude and commercial papain can be found in Flynn (1975).
D. LOSSES OF PROTEOLYTIC ACTIVITY IN PAPAYA LATEX DUE TO DRYING AND DURING STORAGE OF CRUDE AND COMMERCIAL PAPAIN
The proteolytic activity (PA) of commercial papain determines its commercial value in a wide range of applications (Miles,
1985). Thus any losses of PA in crude papain bring about a direct economic loss to the producer.
Papaya latex and crude papain are very unstable products, losing PA and hence commercial value, in relatively short periods of time (Castro, 1981; CONAFRUT, 1973).
Factors involved in the losses during storage and subsequent drying of the papaya latex include: the activity of latex protease inhibitors such as vitamin C and isothiocyanates; microbial degradation of the enzymes; autoproteolysis; irreversible oxidation of cysteine residues; and light sensitivity of the histidine residue in papain, which is essential for activity (Ortiz et al., 1980; Brocklehurst et
-13- al., 1981). There is limited information available about the losses of PA during storage and drying of papaya latex, and during storage of crude papain. Exposure to sunlight (CONAFRUT,
1973; Salunke and Desai, 1984) and extended storage prior to drying (CONAFRUT, 1973; Becker, 1958) are not recommended, but no quantitative data are reported.
Ortiz et al., (1980) investigated the effects of storage prior to drying on the PA of fresh papaya latex. They reported a maximum loss of 20% of the PA after storage for 2 to 24 hr under tropical conditions. Most of the decrease in PA occurred in the first 6 hr of storage. Under commercial practice, the minimum delay after tapping and before drying is likely to be about 2 hr (Ortiz et al., 1980), but often is 6-8 hr (Becker,
1958). Storage under sunlight causes slightly greater losses than storage in the dark (Ortiz et al., 1980). An assay of four samples of fresh latex (4 hr old at time of assay) in comparison with crude papain from the same latex, indicated the
PA loss due to oven drying at 55°C was 7+2% (Ortiz et al.,
1980). Another report (CONAFRUT, 1973) mentions that activity loss during oven drying could be as high as 20%.
Sun-dried crude papain was found to have 20-32% less milk clotting activity as compared with vacuum-dried crude papain (Krishnamurty et al., 1960). Castro (1981) reported that the difference of PA between sun-dried and oven-dried crude papain was only in the order of 10%. The same author found that the loss of PA due to storage of crude papain over a period
-14- of 75 days was 30% and 25% at room and refrigerated temperature, respectively.
Commercial papain is much more stable than crude papain.
According to one manufacturer (Miles, 1985), the losses in PA of commercial papain in sealed containers, stored under cool, dry conditions are normally less than 10% over one year. Storage life can be extended by storing under refrigeration at 5°C.
Dhawalikar and Pandit (1982) reported that the milk clotting activity of a "papain concentrate" (most likely commercial papain) stored for 100 days at 25 or 37QC, decreased by 16%. Most of the loss occurred within the first seven weeks.
E. PROCESS TO IMPROVE THE STABILITY OF CRUDE AND COMMERCIAL PAPAIN
Crude papain has relatively low stability. It must be stored at low temperatures to avoid losses of enzymatic activity.
Crude papain has been shown to lose much of its activity after storage for only a few months (CONAFRUT, 1973).
Many investigators have studied the problem of the high bacterial contamination and low stability of papaya latex and crude papain. Various methods either to improve the processing (i.e. tapping, drying and refining), or to chemically stabilize papain have been suggested (Jones and
Mercier, 1974 ) .
-15- 1. Improvement of tapping and collecting procedures
Tapping and collecting procedures have a direct effect on the quality of the crude papain. Poor tapping and collecting procedures still prevail despite the wide availability of information on the best techniques (Salunke and Desai, 1984).
Common practices in the tapping and collecting procedures that should be avoided are (Salunke and Desai, 1984): (1) tapping by incisions which are too deep, thereby allowing juices and starch from the fruit pulp to contaminate the latex; (2) tapping at times other than the morning or on overcast days, which results in quality reduction of the latex; (3) tapping too soon before maturity, or too long after maturity; (4) leaving the latex too long in the sun, where it can lose enzyme activity and collect foreign matter such as insects and dust.
2. The Boudart Process
The Boudart process, already described, produces a commercial papain with low bacterial contamination, good solubility and high proteolytic activity. Only mechanical methods of purification, such as filtration and centrifugation are used
(Baines and Brocklehurst, 1979; Caygill, 1979; Jones and
Mercier, 1974). The need of modern equipment, somewhat similar to that of a milk drying plant (Jones and Mercier, 1974), limits its applicability to only large scale operations. It is inappropriate to many countries where papain would be considered
-16- a by-product of the fruit on an intermediate scale (Ortiz et al., 1980; CONAFRUT, 1973).
3. Addition of reducing agents
The addition of reducing agents, such as sodium bisulfite or metabisulfite, in combination with a chelating compound (i.e.
EDTA) before drying, has been shown to decrease the losses of proteolytic activity due to drying by 20% as compared to an untreated sample (Ortiz et al., 1980). Krishnamurty et al.
(1960) found that addition of 0.5% metabisulfite to the latex before drying considerably improved the activity of crude papain (i.e. dry latex) when prepared by sun drying, but only slightly when prepared by vacuum drying. Jones and Mercier (1974) mentioned that chemical stabilization of latex brings improvement in retention of proteolytic activity, but the treatment of fresh latex should be done in areas where it is easy to export and import chemicals and biochemical products.
This is not generally the case in papaya growing districts.
Commercial papain usually contains a stabilizing agent such as sodium or potassium metabisulfite (Perez and Lopez-Munguia,
1985). Numerous preparations to stabilize commercial papain solutions have been described in the patent literature. Caygill
(1979) reported more than ten patents on this subject. A typical composition of a preparation to chill-proof beer includes the following (Rommele, 1978): 20-60% sucrose, 1.5-3% sodium metabisulfite and 1-35% papain.
-17- F. SODIUM TETRATHIONATE AS A STABILIZING AGENT OF SULFHYDRYL PROTEASES
Sodium tetrathionate, a symmetrical disulfide, has been shown to reversibly oxidize thiols quantitatively to their corresponding disulfides. The oxidation is promptly reversed by the addition of a reducing agent such as cysteine,
0-mercaptoethanol or dithiothreitol (Means and Feeney, 1971;
Pihl and Lange, 1962).
1. Mechanism of reaction
The reaction of tetrathionate with cysteine proteases can be seen as a controlled oxidation; it proceeds according to the following scheme:
E-SH + R-S-S-R <======» E-S-S-R + R-SH (1)
where E-SH refers to the active sulfhydryl enzyme such as papain, bromelain or ficin; R-S-S-R is the symmetrical disulfide inactivator (i.e tetrathionate); E-S-S-R is the reversibly inactivated enzyme in the form of a mixed disulfide; and R-SH is a low molecular weight thiol (Means and Feeney,
1971).
-18- 2. Reversible inactivation
Upon reaction of proteolytic enzyme with tetrathionate, the protease becomes reversibly inactive. Thus, the process of autolysis is inhibited. Tetrathionate also protects the active cysteine residue in cysteine proteases from irreversible oxidation (Englund et al., 1968). Autolysis and irreversible oxidation are the major causes of irreversible losses of enzymatic activity of cysteine proteases in general, and of papain in particular. It can be expected that the addition of sodium tetrathionate to papaya latex will increase its stability, and minimize loss of proteolytic activity during drying and storage.
Tetrathionate has been used with great success to prevent of autolysis of cysteine proteases during chromatographic purification or separation of: stem bromelain (Takahashi et al.,
1973); ficin (Englund et al., 1968); calotropin, a sulfhydryl protease isolated from the latex of the madar plant (Calotropis qigantea) (Pal et al., 1984); a sulfhydryl protease from sprouting potato tubers (Kitamura and Maruyama, 1986) and more recently sodom apple protease (Aworth, 1987). Use of tetrathionate results in higher yields of enzymes and an increased quality of separation.
Sodium tetrathionate also has an antimicrobial effect against some bacteria (Palumbo and Alford, 1970). It is possible that addition of tetrathionate to papaya latex will decrease the
-19- microbial load of the latex.
The addition of another disulfide, 2,21-dipyridyl disulfide
(2PDS) to fresh papaya latex in order to increase its stability has been reported (Brocklehurst et al., 1985).
The reversible inactivation of papain, with tetrathionate, was used by Kim Kam et al. (US Patent 3,818,106 (1974)) to prepare an enzyme formulation for injection ante mortem into animals as a meat tenderizer. Proteolysis occurred without causing severe physiological reactions in the injected animals, which might otherwise result in condemnation of the carcasses by governmental inspectors. In the Proten process
(developed by Swift and Company Limited), a concentrated solution of tetrathionate-inactivated papain is injected into the jugular vein, ten to thirty minutes before slaughter.
Continuing glycolysis depletes oxygen from the muscle of the freshly slaughtered carcass, resulting in an accumulation of free thiols and other reducing agents. Under these conditions, the oxidized papain may become reactive. The conversion is slow in chilled meat, but is completed rapidly on warming (Dransfield and Etherington, 1981). This process has been fully approved by the Meat and Poultry Inspection Program of the US Department of
Agriculture (Smith et al., 1973).
According to Bradley et al. (1987) in the United Kingdom, some hundred thousand cattle are treated annually by the
Proten process in 21 abbattoirs.
Recently it was confirmed (Bradley et al., 1987) that the
-20- Proten process does not cause behavioral or other clinical abnormalities in cattle following treatment. Carcasses were examined by gross visual assessment. It was concluded that the
Proten process did not cause detectable hepatocellular or renal damage. In another study, the Proten process significantly
increased the tenderness and overall satisfaction ratings of bullock steaks, in comparison to steaks from untreated bullocks
(Smith et al., 1973)
G. CHEMICAL PROPERTIES OF TETRATHIONATE
Compounds of the composition H2S*06 have been known for a long
time in the form of salts that are rather unstable in
1 2 J aqueous solution. This holds for the ions SJOG" , StOs' , SS06"
and SsOs'% which have been designated as polythionic acid
(Schmidt, 1972). Although salts of these compounds are usually
designated as polythionates, Na2S30s and Na2S40s are known as
sodium trithionate and tetrathionate, respectively. The proper
names of sulfane disulfonates, for example monosulfane disulfonate
fortrithionate, and disulfane disulfonate for tetrathionate,
should replace the established and customary older names
(Schmidt, 1972).
According to Schmidt (1972), the older literature on
these polythionic acids is overwhelming. The problems connected
with their chemical nature, reactions, formation, and
decomposition were for a long time one of the great
-21- puzzles in inorganic chemistry. The same author indicates that many of the old publications are frequently contradictory, which shows that no real understanding existed on the nature of the compounds.
1. Some properties of tetrathionate
Sodium and potassium tetrathionate are colorless, with platelike or prismatic crystals (Feher, 1963). Tetrathionate is readily soluble in water up to 12.6% and 23.2% at 0 and 20°C, respectively. They are insoluble in absolute alcohol (Feher,
1963). Aqueous solutions of the salts decompose into trithionate and sulfur if heated (Haff, 1970).
The pure dry material is stable for a long time without change, but decomposes if impurities (i.e. thiosulfates or sulfites) are present (Feher, 1963).
Tetrathionate is stable in cold concentrated acid (Schmidt,
1972). Fordos and Gelis (1842) reported that the diluted aqueous solution could be boiled without decomposition, but the concentrated solution decomposed into sulfur, sulfur dioxide and sulfuric acid.
Tetrathionate is very unstable in alkali solution, decomposing into sulfate and sulfite. Dilute aqueous solutions of tetrathionate decompose with time at room temperature (Schmidt,
1972) .
-22- 2. Structure of tetrathionate
Using X-ray crystallography for structure determination, Foss
(1960) reported that tetrathionate ions consist of two distorted
2 S203~ tetrahedra joined by a covalent bond:
Two types of sulfur-sulfur bonds occur in this compound,
between two divalent sulfur atoms in the middle of the chain and between one divalent and one sulfonate sulfur atom in the
ends.
3. Uses
Tetrathionate has been reported to be used as a fermentation aid in the production of penicillin (Peterson, 1959). It has also been reported to act against cyanide poisoning (SSrbo,
1972), as it reacts spontaneously with cyanide, the latter being converted to thiocyanate. Although an antidote effect has been established, tetrathionate is inferior to thiosulfate in this respect (Sorbo, 1972). Since tetrathionate has bactericidal action it has been used, to a limited extent, for medical formulations, especially lotions and creams (Sorbo,
1972) .
-23- Tetrathionate, produced In. situ - is widely used in a selective enrichment medium (Tetrathionate broth) for Salmonella (Poelma et al., 1984).
Calcium tetrathionate showed promising results as a fungicide against powdery mildew of cucumbers (Golyshin et al., 1967). An organic derivative of tetrathionate, bis(diisobutylamine) tetrathionate, has been used as a fungicide for powdery mildew
(Abylgaziev, 1967), and hydrazine tetrathionate has been shown to be useful for combating a parasitic fungus (Erysiphe graminis) of cucumber (Sanin et al., 1966).
Tetrathionate was also used to produce immunoglobulin preparations for intravenous use with decreased anti-complementary activity (Yoshida et al., 1980; Masuyasu and
Tomibe, 1979).
Damodaran (1986) reported a simple method of removal of nucleic acids from yeast nucleoprotein complexes by sulfitolysis.
This method involved the treatment of yeast nucleoproteins with sodium sulfite followed by sodium tetrathionate. This treatment caused destabilization and dissociation of the nucleoprotein complexes. Subsequent precipitation of proteins at pH 4.2 resulted in a protein preparation with low levels of nucleic acids (Damodaran, 1986).
In the area of biotechnology, tetrathionate was used to regenerate disulfide bonds in a genetically engineered microbial rennet (Hayenga et al., 1982).
-24- 4. Toxicity
The toxic action of tetrathionate is due to the fact that it rapidly oxidizes, in. vivo, sulfhydryl compounds to disulfides,
itself being reduced to thiosulfate (Sorbo, 1972; Gilman et al.,
1946a) .
Tetrathionate has been found to produce a strong nephrotoxic action causing complete anuria in dogs within 30 to 60 min
(Gilman et al., 1946a). The lethal dose of tetrathionate when injected intravenously (i.v.) into rabbits was 100 mg/kg of
body weight. Pathological examination of the kidneys revealed
necrosis of the cells of the proximal renal tubes (Gilman et
al., 1946a).
An impairment of kidney function has also been demonstrated
in human patients who received relatively large doses
of tetrathionate (Sorbo, 1972).
No LDso value has been reported for sodium tetrathionate. For
bis(diisobutylamine) tetrathionate LDso values obtained by
intramuscular injection were reported as 6,750 mg/kg for rats,
and 2,169 mg/kg for mice. Clonic spasms and death in the first day with inhibition of breathing were found to occur in both
species (Abylgaziev, 1967).
Comparison of the toxicities of tetrathionate and bisulfite, a compound commonly used as a protecting agent of the PA of papaya
latex, suggest that tetrathionate is not as toxic as bisulfite;
the i.v. LDso for bisulfite for rabbit is 65 mg/kg (FAO, 1974),
-25- compared to an approximate value of 100 mg/kg for tetrathionate.
Although LDB0 and lethal doses are expressions of the toxicity of a chemical compound, a more realistic procedure to assess the potential risk of a food additive must involve a comparison of the most probable consumption level to various expressions of toxicity (e.g. LDso or lethal dose values) (Vanderstoep, 1988).
In order to estimate the probable daily intake of tetrathionate by man, the following assumptions were made:
1) Commercial papain is the sole source of tetrathionate in
the diet;
2) For the calculation of the daily intake of tetrathionate
only the two major food uses of commercial papain, meat
tenderization and chillproofing of beer, are considered;
3) The per capita consumption of beer and red meat were
taken as 217.5 mL/day and 195.5 g/day, respectively
(Statistics Canada, 1987);
4) All beer was assumed to be chillproofed at a level of
8 ppm (8 mg/L) (Reed, 1966) using a commercial product
that had 16% of commercial papain (Perez and Lopez
Munguia, 1985), and a level of tetrathionate in the
commercial papain was taken as 1%;
5) Only 10% of the meat was tenderized with papain. The level
of tenderizer added to meat was taken as 12 g/kg meat
(Underkofler, 1972). The concentration of commercial papain
in the tenderizer was assumed to be 2% (Underkofler, 1972),
and the level of tetrathionate in the papain was again
-26- taken as 1%.
Taking these assumptions into consideration we have:
-Consumption of tetrathionate due to beer intake:
0.217 L/day x 8 mg/L x 0.16 x 0.01 = 0.0028 mg/day
-Consumption of tetrathionate due to meat intake:
0.02 kg/day x 0.012 kg/kg x 0.02 x 0.01 = 0.048 mg/day
Total consumption of tetrathionate = 51 \lq/ day
Using the i.v. lethal dose of 100 mg/kg for rabbits as being applicable to man a lethal dose of tetrathionate for the average man is 7,000 mg (70 kg x 100 mg/kg). The intake of 51 Hg/day is
0.00073% of the assumed lethal dose. This would suggest that the use of tetrathionate as an additive in commercial papain does not represent a health hazard.
Although tetrathionate is not a food additive, it is expected to be found in those products where sulfur dioxide (SO*) is used,
since S02 is very likely to be oxidized to tetrathionate and to other compounds by reacting with food constituents (Eckohoff and
Okos, 1986).
H. PREPARATION OF TETRATHIONATE
Since tetrathionate is the first oxidation product of thiosulfate, most preparation methods are through an oxidation reaction of thiosulfate.
Table 3 reports the standard electrode potential of some
-27- Table 3. Standard electrode potential (E°) for some chemical reactions*'8.
Half reaction E<>, v
S«CVS + 2e" 2SjO,-a +0.170
la + 2e" 21" +0.535
H»02 + 2H* + 2e- 2H20 +1.776
Fe*' + e- Fefl +0.771
Cu" + e" Cu» +0.167
VCV1 + 2H* + e" VO*2 + H»0 +0.361
*-Half reactions are written as reduction
BSource: Fritz and Schenk (1972)
-28- oxidizing agents that have been used to synthesize tetrathionate from thiosulfate. The standard electrode potential for the oxidation of thiosulfate to tetrathionate is only -0.17 V which indicates weak oxidants should be used to synthesize tetrathionate, in order to prevent further oxidation of thiosulfate to higher valences (i.e. sulfates). Some chemical reactions in the synthesis of tetrathionate are shown in
Table 4. These reactions are discussed in detail in the following section.
1. Iodine oxidation
Oxidation of a thiosulfate with acidic iodine yields tetrathionate quantitatively (Schmidt, 1972).
According to Awtrey and Connick (1951) the mechanism is as follows:
2 I-I + :SSOr > I:SS03- + I" (2)
2 2 SS03:" + I:SS03 > 03SS:SS0r + I" (3)
It should be noted that transfer of the two electrons occurs in the second step, when a second thiosulfate ion attacks the iodine thiosulfate intermediate. The two electrons, bonding iodine to sulfur in the intermediate, are transferred to iodide as it is displaced from the intermediate by the second thiosulfate. This is a typical example of a bimolecular nucleophilic displacement
reaction (SN2) (Fritz and Schenk, 1972). This reaction is extensively used in analytical chemistry for oxidation-reduction
-29- Table 4. Chemical reactions in the synthesis of tetrathionate.
Oxidant Reaction
Iodine 2Na2S20i + I2 • 2NaI + Na»S40-
Hydrogen Peroxide 2Na2S203 + H202 * Na2S«06 + 2NaOH
Ferric chloride 2Na2S20- + 2FeCli » Na2S«0< + 2FeCl2 + 2NaCl
Cupric sulfate 3Na2S20j + 2CuSO« » Na-SiOs + 2Na2S04
+ Cu2S203
J J 1 Sodium vanadate*- 2S20r + VOl-i » S«0-- + VOJ"
*This reaction is only for the chemical species involved in the oxidation-reduction
-30- titration. Gilman et al. (1946a) reported in detail the method
of preparation of sodium tetrathionate with iodine. They
reported that the tetrathionate obtained was 98.8 to 102% pure,
with an overall yield of 65% of the theoretical yield. A similar
iodine method was reported by Feher (1963).
2. Oxidation with other compounds
Oxidizing agents other than iodine also produce
tetrathionate from thiosulfate. Mellor (1930), in his classical
book, mentioned many of the reactions, reported until that
time, to synthesize tetrathionate. Since many of the original
references are from the beginning of this century it was
impossible, in many cases, to review the original papers.
(a) Oxidation with hydrogen peroxide
Nabl (1900) in a short "correction" indicated that hydrogen peroxide oxidized thiosulfate to tetrathionate provided that
the sodium hydroxide was neutralized as it was formed.
Otherwise the hydroxide decomposed the tetrathionate into thiosulfate, sulfate and sulfite. This "correction" was very superficial and no detailed procedure was reported.
Abel (1907) reported that the second order rate constant for the reaction of hydrogen peroxide with thiosulfate was 1.53. No units were indicated. Assuming the standard units (M"'sec"M for the aforementioned constant, it can be calculated that in approximately 35 min, the reaction should reach 90% completion.
-31- Since hydrogen peroxide is a very strong oxidizing agent, care has to be taken to control the reaction, in order to avoid oxidation of the thiosulfate to sulfite or sulfate.
(b) Oxidation with metals salts
Fordos and Gelis (1842) reported the synthesis of tetrathionate using ferric chloride as the oxidizing agent. More recently, the same oxidation reaction was reported to be rather
slow, and to be catalysed by copper salts (Lar and Singh, 1956).
The reduction of cupric to cuprous salts can be used to
oxidize thiosulfate to tetrathionate (Zeltnoff, 1867; Raschig,
1920). The standard potentials for ferric and cupric oxidation,
shown in Table 3, suggest that both ions are mild oxidizing agents.
(c) Oxidation with vanadate
Gowda et al. (1955) developed a method to estimate thiosulfate
with sodium vanadate. In this method, sodium thiosulfate was
quantitatively oxidized to tetrathionate in five minutes at room
temperature with excess sodium vanadate. The reaction occurred in
a medium containing sulfuric or acetic acid and a small amount of
copper sulfate as a catalyst. Further oxidation did not occur
even when the mixture was heated to boiling. After the reaction
was complete, the excess vanadate was titrated with a standard
solution of ferrous ammonium sulfate.
Since quantitative oxidation of thiosulfate to tetrathionate
-32- Since quantitative oxidation of thiosulfate to tetrathionate was reported, it is possible to use this reaction to synthesize
tetrathionate.
3. Other methods of synthesis of tetrathionate.
The reaction of sulfuryl chloride with thiosulfates results in
practically quantitative yields of pure tetrathionate and sulfur dioxide (Schmidt, 1972).
Tetrathionate can also be prepared by the reaction of sulfur chloride with the rather complicated mixture that is formed
when sulfur dioxide is dissolved in water (Feher, 1963). This
reaction mixture is called "sulfurous acid" by tradition, without
having a definitive composition (Schmidt, 1972). This method of preparation is somewhat difficult to carry out and although good
yields and purity are reported for the tetrathionate obtained,
its application is limited to laboratory scale production.
Since the sulfur bacterium Ectothiorhodospira shaposhnikovi has been reported to oxidize thiosulfate to tetrathionate, it
is possible to use this bacterium to synthesize tetrathionate
(Gogotava and Vainshteim, 1981).
Pure cultures of Thiospira accumulate tetrathionate when grown
on a medium containing organic compounds (Dubinins and Selvich,
1983) .
-33- Another report indicated that an autotrophic, acidophilic
Thiobacillus metabolized thiosulfate to tetrathionate during growth and could not reoxidize this product (Reynolds et al.,
1981).
-34- MATERIALS AND METHODS
A. MATERIALS
Since papaya latex is commercially unavailable, crude papain,
Type I (Sigma Chemical Co. St. Louis, MO) was used instead.
Sodium tetrathionate was obtained from ICN Pharmaceuticals, Inc.
(Life Science Group, Plainview, NY) and sodium metabisulfite, anhydrous powder, was purchased from Matheson Coleman & Bell
Manufacturing Chemists, Norwood, OH. Cysteine hydrochloride and
EDTA were from Sigma Chemical Co. St. Louis, MO. Casein, according to Hammersten was from BDH Chemicals, Vancouver B.C.
Glass distilled deionized water was used in the preparation of all solutions and buffers. All other chemicals were of analytical grade. All pH-values were measured using a Fisher model 420 pH-meter (Fisher Scientific Co., Pittsburgh, PA).
B. REHYDRATION OF CRUDE PAPAIN
In order to simulate a system similar to fresh papaya latex, crude papain was rehydrated with distilled water to give a 20% solution (w/v), a solid ratio similar to that of fresh papaya
latex (Ortiz et al., 1980). The rehydration was as follows: crude papain was mixed with cold distilled water and left at 4°C for 2 hr with slow agitation. This rehydrated crude papain will be referred to as papaya latex.
-35- C. DRYING CHARACTERISTICS OF PAPAYA LATEX
The effect of temperature (55, 80 and 100°C), drying load
(1,190, 2,381, and 4,792 g/ra* of drying area), and addition of
sulfite or tetrathionate (both at 1% (w/v)), on the drying rate
of papaya latex was investigated. An electrically heated, mechanical convection, forced horizontal air-flow tray drier was used (Blue M Stabil-Therm Oven, Model OV-490a-2, Blue M Electric
Company, Blue Island, IL).
The samples were dried in aluminum dishes (i.d. 2.3 cm and
0.5 cm deep). The oven was preheated for at least one hour at
the defined temperature before the start of the drying experiments. In order to obtain the drying curves, samples were withdrawn from the oven at various time intervals, and the moisture content of all samples was determined as the weight loss
after 12 hr at 80°C under vacuum.
D. DETERMINATION OF INFLUENTIAL FACTORS ON THE LOSSES OF PROTEOLYTIC ACTIVITY OF PAPAYA LATEX
The fractional factorial design LIT (3") of Taguchi (1957) was used to determine the factors which may significantly affect the losses of proteolytic activity of papaya latex due to drying.
By using a fractional factorial design it was possible to determine if tetrathionate protected the PA of papaya latex
during drying.
Protection of the PA provided by tetrathionate was compared to
-36- that produced by sodium metabisulfite, a compound commonly used in the commercial drying of papaya latex.
The factors evaluated, together with their assigned upper and lower limits are shown in Table 5 . The scheme used is presented in Fig. 1.
The pre-drying additive, sodium metabisulfite or sodium tetrathionate, was added directly to the latex. The treated or control latex was stored at room temperature (20-22°C) prior to subjecting it to drying under the conditions specified by the fractional factorial design. After the storage period the samples were dried in the electrically heated, forced air oven, at the temperature specified by the fractional factorial design.
Drying was continued until the latex hardened and crumbled readily when pressed by the fingers. This usually occurred when the residual moisture was 6+2%.
Dried samples were sealed in polyethylene bags. The sealed bags were placed inside amber glass jars with dessicant
(drierite) and stored at -20°C until PA determination (within three weeks). Preliminary experiments showed that no losses of
PA occurred during storage of crude papain at -20°C. PA determination was carried out using the method reported below.
The values of PA from the 27 drying treatments (in duplicate) were analyzed using a Taguchi's factorial analysis of variance computer program written in IBM-Basic (Arteaga, 1986).
-37- Table 5. Factors, and their assigned levels investigated for their possible influence on the losses of proteolytic activity of papaya latex.
Level
Factor 1 2 3
Predrying treatment* TT MBS CON
Drying temperature, °C 55 70 100
Storage prior to drying, h 2 12 24
Drying load, g/m* 1,190 2,381 4,762
Type of storage prior to drying11 D D/L L
*Predrying treatments: TT = addition of 1% sodium tetrathionate. MBS = addition of 1% sodium metabisulfite. CON = no additives (control treatment).
BType of storage prior to drying: D = storage under dark conditions. D/L = first half of the storage time under dark and other half under light conditions. L = storage under light conditions.
-38- Drying temperature
Storage time Treatment prior prior to drying to drying
Drying load
Type of storage prior to drying
Figure 1. Scheme used in the Taguchi L21 fractional factorial experiment (311).
-39- E. EFFECT OF DIFFERENT TYPES OF DRYING AND ADDITIVES
ON THE LOSSES OF PROTEOLYTIC ACTIVITY OF PAPAYA LATEX
The effect of three types of drying, namely, sun drying,
vacuum drying and oven (air) drying, on the losses of PA of
papaya latex, with or without additives was determined using a 3
x 3 full factorial experimental design. The two factors, and the
corresponding levels were: type of drying (sun, vacuum, and air
(oven) drying) and treatment prior to drying (addition of 1% metabisulfite, or tetrathionate, and no addition of additives).
The additives were added directly to the latex. All samples
were dried in aluminum dishes with a drying load of 1,190 g/m*.
Each treatment was done in triplicate.
Sun drying (20-25°C) was carried out for 6 hr. After this
period, the moisture content of the samples was still high (30%),
so the samples were vacuum dried for 3 hr at 55°C, 27 inches of
vacuum, to a moisture content of 6+2%. Oven drying was done at
55<>C for a period of 1.5 hr, to reach a final moisture content of
6±2%. Vacuum drying was carried out at 55<>C with a vacuum of 27
inches, for 5 hr; the final moisture of the product was also
6±2%. Dried samples were sealed in polyethylene bags, and the sealed bags were placed inside amber glass jars with dessicant.
The jars were stored at -20°C until PA determination (within three weeks). PA determination was carried out using the method
reported below.
-40- F. LOSSES OF THE PROTEOLYTIC ACTIVITY OF CRUDE PAPAIN DURING STORAGE
Papaya latex with 1% metabisulfite or 1% tetrathionate was oven dried at 55°C for 1.5 hr. The resulting crude papain was sealed in polyethylene bags, and the sealed bags were placed in transparent glass jars and stored at room temperature.
At 1 wk intervals for up to 13 wk the PA was measured in duplicate, using the method reported below. A control sample
(i.e. crude papain with no additives added) was stored and analysed in the same manner.
G. PROTEOLYTIC ACTIVITY ASSAY
The method of Hanada et al. (1978) with modifications was used. This method is based on the quantitation by u.v. spectrophotometry of trichloroacetic acid (TCA)-soluble peptides following casein hydrolysis.
The crude papain samples were finely ground with a pestle and mortar. Approximately 5 mg of powder was transferred to a 25 mL volumetric flask and the flask was made to volume with freshly prepared activating buffer (40 mM cysteine HCl and 20 mM EDTA in
0.05 M phosphate buffer, pH 6.8). This solution was incubated in a water bath at 40°C for 15 min. An aliquot of 0.5 mL of the activated enzyme solution was added to 5 mL of 1% Hammarsten-type casein solution in 0.05 M Tris-HCl buffer (pH 8), preequilibrated at 40°C. After addition of the enzyme solution the test tube
-41- contents were immediately mixed with a vortex mixer and incubated at 40°C for 10 min. Then 5 mL of 0.44 M TCA solution was added to the mixture and the contents of the test tube were shaken vigorously. The test tube was placed again in the water bath at
40°C for 30-40 min to let the precipitated protein fully coagulate. Samples were centrifuged (10,000 xg, 15 min, 4<>C
Sorvall R2-B) and filtered under vacuum through Whatman No.42 filter paper. All filtrates were completely clear. The absorbance of the filtrate was measured at 280 nm with a Cary 210 spectrophotometer (Varian Associates Inc., Palo Alto, CA).
Proteolytic activities were expressed as international units i.e
Jimols of tyrosine liberated min"1mg"1 of sample, under assay conditions. A standard curve prepared with L-tyrosine plus casein solution, under the conditions for assay was used to convert absorbance to Hmols of tyrosine.
The 1% casein solution was prepared as described in the Food
Chemical Codex (FCC III, 1981), with the modification that
Hammarsten-type casein was dissolved in 0.05 M Tris-HCl buffer
(pH 8.0) instead of in phosphate-citrate buffer, pH 6.8. The use
of the Tris buffer enabled the storage of the casein solution for periods of more than one week (Arnon, 1970).
Each sample evaluation consisted of four tubes; two incubated active enzyme/substrate mixtures and two substrate mixtures to which active enzyme was added after the TCA solution.
-42- H. PREPARATION OF SODIUM TETRATHIONATE
I. Iodine oxidation
The method reported by Gilman et al. (1946a) was used without modification. Tetrathionate was prepared by the dropwise addition of a concentrated solution of sodium thiosulfate (250 g of
NaiSaOj^SHjO in 250 mL of water) to an ice-cooled alcoholic solution of iodine (127 g of I* and 50 g of Nal in 500 .mL of absolute ethanol). The reaction mixture was vigorously stirred and maintained below 20°C during the addition of thiosulfate.
Precipitation of tetrathionate began when approximately half of the thiosulfate had been introduced.
After the reaction was completed, one liter of absolute ethanol and 500 mL of anhydrous ethyl ether were added to the reaction mixture, in order to separate tetrathionate. The mixture was left at 4°C for approximately 12 hr. The precipitate was collected on a buchner funnel and washed with small portions
of absolute ethanol to remove excess iodine. The precipitate was then dried at room temperature, in vacuo, over drierite. The solid was stored at 4°C in amber bottles until analysed.
2. Hydrogen peroxide oxidation
After a series of preliminary experiments, the final method
adopted was as follows: 16 g of Na2Sj03»5H20 was dissolved in 50
mL of distilled water. To this solution was added dropwise, 50 mL
-43- of a 3% solution of hydrogen peroxide at an approximate flow rate of 1.5-2.0 mL/min. The solution was mixed with a magnetic stirrer and the pH was continuously monitored with the use of a pH-meter. With each drop of hydrogen peroxide solution added, it was necessary to add one or two drops of either 0.10 N HaSOi or
0.50 M acetic acid, to maintain the pH near neutrality (pH 6-8).
Two reaction temperatures were tested: 10 and 20°C.
After all the hydrogen peroxide was added, the reaction mixture was allowed to stand for 20 min. Crystallization with ethanol and ether, drying and storage were carried out as described for the iodine oxidation method.
3. Ferric oxidation
The final technique used was as follows: 6 g of ferric
chloride (FeCl-»6H20) was dissolved in 25 mL of distilled water.
Upon dissolution of the ferric chloride, the pH of the mixture dropped to approximately pH 3.5. One set of experiments was done without pH adjustment and in the other the pH was adjusted with 0.1 N NaOH to approximately pH 7. To this solution was added dropwise 20 mL of a solution of thiosulfate (5 g of
Na2S203»5HaO in 20 mL of distilled water) at a flow rate of 1.5-
2.0 mL/min. Continuous agitation was carried out by using a magnetic stirrer.
Copper salts have been reported to act as catalysts in the oxidation of thiosulfate with ferric ions (Lar and Singh, 1956).
Thus, in a series of experiments, 1 mL of a 10% solution of
-44- cupric acetate was added to the reaction mixture just before
addition of the thiosulfate solution.
After the thiosulfate was added, the reaction mixture was
allowed to stand for one hour. Crystallization with ethanol and
ether, drying and storage were carried out as described for the
iodine oxidation method.
4. Cupric oxidation
The final method used was as follows: 5 g of cupric sulfate
(CuS0«»5Hj0) was dissolved in 25 mL of distilled water. A drop in
pH to about 3.5 was observed upon dissolution of the salt. In
some experiments, as in the ferric oxidation, the pH was
adjusted with 0.1 N NaOH to pH 7. To this solution was added
dropwise 20 mL of a thiosulfate solution (7.5 g of Na2S203»5H20
in 20 mL of distilled water). The solution was agitated with a magnetic stirrer.
After the thiosulfate was added, the reaction mixture was allowed to stand for one hour. Crystallization with ethanol and
ether, drying, and storage were carried out as described for the
iodine oxidation method.
5. Vanadate oxidation
The technique reported by Gowda et al. (1955), for estimation
of thiosulfate via reaction with vanadate was modified for
preparation of tetrathionate from thiosulfate.
To a solution of sodium vanadate (1.5 g NaV03»2H20 dissolved
-45- in 10 mL distilled water) were added 5 mL of a 1% solution of cupric acetate and 0.7 mL of 7 M sulfuric acid, followed by the dropwise addition of 10 mL of a sodium thiosulfate solution (10
g NajS203»5H20 in 10 mL of water) at a flow rate of 1-1.5 mL/min.
According to Gowda et al. (1955) the reaction between vanadate and thiosulfate is completed in five minutes at 28°C. The mixture was allowed to stand for 10 min after the thiosulfate solution was added. Crystallization with ethanol and ether, drying, and storage was carried out as described for the iodine oxidation method.
I. DETERMINATION OF PURITY AND YIELD
Many methods have been reported for the determination of polythionates (for a review see Williams, 1979). An HPLC method has been reported by Chapman and Beard (1973). As mentioned by
Williams (1979), determination of individual pure polythionates is relatively easy but analysis of a mixture is more difficult.
After the solvent crystallization and drying of the tetrathionate-containing precipitate, its weight was recorded.
The % tetrathionate in the solid was determined by the following two methods.
1. Iodate-iodine titration
This method was first reported by Gilman et al. (1946a,b) and
-46- is a relatively simple and straightforward procedure. The method is an indirect iodine titration; the general form for this type of reactions is (Fritz and Schenk, 1972):
Aox + I"(excess) ^ ABBD + 12 (4)
I2 + 2SjCV2 > 21" + S«CV2 (5) where Aox and ABBD are the oxidized and reduced states of A, respectively. The iodine formed in reaction (4) is equivalent to the amount of Aox in the sample to be analysed, and is titrated with standard thiosulfate with starch as an indicator (reaction
5) .
The analysis of tetrathionate with iodate is based on the
reaction in which iodate (I03) quantitatively oxidizes tetrathionate in the presence of HC1 to sulfate (reaction 6).
The unreacted iodate reacts with an added excess of iodide to give free iodine (reaction 7), which was then titrated with 0.10
N or 0.05 N thiosulfate (reaction 8).
2 + 2 2SAOS- + 7I03- + 2H + 7C1" > 7IC1 + 8SOr + H20 (6)
IOr + 51- + 6H* > 3I2 + 3HzO (7)
I» + 2S2O3-2 > 21- + S For the determination of tetrathionate 25 mL of 0.1 N KI03 was added to 25 mg of the solid, followed by 5 mL of 2 N HC1. After standing a minimum of 5 min, 5 mL of 10% KI were added to convert IC1 and unreacted ICV to free iodine which was then titrated with 0.1 N thiosulfate. A blank was run parallel to the samples. -47- Since thiosulfate was also oxidized with iodate to sulfate- it was necessary to determine in the same sample the amount of thiosulfate. For the determination of thiosulfate a similar indirect iodine method was used. Both ions- tetrathionate and thiosulfate, were oxidized by iodate to sulfate. Only thiosulfate was oxidized , however, by iodine (Gilman et al., 1946b). In the analysis of thiosulfate 25 mg of the solid was dissolved in 5-10 mL of water. One drop of phenolphthalein was added and the solution was made alkaline (pink color) with 0.1 N NaOH. To this solution 25 mL of 0.1 N KI03, 5 mL of 10% KI and 5 mL of 2 N HC1 were added in the order named. Under alkaline conditions, iodate reacts with iodide to produce iodine (reaction 9) , without reacting with the thiosulfate present. Upon addition of the acid, the iodine formed oxidized thiosulfate (reaction 10) . The excess iodine was then titrated with 0.1 N thiosulfate (reaction 11). A blank was run parallel to the samples. IOr + I" * I2 (9) 2 S-03 + I- (excess) -» 21" + S 2 S203 + Ii r 21- + S«Os- (11) The following equations were used to calculate the amount of thiosulfate and tetrathionate in the sample: -48- Iodate reaction mg Na2S203= DELTA * N * 158 (12) where DELTA = (mL titration blank - mL titration sample) N = normality of thiosulfate Iodine reaction mg Na2S203= DELTA * N * (158/8) (13) where DELTA = (mL titration blank- mL titration sample) N = normality of thiosulfate mg Na2S«Ot = mg Na2S203 (iodine) - mg Na2S203 (iodate) (14) 2. Alkaline cyanolysis The second method used for the determination of tetrathionate and thiosulfate was the colorimetric method reported by Nor and Tabatabai (1975). This method involves alkaline cyanolysis of thiosulfate in the presence of cupric ions or alkaline cyanolysis of 57% of tetrathionate in the absence of cupric ions. Colorimetric determination of the thiocyanate is based on the formation of a ferric thiocyanate complex. The reactions in this case were: 2 1 S20r + CN" + Cu" SCV + CNS* (15) 2 2 2 S«0t- + CN" + HsO • S203" + SOr + 2HCN + CNS* (16) CNS* + Fe° • Fe-CNS complex (17) For the determination of thiosulfate + tetrathionate the method was as follows (Nor and Tabatabai, 1975): an aliquot (1-2 mL) of sample containing 25-200 ug of S as tetrathionate and thiosulfate was placed in a 25 mL volumetric flask. One milliliter of 0.1 M KCN was added, and the flask was swirled to -49- mix the contents. After 15 min 2 mL of 0.033 M CuCl2 and 1 mL of a solution of ferric nitrate-nitric acid solution (0.25 M Fe(NO-)3»9H20:3.1 M HN0-) was added. The solution was made to volume with distilled water, and the flask was inverted several times to mix the contents. After two minutes, the absorbance at 460 nm of the reddish-brown color ferric thiocyanate complex was measured. For the determination of only tetrathionate the procedure described above was repeated with the omission of the addition of 2 mL of 0.033 M CuCl2. The tetrathionate and thiosulfate S or tetrathionate S content of the aliquot was calculated by referring to a calibration curve based on the results obtained with standards containing 0, 50, 100, 150, 200 and 250 jUg of thiosulfate S. 3. Melting point determination The melting point of the tetrathionate-solids obtained was determined using a Perkin-Elmer DSC-2 System (The Perkin-Elmer Corporation, Norwalk, CT). Samples of the solid (5-7 mg) were weighed into tared aluminum pans, and the pans were sealed. The pans were held at 20<>C for 10 min, then heated to 200<>C at a rate of 10°C/min. An empty pan was used as a reference. The thermograms were automatically recorded and the melting point was taken as the maximum temperature of the endothermic peak. -50- 4. Calculation of purity and yield Purity and yield were calculated using the following formulas: Purity (%) = mg of tetrathionate in sample X 100 (18) mg sample Yield (%) = q precipitate X purity X 100 (19) # theoretical yield g NajS«06 2HaO Theoretical yields were calculated on the basis of the stoichiometric reactions reported in Table 4. J. COST EVALUATION In order to determine the most cost-efficient method for synthesis of tetrathionate, the cost of the tetrathionate obtained using the different methods was calculated. The calculations were based on the prices of the particular chemicals used in the synthesis and the yield obtained for each method. The prices of the chemicals were those from Sigma Chemical Company (1988). K. INACTIVATION/ACTIVATION EFFICIENCY OF THE SYNTHESIZED TETRATHIONATES Each tetrathionate preparation was tested for its ability to reversibly inactivate papaya latex. For this determination crude papain was rehydrated with water to give a 20% (w/v) solution as previously described in section B. One milliliter of this papaya -51- latex was transferred to a 25 mL volumetric flask and 1 mL of a solution of the tetrathionate to be tested (0.5 mg/mL in 0.05 M phosphate buffer pH 6.8) was added. After gently shaking the flask to mix the contents, the mixture was incubated at room temperature for 15 min. The solution was made to volume with 0.05 M phosphate buffer, pH 6.8, 0.02 M EDTA (inactivation), or with the same buffer containing 0.05 M cysteine (reactivation). After 10 min at room temperature, duplicate 1 mL aliquots were used to determine the PA, using the method reported in section G. A control sample, to which no tetrathionate was added, was run parallel to each experiment. Inactivation was taken as the ratio of the difference between the PA of a control sample (no tetrathionate added) and the PA of the tetrathionate treated sample, to the PA of the control sample. Reactivation was calculated as the ratio of the PA activity of the tetrathionate treated samples diluted in the cysteine containing buffer to the PA of the control sample (no tetrathionate) diluted with the same buffer. The inactivation efficiency of the solid was expressed as the percentage inactivation of a sample of pure tetrathionate. Similarly, the reactivation efficiency of the solid was taken as the percentage reactivation of a sample of pure tetrathionate. -52- RESULTS AND DISCUSSION A. DRYING RATES OF PAPAYA LATEX The effects of temperature and drying load on the drying rates of papaya latex, without additives, are shown in Fig. 2 and 3. As expected, the shortest time for reaching a constant weight was needed at the highest drying temperature (100°C), or the lowest drying load (1,190 g/m*). At 55<>C, with a drying load of 2,381 g/m*, it took approximately 150 min (2.5 hr) to reach a constant weight (moisture content of 6+2%). Ortiz et al. (1980) reported that fresh papaya latex required approximately 240 min (4 hr) to dry at 55<>C. The difference in drying time is likely due to the different type of oven used. The shorter drying time for rehydrated crude papain, however, could indicate that drying of papaya latex caused changes in some of the components of fresh papaya latex, with the result that the rehydratation water added to the crude papain was not as tightly bound as the original water in the fresh papaya latex. The effect of addition of sodium metabisulfite or sodium tetrathionate on the drying rates, at 55°C, of papaya latex is shown in Fig. 4. Addition of these compounds at a 1% level, did not change the drying rate compared to a control papaya latex. Similar results were found for the other temperatures. -53- Figure 2. The effect of three different drying temperatures on the drying rate of papaya latex at a drying load of 2,381 g/m-. Each point represents the mean of three determinations. In all case the coefficient of variation was less than 5%. -54- Figure 3. The effect of drying load on the drying rate of papaya latex at a drying temperature of 80°C. Each point represents the mean of three determinations. In all cases the coefficient of variation was less than 5%. -55- 100-, 8 8 8 S 75- O • § o Drying Temperature 55°C 50- o Control o 1* Metabisulfite e A 1* Tetrathionate 25- T—i—r I i i—i—i—I—i—i—i—i—|—i—i—i—i—j—i—i—i—i—| 50 100 150 200 250 Drying Time (min) Figure 4. The effect of the addition of 1% tetrathionate or metabisulfite on the drying rate of papaya latex at 55 °C and drying load of 2,381 g/m«. Each point represents the mean of three determinations. In all cases the coefficient of variation was less than 5%. -56- B. DETERMINATION OF INFLUENTIAL FACTORS ON THE LOSS OF PROTEOLYTIC ACTIVITY DUE TO DRYING OF PAPAYA LATEX 13 By using the fractional factorial design L27 (3 ) of Taguchi (1957), it was possible to determine which factors affected the losses of PA during drying of papaya latex. The factors examined were addition of additives (metabisulfite or tetrathionate), type (light or dark conditions) and length of storage prior to drying, drying temperature, and drying load, as well as possible interactions among the factors. Data collected from the 27 drying treatments, following the 13 fractional experimental design L27 (3 ) of Taguchi (1957), were analyzed by analysis of variance to determine the significance of the factors and the possible interactions on the proteolytic activity losses of papaya latex during drying. Table 6 shows the analysis of variance of the three level factorial design. The following main effects were computed to be highly significant (p<0.01): treatment prior to drying, drying temperature and, drying load. The type of storage prior to drying (light or dark conditions) was found to have a significant effect at p<0.05. The interaction (treatment prior to drying) x (drying temperature) was highly significant (p<0.01). The other interactions were non significant at p=0.05. The effect curves in Fig. 5 illustrate the fact that a significantly higher mean PA retention was achieved with the addition of 1% tetrathionate than with the addition of metabisulfite at the same level, at a drying temperature of 55°C. -57- 13 Table 6. Analysis of variance (Taguchi's L27 3 ) of proteolytic activity values of papaya latex obtained from 27 drying experiments. Source of variation DF Mean Square F-value Predrying treatment (PT) 2 730.91 13.64" Drying temperature (DT) 2 9959.86 185.87" Storage prior to drying 2 146.49 2.73 n.s. Drying load 2 421.51 7.87" Type of storage prior to drying 2 270.96 5.06* PT X DT 4 311.17 5.81" Errors- 12 53. 59 Total 26 'significant at p < 0.05 • "significant at p < 0.01 n.s. not significant at p > 0.05 *-The sums of square values for the interactions that do not appear in the ANOVA table were very low and, therefore, were incorporated into the error sums of squares. -58- Figure 5. Effect curve for the interaction between drying temperature and treatment prior to drying on the proteolytic activity retained by crude papain. (Mean+Confidence limits calculated at p<0.05). -59- Although the mean value of the PA after drying at 70<>C was slightly higher when the latex was treated with tetrathionate than the corresponding value with the treatment of metabisulfite, overlapping of the confidence limits (p<0.05) indicated no significant difference between the two treatments. Duncans multiple range test confirmed the above conclusion. At a drying temperature of 100°C neither treatment protected the PA of papaya latex. Commercial drying of papaya latex is usually done at 50-55°C (Ortiz et al., 1980). Since tetrathionate has been found to be significantly superior to metabisulfite as a protecting agent of the PA of papaya latex at 55°C, it can be concluded that tetrathionate has potential commercial application as an agent for protecting the PA of papaya latex. The effect curves in Fig. 6 show that highest mean retention of the PA of papaya latex occurred when the drying load was at the minimum level tested (1,190 g/m* of drying area) for the control papaya latex and the latex treated with either additive. Overlapping of the confidence limits (not shown in the graph), however, indicated no significant differences between the retention of PA at the different drying loads for the papaya latex treated with tetrathionate or metabisulfite. -60- 100-i Treatment o 156 tetrathionate o 158 metabisulfite A Control 80 Q) C 0) C£ 60 >s o < 40- o CD -+-» o CL 20 1,190 2,381 4,762 Drying load (g/m2) Figure 6. Effect curve for the interaction between drying load and treatment prior to drying on the proteolytic activity retained by crude papain. Only the confidence limits (at p<0.05) for the latex treated with tetrathionate are shown. -61- C. EFFECT OF DIFFERENT TYPES OF DRYING AND ADDITIVES ON LOSS OF PROTEOLYTIC ACTIVITY OF PAPAYA LATEX As previously reported (Krishnamurty et al., 1960), sun drying caused higher losses of PA in papaya latex as compared to the other drying methods (Table 7). For sun drying the addition of tetrathionate or metabisulfite did not significantly decrease the loss of PA as compared to an untreated latex. It is thought that a major cause of loss of PA during sun drying is the effect of UV radiation on a histidine residue essential for PA in papain (Brocklehurst et al., 1981). Since neither tetrathionate nor metabisulfite protect this histidine residue from UV radiation, a major decrease in the losses of PA is not likely to occur when tetrathionate or metabisulfite is added. As expected, vacuum drying produced the least loss of PA in papaya latex (Table 7). Although oxidation was already prevented with the use of hypobaric conditions in this drying method, the addition of tetrathionate or metabisulfite to the rehydrated papaya latex caused significant improvement in the PA retention. In the case of fresh papaya latex Krishnamurty et al. (1960) reported that addition of metabisulfite did not increase the retention of PA during vacuum drying as compared with untreated latex. -62- Table 7. Mean retention of the proteolytic activity*- (PA) of the crude papain resulting from papaya latex treated with 1% tetrathionate or 1% metabisulfite and dried using different methods3'0. Type of drying1 Treatment Sun Vacuum Oven Control* 61.3 + 2.63- 78.7+3.51 65.6+3.51 Metabisulfite 64.4 + 3.21 86.6+3.02 84.0 + 3.92 Tetrathionate 63.7+3.I1 96.2+4.02 100.0+3.83 *-PA expressed as % of the original PA present in the latex before drying BEach value is the mean of three determinations. Mean +S.D °Means in the same column which are not followed by the same number are significantly different at p<0.05 level ^Drying conditions are reported in Materials and Methods "Latex with no added additives -63- D. EFFECT OF ADDITIVES ON LOSS OF PROTEOLYTIC ACTIVITY OF CRUDE PAPAIN DURING STORAGE Addition of 1% tetrathionate to papaya latex protected the PA of the resulting crude papain during storage at room temperature. This protection was greater than that provided by the same level of metabisulfite (Fig. 7). The addition of metabisulfite also decreased the losses of PA of crude papain, but to a lesser extent than tetrathionate. Relatively high losses of PA occurred during storage of the control sample; almost 40% of the original PA was lost in 13 wk (91 days) (Fig. 7). Castro (1981) reported losses of PA of 30% for crude papain stored 75 days at room temperature. Since crude papain is usually exported from the producing to the refining countries (Leung, 1980; Flynn, 1975), it is very likely that it is stored for relatively long periods with consequent losses of PA. By adding tetrathionate to the latex before drying, it is possible to minimize the losses in PA, thus, increasing the stability of the crude papain. For the control latex (no additives added), the decrease in PA with time followed a non-linear relationship (Fig. 7). The empirical reaction order of the PA loss over time for this latex, determined using the linearization procedure of Durance et al. (1986), was found to be 1.5. Since loss of PA of crude papain are due to a number of different factors (i.e. oxidation, microbial degradation, autodigestion, interaction of protein with the carbohydrates present in the latex, etc.) it is -64- 110-i 100- "O c 90- CD LY. 80 o < 70H o Treatment O Control 6C • \% Metabisulfite 8 ^ A 1* Tetrathionate O 0_ 50 0 1 ~i 1 1 1 r i 1 1 1 1 1 Storag3 4 e5 Tim6 e7 (weeks8 9 1)0 11 12 13 Figure 7. Change in the proteolytic activity of crude papain with or without additives during storage at room temperature. Each point is mean of duplicates determinations. -65- expected that the overall kinetics o£ degradation are complex. For the samples of crude papain with either additive, the decrease in PA was linear over the storage period analysed, which indicated a zero order reaction. Although no objective measure of the color of the crude papain was done, no change in this parameter was observed during the storage time tested for all samples. E. COMPARISON OF DIFFERENT METHODS TO SYNTHESIZE TETRATHIONATE Yields and purity of tetrathionate synthesized by the different methods used are shown in Table 8. Two methods, the iodine-iodate reaction and the alkaline cyanolysis, were used to determine % tetrathionate in the prepared samples. Analysis of variance of the results of both methods showed no significant difference between them. As expected, iodine oxidation gave tetrathionate of high purity with good yield (65 %). The solid obtained was practically pure tetrathionate. Different conditions were tested in the hydrogen peroxide oxidation method. Decreasing the temperature from 22 to 10<>C increased the amount of tetrathionate obtained. Addition of copper salts had the same effect. Copper salts have been reported to act as catalysts in the oxidation of thiosulfate with ferric ions (Lar and Singh, 1956). Two different acids were used to neutralize the sodium hydroxide that was formed -66- Table 8. Yields*- and purity of tetrathionate slnthesized by the different methods. Conditions Temp. Yield* Purityc Method pH (°C) (%) (%) Iod ine N,,D. D 4 65 98 Peroxide™ 6,. 8 10 35 50 Peroxide™ 6.. 8 22 41 60 Peroxide** 6,. 8 10 40 60 Peroxide11'. a 6.. 8 10 41 68 Peroxide*-'. o 6.. 8 10 43 70 Ferric 3,, 0 22 15 15 Ferr ic 6,. 8 22 18 20 Ferric0 6.. 8 10 25 17 Cupr ic 3.. 0 22 30 28 Cupr ic 6.. 8 22 30 30 Cupr ic 6.. 8 10 35 20 Vanadate™' a N.,D. D 22 40 69 Vanadate*"'. a N.,D. D 22 35 88 ^-Values were calculated using the mean values of duplicate determinations BYield expressed as the % of theoretical yield °Purity expessed as the % tetrathionate in precipitate measured using the iodate-iodine reaction DN.D.= Not determinated "Sulfuric acid used ""Acetic acid used aCupric sulfate was added as a catalyst -67- during the reaction of peroxide with thiosulfate. It was found that the weak acid (acetic) gave slightly higher yields of tetrathionate than the strong acid (hydrochloric). Under the conditions tested, ferric sulfate did not give good yields of tetrathionate. It is possible that the precipitate obtained was mainly unreacted thiosulfate and ferrous salts. Somewhat higher yields were obtained using cupric chloride as the oxidizing agent, however, they were smaller than the yields for the peroxide or vanadate oxidation. The vanadate oxidation method resulted in similar yields to the peroxide oxidation. Gowda et al. (1955) reported that acetic acid was preferred to sulfuric acid in the determination of thiosulfate with vanadate. In these experiments use of sulfuric acid produced higher yields of tetrathionate. Overall only the peroxide and vanadate methods gave adequate yields, which were still lower than the yield obtained with the iodine method. It is possible that with further research, the yields of these methods can be increased to make them a more economical alternative for the synthesis of tetrathionate. F. COST EVALUATION OF THE METHODS OF TETRATHIONATE SYNTHESIS To determine the most cost effective method of synthesis of tetrathionate a cost evaluation of each method was performed. The price of the chemicals used for the synthesis of tetrathionate -68- by the different methods, together with the prices of sodium metabisulfite and sodium tetrathionate are shown in Table 9. Sodium tetrathionate is roughly 24 times more expensive per gram than metabisulfite, at current commercial prices. Since purity has a major effect on the price of a chemical compound, in order to standardize cost, the grade of the chemicals reported in Table 9 is ACS Grade (American Chemical Society Grade). Taking into consideration the cost of the different chemicals, with or without solvents, the cost of synthesis of sodium tetrathionate using the different methods was calculated (Fig. 8a and 8b). This analysis indicated that the tetrathionate obtained with the cupric oxidation method had the highest calculated price. In the case of the iodine oxidation, the cost of the iodine represented a high proportion (24.3%) of the total cost of tetrathionate obtained (Fig. 9). For the peroxide method, the cost of the oxidant represented only 3.7% of the total cost. Ethanol and ethyl ether contributed to a major proportion of the total cost of the tetrathionate (Fig. 9). Although, in these experiments, the volumes and proportions of the solvents were kept constant, it could be possible to decrease the total cost of tetrathionate by either decreasing the volumes of solvent used or by using less expensive solvents to crystallize the tetrathionate. The hydrogen peroxide method for tetrathionate synthesis was 18% less expensive than the iodine oxidation (solvents -69- Table 9. Commercial price of tetrathionate, metabisulfite and chemicals used to synthesize tetrathionate. Price* Compound Formula (USD/g) Sodium tetrathionate Na2S«0«»2H20 0.600* Sodium metabisulfite Na-SiOs 0.025° Sodium thiosulfate NajS-Oj»5H20 0.013 Iodine Ii 0.141 Sodium iodide Nal 0.082 1 Hydrogen peroxide * H202 0.117 Ferric chloride FeCl,»6H20 0.006 Cupric sulfate CuSO-»5H-,0 0.024 Sodium vanadate NaV0i»2H20 0.060 *Prices according to Sigma Chemical Comp. except when indicated BPrice from Aldrich Chemical Comp. aPrice from Fisher Scientific D Price per g of H202 based on a concentration of 30% H202 in the commercial solution -70- 2.00 1.80 - Iodine Peroxide Cupric Ferric Vanadate Method Figure 8a. The cost of synthesis of tetrathionate for the different methods evaluated. Cost including that of the solvents. Note: The experimental yields obtained were used for the calculation of the cost. -71- 0.40 i r Iodine Peroxide Cupric Ferric Vanadate Method Figure 8b. The cost of synthesis tetrathionate for the different , methods evaluated. Cost without including that of the solvents. Note: The experimental yields obtained were used for the calculation of the cost. -72- Figure 9. Distribution of the cost of synthesis of tetrathionate using the iodine (A) and the hydrogen peroxide (B) methods. included), while the vanadate oxidation method was 16% less expensive than the iodine method (solvents included). G. MELTING POINT DETERMINATION Determination of the absolute purity of materials by differential scanning calorimetry (DSC) has been an accepted technique in the pharmaceutical and chemical industries since the development of DSC in the early 1960s (Brennan et al., 1984). The melting point of a commercial sample of sodium tetrathionate (102.0±5% pure as measured by iodine-iodate titration), determined by DSC, was 115±2°C (mean of three determinations). The thermogram of the commercial tetrathionate gave, as expected, only one endothermic peak, with a temperature range between 110-120<>C (Fig. 10). Tetrathionate synthesized by the iodine method (98% pure as measured by iodine-iodate titration) gave very similar results (Fig. 11). When samples of tetrathionate synthesized by the other methods were subjected to DSC analysis, the characteristic endothermic peak of commercial tetrathionate was not observed. Instead, an endothermic peak at a lower temperature appeared (Fig. 12-15), except for tetrathionate synthesized by the vanadate method using acetic acid (Fig. 16). Even when the sample contained 50% tetrathionate (measured by the iodine-iodate titration), the characteristic peak of tetrathionate did not appear. Since melting point determination is very sensitive to -74- Figure 10. Typical DSC thermogram of a commercial tetrathionate sample from ICN Pharmaceutical, Inc. Tetrathionate content (%) determined using the iodate-iodine reaction (Mean±S.D n = 3)= 102.0+.5% -75- Figure 11. Typical DSC thermogram o£ tetrathionate synthesized by the iodine reaction. Tetrathionate content (%) determined using the iodate-iodine reaction (Mean+S.D n=3)= 98.31±6% -77- Figure 12. Typical DSC thermogram of tetrathionate synthesized by the peroxide method. Tetrathionate content (%) determined using the iodate-iodine reaction (Mean+S.D n=3)= 37.9+6% -79- Figure 13. Typical DSC thermogram of tetrathionate synthesized by the cupric method. Tetrathionate content (%) determined using the iodate-iodine reaction (Mean+S.D n=3)= 20.4+8% -81- 15.0 CUS04 PH-6 0.0 TEMPERATURE -83- Figure 15. Typical DSC thermogram of tetrathionate synthesized by the vanadate method with hydrochloric acid. Tetrathionate content (%) determined using the iodate-iodine reaction (Mean+S.D n=3)= 38.6+7% -85- 15.0 VANADATE HAC 0.0 iU.O " '• 70.0 • 150.0 TEMPERATURE -87- 8.00 VANADATE HCL WT» 11. 45 mg MAXi 104.44 SCAN RATE, 20.00 d A Q Q PEAK FROMi 64.22 Z TOi 118.08 ONSETi 05.24 CAL/GRAMi 10.47 U LU cn x 4.00 + CO < CO u 150.00 3O0 50.00 90.00 110.00 1307 DSC TEMPERATURE CO the purity of the compound, it was thought that impurities present in the solids (i.e. thiosulfate, sulfite, sulfates and/or other salts) interfered with the melting point determination. A sample of pure thiosulfate run on the DSC gave a melting point of 55±3°C (Fig. 17) (mean of three determinations), similar to previously reported literature values (Weast, 1987). It is interesting to note that the endothermic peak of pure thiosulfate occurred in the same temperature range (50-55<>C) as the peak of the solids obtained with the other methods of tetrathionate synthesis which contained 30-50% tetrathionate. In order to determine the effect of impurities on the melting point of tetrathionate, pure tetrathionate was mixed with different proportions of thiosulfate. The results indicated that even small amounts of thiosulfates (10% w/w) had a dramatic effect on the thermogram of tetrathionate. When thiosulfate was mixed at levels of 35-40% (w/w) or higher with pure tetrathionate the sharp endothermic peak at 110-115°C disappeared (Fig. 18). Addition of thiosulfate to tetrathionate decreased the area of the endothermic peak at 110-115°C and increased the area of the endothermic peak at 50-55°C (Fig. 19). A decrease in the melting point of tetrathionate also occurred with the addition of thiosulfate (Fig. 20). These results confirm the fact that the determination of purity by DSC becomes unreliable as the proportion of contaminants increases. A similar effect, where impurities in the compound cause -89- Figure 17. Typical DSC thermograms of pure thiosulfate from Fisher Scientific Comp. -90- 20.0 T- THIOSULPHATE WTi 4.50 mg SCAN RATEi 20.00 deg/min MAXi S3.33 A a a 'PEAK FROM. 47 ONSETi 49. 13 CAL/GRAMi. 513, 34 u CO I 10. ut U5 -J < u '.'n 1 ' i ^— +— ~«—: ~4;- 20.0 . " " " 110.0 " 230.0 TEMPERATURE CO DSC Figure 18. Typical DSC thermogram of a mixture of pure thiosulfate and tetrathionate at a 1:1 (w/w) ratio. -92- 10.0 130.0 220.0' TEMPERATURE % Thiosulphate in Mixture (w/w) Figure 19. Effect of addition of thiosulphate on the area of two endothermic peaks of tetrathionate: Endothermic peak with temperature range 47-66°C (solid line); endothermic peak with temperature range 104-137<>C (broken line). Mean + S.D (n = 3). -94- 120.00 -i 110.00 H O o 100.00 H o Q_ cr> 90.00 H c 80.00 H 70.00 10 20 30 40 % Thiosulphate in Mixture (w/w) Figure 20. Effect of addition of thiosulfate on the melting point of tetrathionate. -95- dramatic changes in the thermogram, was reported for many compounds (Brennan et al., 1984). H. INACTIVATION AND ACTIVATION EFFICIENCY OF THE SYNTHESIZED TETRATHIONATE As expected, for most samples, the higher the proportion of tetrathionate in the sample, the higher the inactivation efficiency (IE) of the solid (Table 10). Tetrathionate synthesized by the iodine method gave 100% IE, which indicates that it inactivated the PA of papaya latex to the same extent as a commercial sample of tetrathionate. In the case of the solid prepared by the ferric oxidation method, the IE was higher than expected, considering its low tetrathionate content. This result suggests that other compounds present in the solid (e.g. ferrous salts) may also have an inactivation effect on the PA of papaya latex. In most cases the activation efficiency (AE) was nearly 100%, with the exception of the solid prepared with the ferric method. Since the proteolytic enzymes in papaya latex are inactivated by metal ions (Fukal et al., 1984), and this inactivation is not reversed by addition of cysteine, these results suggest that this solid contained a large amount of ferrous salts which react with the proteases of papaya latex. This explanation is supported by the fact that increasing the concentration of EDTA in the buffer in order to chelate more metal ions, increased the activation efficiency (Table 10). -96- Table 10. Inactivation (IE) and activation (AE) efficiency* of tetrathionates synthesized using different methods. Efficiency* Method of % tetrathionate in IE AE synthesis sample0 (%) (%) Control0 100 100 100 Iodine 100 100 100 Peroxide 43 40 95 Ferric 23 38 60 Ferric 23 18= 100 Cupric 30 23 85 Vanadate 42 34 100 *The definitions of inactivation (IE) and activation efficiency (AE) are given in the Methods section *Values were calculated using the mean values of duplicate determinations for both inactivation and activation efficiencies c% tetrathionate reported is the mean value of duplicate determinations using the iodate-iodine reaction ^Commercial sample from ICN Pharmaceuticals, Inc. BThe concentration of EDTA in the buffer was increased to 50 mM -97- CONCLUSION The main objective of this study was to determine if addition of tetrathionate to papaya latex would protect its proteolytic activity (PA) during drying, as well as during storage of crude papain. The results obtained indicate that addition of tetrathionate, at a 1% level, to papaya latex completely inhibited the losses of PA when the drying temperature was low (50-55<>C). Since, in commercial practice, 55<>C is the commonly used drying temperature for papaya latex, addition of tetrathionate has potential industrial application. Addition of 1% metabisulfite also protected the PA of papaya latex during oven drying, however, its protective effect was significantly lower than that one given by tetrathionate. A fractional factorial experimental design showed that the best conditions (minimum PA losses) for drying papaya latex were: addition of 1% tetrathionate; a drying temperature of 55<>C; a drying load of 1,190 g/m2, and if storage prior to drying was necessary, it had to be under dark conditions. The effects of three different drying methods (sun, oven, and vacuum) on the PA of papaya latex were also investigated. As reported by other researchers, vacuum drying produced the smallest PA losses. In this case addition of tetrathionate or metabisulfite, both at a 1% level, was found to be equally effective in protecting the PA of papaya latex. The greatest PA losses occurred during sun drying, and for this drying method, neither tetrathionate nor metabisulfite minimized the PA losses in papaya latex as compared with an untreated latex. The second part of this study was aimed at investigating different chemical methods for synthesis of tetrathionate and to compare the yields and costs of the tetrathionate obtained. Tetrathionate yields (as % of the theoretical yield) ranged from 65% for the iodine reaction to 15% for the ferric oxidation method. Overall, only the peroxide and vanadate methods gave yields comparable to the one obtained by the iodine method. A cost analysis indicated that the solvents needed for crystallizing tetrathionate contributed significantly to the overall cost of the compound. Both the peroxide and vanadate methods were easier to performed on a laboratory scale, and both methods gave tetrathionate at about 20% lower costs than the iodine method. In order to characterize the tetrathionate obtained from the different synthesis methods, DSC was used to determine the melting point of the solids. The melting point of pure tetrathionate was found to be 115<>C. However, due to the high proportion of compounds other than tetrathionate in the solids, the melting behavior of the solids was completely different than the one found for pure tetrathionate. -99- CHAPTER 2. Chemical modification of papain by tetrathionate. LITERATURE REVIEW A. CHEMICAL MODIFICATION OF PROTEINS Modification of a protein usually refers to physical, chemical, or enzymatic treatments changing its conformation, its structure, and consequently its physicochemical and functional properties (Chobert et al., 1988); Chemical modification is generally referred to as the intentional alteration of a protein structure, or conformation, by chemical agents (Means and Feeney, 1971). In general, it involves derivatization, by specific reagents, of some reactive side chain groups in the protein molecule, such as charged anionic and catlonic groups, hydroxyl, amide and thiol residues. The amino acid side chains of proteins most often modified are probably the e-amino group of lysine, the sulfhydryl group of cysteine, or its oxidized product the disulfide group of cystine (Feeney, 1977). Chemical modification of a protein is a technique widely used in fundamental studies in various areas of protein research including enzymology, immunochemistry, X-ray crystallography and purification of proteins. The techniques have advanced so rapidly that almost every journal of biochemistry has an article which reports chemical modification, many of these using several different methods for different side chains (Feeney, 1987). With the present availability of -100- specific chemical reagents and sophisticated analytical techniques, chemical modification has become a powerful tool for protein chemists, in the study of structure and function of biologically active proteins (Feeney, 1987; Feeney, 1977; Means and Feeney, 1971). Chemical modification is routinely used to investigate the roles of individual amino acid chains in relation to the physical, chemical, and biological properties of proteins and to determine the active-site residues in enzymes. The very powerful methods of in vitro mutagenesis and direct chemical synthesis are achieving results beyond those capable with chemical modification, particularly for the determination of the roles of individual residues in protein structure and function. Yet chemical modification is still a good adjunct for these methods (Feeney, 1987). Feeney (1987) indicated that "chemical modification of proteins should continue to serve as an important method in protein chemistry. Although there are now better methods for attacking some of the problems, these methods still require chemical procedures for many of their analysis and applications". This author concluded with a list of 24 areas where chemical modification should continue to be important in protein research. -101- B. CHEMICAL MODIFICATION OF FOOD RELATED PROTEINS Commercial applications of chemical modification of proteins have a long history related to the pharmaceutical, dyeing, and textile industries (Means and Feeney, 1971). Although the addition of chemicals to food has long been practiced, the intentional chemical modification of food proteins is still largely found only in the patent literature and is practiced to only a limited extent. Obvious barriers to the chemical modification of food proteins for human usage include esthetic, cultural, legal, and medical aspects (Feeney, 1977). The three general areas of application of chemical modification of food proteins are : 1) blocking of deteriorative reactions; 2) improvement of physical properties, and 3) improvement of nutritional properties (Feeney, 1977). Different deteriorative reactions such as oxidation, Maillard reactions, and cross linking affect food proteins during processing and storage. In this case, the general purpose is to modify the proteins to either prevent a chemical reaction or greatly retard its rate. These objectives can be accomplished by blocking a protein group which undergoes a reaction, or by changing the conditions, thus greatly retarding the deteriorative reaction (Feeney, 1977). Improvements in physical properties can be obtained by chemical modification. Modification in this case is aimed at -102- changing the gross physical and chemical interactions of the molecule which are important to functional properties such as foaming and whipping capabilities (Feeney, 1977). Improvement of the nutritional quality by chemical modification may prove to be the most important use from the standpoint of society's fundamental needs. This objective might be brought about by increasing the digestibility of the protein, inactivating toxic or inhibitory substances, or attaching essential nutrients to the proteins. An example of this approach is the covalent incorporation of essential amino acids to soy proteins by means of the carbodiimide reaction (Voutsinas, 1978). Attachment of coloring or flavoring agents to proteins might also improve their acceptability (Feeney, 1977). C. MODIFICATION OF SULFHYDRYL GROUPS IN PROTEINS The ease and specificity with which the sulfhydryl or thiol (-SH) group can be modified makes it a prime target for experimental study, and presumably accounts for its widespread involvement in biological phenomena (Brocklehurst, 1979). Proton dissociation provides the thiolate ion (RS-), probably the most powerful nucleophile found in biological materials (Brocklehurst, 1979). The RS~ ions are over 500 times more nucleophilic than the corresponding oxygen analogue RO~ (Jocelyn, 1972). -103- A large number of reagents that react readily with -SH groups have been described (Brocklehurst, 1979; Friedman, 1973; Jocelyn, 1972; Means and Feeney, 1971). Although the high intrinsic nucleophilicity of the thiolate ions is often sufficient to ensure their specific modification by many types of electrophilic reagents, this cannot always be guaranteed when the -SH groups reside in proteins (Brocklehurst, 1979). The number of reactive sulfhydryl groups reported for proteins frequently differs because of the use of different reagents. The hydrophobic character of sulfhydryl groups, and their presence, in many cases, in hydrophobic environments in conjunction with the varied ability of reagents to penetrate hydrophobic regions partly account for the differences in reactivity of -SH (Means and Feeney, 1971). The two reactions most commonly employed to modify the -SH groups in proteins are oxidation and alkylation. 1. Oxidation Perhaps the most biologically interesting property of -SH groups is that they can be oxidized. The -SH group is relatively easily oxidized by a wide variety of oxidizing agents, but the majority of these substance (i.e. HaOa, Ia) are not regarded as specific reagents for -SH groups. Since sulfur has valences ranging from -2 to +6, several types of oxidation products are possible. Table 11 shows the most common oxidation products of -SH groups. The most easily formed product is the disulfide -104- Table 11. Oxidation products of sulfhydryl groups. Sulfur Formula Name valence R-SH sulfhydryl -1 R-SOH sulfenic -1 R-S-S-R disulfide 0 R-SO2H sulfinic + 3 R-SO3H sulfonic + 5 Source: Friedman (1973) -105- (-S-S-) (Jocelyn, 1972). Disulfides are much less active than -SH groups and may function to stabilize protein structure. However various reagents can cleave them converting them back to -SH groups or other derivatives (Friedman- 1973; Jocelyn, 1972). The most specific reagents for oxidizing sulfhydryl groups to disulfides are probably disulfides themselves. Their specificity for -SH groups may be considered absolute for practical purposes (Brocklehurst, 1979; Dixon and Webb, 1964). This reaction between sulfhydryl groups with disulfides is commonly known as thiol disulfide interchange (Jocelyn, 1972) . (a) Modification by aromatic disulfides The compound 5,51-dithiobis (2-nitrobenzoic acid) (DTNB) reacts with free -SH groups of proteins, forming thionitrobenzoate-protein and liberating one mole of thionitrobenzoate anion for each -SH group (Brocklehurst, 1979; Means and Feeney, 1971). Although its main application has been in the measurement of -SH groups of proteins (Habeeb, 1972). DTNB has also been used to modify -SH groups in many proteins, such as streptococcal dihydrofolate reductase (Warwick and Freisheim, 1975) and cytolysin, a thiol-activated exotoxin produced by Clostridium perfringens type A (Iwamoto et al., 1987). In some cases the modification is reversible by treatment with G-mercaptoethanol. However, in some proteins, a -106- secondary change takes place during storage that eliminates the ability to be reactivated. This secondary inactivation appears to result from disulfide interchange with unreacted sulfhydryl groups in the partially modified enzyme (Means and Feeney, 1971). Another type of aromatic disulfide is the pyridyl disulfide such as the symmetrical reagents, 2,2' dipyridyl disulfide (2PDS) and 4,4' dipyridyl disulfide, which are usually referred to as two protonic state electrophiles. These reagents have been used to modify -SH groups with low pKa's (Brocklehurst, 1979). Titration with 2PDS at pH 4 and 8 has been shown to permit detection of chymopapain contaminants in preparations of papain (Baines and Brocklehurst, 1979). (b) Modification by tetrathionate Sodium tetrathionate (Na2S»06) is another disulfide that has been used to modify sulfhydryl groups of proteins. As mentioned before (see CHAPTER 1) tetrathionate rapidly oxidizes simple thiols to corresponding disulfides. In the case of some sulfhydryl proteins, rather stable sulfenylthiosulfate intermediates have been observed (Means and Feeney, 1971). The reaction of tetrathionate with sulfhydryl proteins is rapidly reversible upon addition of excess thiol (e.g. cysteine) (Liu, 1967). -107- Sodium tetrathionate has been used in protein chemistry in three different areas: I. As a stabilizing agent of sulfhydryl protease This application has already been reviewed (see CHAPTER 1). Briefly, sodium tetrathionate, by reversibly inactivating sulfhydryl enzymes, increases the storage stability of the proteins, minimizing activity losses due to autolysis and irreversible oxidation. II. As a blocking agent of cysteine residues The reversibility of the reaction of tetrathionate with cysteine residues has been utilized to protect (block) cysteine residues in proteins from modification, thus permitting the selective modification of less reactive residues. The first paper describing the use of tetrathionate as a blocking agent was published by Liu (1967). Using this method Liu (1967) demonstrated the presence of a histidine residue at the active site of streptococcal proteinase. A similar approach was used by Gleisner and Liener (1973) to demonstrate the presence of a histidine residue at the active site of ficin. Murachi and Okumura (1974) also used tetrathionate as a blocking agent during the photooxidation of histidine in bromelain and papain. The use of reversible blocking of cysteine residues with tetrathionate has not been restricted only to sulfhydryl enzymes. -108- Nakai et al. (1973) investigated the effect of histidine modification of K-casein, with different compounds, on the stabilizing ability of this protein on a3i-casein. In order to prevent modification of the cysteine residues of K-casein, sodium tetrathionate was used as a blocking agent. In this case, the amino groups of the protein were also blocked by acylation with citraconic anhydride. With the use of these blocking reactions, it was found that the histidine residues of K-casein are not essential for its stabilizing ability on otsi-case in. Rothenbuhler and Kinsella (1986), working on the effect of disulfide reduction and molecular dissociation of soy glycinin in its rate of proteolysis, used tetrathionate to prevent reoxidation of the free SH groups formed following reduction of the disulfide groups. III. As a chemical modification agent of cysteine residues Since tetrathionate reacts with sulfhydryl groups in a highly specific way, it has been widely used to modify them. The role of the sulfhydryl groups in D-glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle was studied by Pihl and Lange (1962) using chemical modification of the cysteine residues with different reagents, including tetrathionate. Tetrathionate was found to inhibit the enzyme rapidly and completely and the inhibition was promptly reversed by cysteine. Approximately 3 moles of tetrathionate were needed -109- to inhibit the enzyme completely. Further determinations indicated that the binding of radioactive sulfur (3SS) was associated with the disappearance of an equivalent number of enzyme -SH groups, thus ruling out the formation of intra- or inter-molecular disulfide bonds. The inhibition was found to be due to the fact that tetrathionate reacted stoichiometrically with three particularly reactive -SH groups of the enzyme, resulting in the formation of protein sulfenyl thiosulfates. Parker and Allison (1969) working with the same enzyme, but from a different source (pig muscle), reported similar results. They found that tetrathionate reacted stoichiometrically with the catalytically active -SH groups of the enzyme to form sulfenyl thiosulfate derivatives. These enzyme derivatives were found to be stable at 0<>C but decomposed at higher temperatures. In a more recent article Tsou et al. (1982), reported that tetrathionate or iodoacetic acid modification of the active site, cysteine-149, of D- glyceraldehyde-3-phosphate dehydrogenase caused conformational changes in the enzyme. Tetrathionate was also used to modify the -SH groups of the enzyme D-amino acid oxidase and of hemoglobin (Neims et al., 1966). Chung and Folk (1970) studied the mechanism of inactivation of guinea pig liver transglutaminase by tetrathionate. They reported that treatment of this enzyme with 2 moles of tetrathionate resulted in almost complete loss in enzymic activity, and that this loss was accompanied by a -110- concomitant disappearance of four sulfhydryl groups in the enzyme. The native enzyme contained 17-18 sulfhydryl groups per molecule and no disulfide bonds. However, it was found that less than 0.15 moles of "S-tetrathionate were bound per mole of enzyme. These authors concluded that the enzymatic changes which occurred upon reaction of transglutaminase with 2 moles of tetrathionate were a result of intramolecular disulfide bond formation and that these catalytic changes were associated with a single disulfide bond in the enzyme molecule. The activities of lactate dehydrogenase, glutamate dehydrogenase, aspartate amino transferase, (3-galactosidase, N-acetyl-p-D-glucosamidase, leucine aminopeptidase, Y- glutamyltransferase and alkaline phosphatase in renal tissue and in urine of rats were decreased by tetrathionate (Kunovic et al., 1981). Pyruvate kinase from human erythrocytes was also modified by tetrathionate. Although inhibition occurred, no changes in conformation or thermolability occurred (Valentine and Paglia, 1983). Sodium tetrathionate was found to cause conformational changes in the glucose-transporting protein of human erythrocytes (Krupka, 1985). In a recent article, Prasad and Horowitz (1987) investigated the chemical modification of bovine liver rhodanase with tetrathionate. Rhodanase catalyses the transfer of the outer sulfur atom of thiosulfate to the nucleophilic acceptor -111- substrate, cyanide (Prasad and Horowitz, 1987). This enzyme is a single polypeptide chain containing 293 amino acid residues of which four are cysteine residues. One of the cysteine residues, Cys-247, is at the active site. There are no disulfide bonds present in the enzyme. These researchers reported a linear relationship between loss of enzymatic activity and amount of tetrathionate used for the modification. Tetrathionate modification of rhodanase could be correlated with the changes in intrinsic fluorescence or with the binding of the active site reagent 2-anilinonaphthalene-8- sulfonic acid. Circular dichroism spectra of the protein suggested increased ordered secondary structure in the protein after reaction with tetrathionate. The authors suggested that tetrathionate modification of rhodanase may proceed through formation of sulfenyl thiosulfate intermediates at sulfhydryl groups, close to, but not identical, with the active site sulfhydryl group, which then can react further with the active-site sulfhydryl group to form disulfide bridges. (c) Modification by Iodobenzoates and mercurials Iodobenzoates have become widely used enzyme inhibitors. Their principal reaction with proteins is the oxidation of -SH groups to disulfide bonds. Oxidation of other residues has not been detected but may be possible, especially the oxidation of methionine residues (Brocklehurst, 1979; Means and Feeney, 1971). This modification is usually reversible with the -112- addition of cysteine or dithiothreitol (Means and Feeney, 1971). Mercurials are widely used to quantitate and determine the effect of substitution of sulfhydryl groups (Means and Feeney, 1971). The reaction of mercurials with proteins was thoroughly reviewed by Dixon and Webb (1964). The most commonly used inorganic mercurial is mercuric chloride. Organic mercurials such as p-chloromercuric benzoate are also widely used (Storey and Wagner, 1986). 2. Alkylation Compounds with -SH groups are alkylated, under mild conditions, by various stable and water soluble halogenated acids or their salts (R'-Hal), such as iodoacetic acid or iodoacetamide, to give sulfides (R-S-R') (Jocelyn, 1972). Like most other reactions of thiols, the reactive species in such alkylation reactions is the thiolate anion (RS~). Hence, the rate of reaction decreases with decreasing pH of the medium, and also, due mainly to pK differences, with the nature of the thiols themselves (Jocelyn, 1972). Neighboring groups can either enhance or suppress the reactivity of a group. For example, the -SH in streptococcal proteinase is 50 to 100 times more reactive than that of glutathione (Jocelyn, 1972). A similar activated -SH has been observed in papain (Sluyterman, 1967a) . Reactivities of -SH groups of proteins can be either enhanced or suppressed by substrates, allosteric effectors and other -113- specifically interacting substrates. In the case of both papain and ficin, the presence of certain substrates caused a several-fold stimulation of their reactivity to haloacetates (Whitaker, 1969). All the haloacetate compounds modify -SH groups in an irreversible way. N-Ethylmaleimide (NEM) and its derivatives are other widely used sulfhydryl reagents. NEM has been used to determine the effects of -SH modification and the number of sulfhydryl groups in a wide variety of proteins. It also reacts under certain conditions with amino groups (Means and Feeney, 1971). Long chain alkyl maleimides are sometimes more effective reagents than NEM for reactions with -SH in nonpolar environment (Jocelyn, 1972) . D. PAPAIN 1. Definition and isolation. Pure papain (E.C.3.4.22.2) is a sulfhydryl protease (or proteinase) isolated from Caricla papaya latex (Brocklehurst et al., 1981). Papain was first isolated in crystalline form from fresh papaya latex by Balls and co-workers in 1937 but is more conveniently isolated from commercially dried latex by the procedure of Kimmel and Smith (1957), or by the method of Baines and Brocklehurst (1979) if dried papaya latex of high solubility is used. -114- Papain prepared using the mentioned procedures is usually present in three forms: active papain, reversibly oxidized and irreversibly oxidized papain (Brocklehurst et al., 1981). Active papain (50%-80% of the total enzyme content) contains one free cysteine group per molecule, that of cysteine-25, which is part of the catalytic site of the enzyme. In reversibly oxidized papain, this catalytic site -SH group is linked in a disulfide bond with that of a free cysteine molecule, while in irreversibly oxidized papain, the catalytic site -SH group has been oxidized to a sulfinic acid group (Brocklehurst et al., 1981) . Fully active papain, containing one mole SH per mole of protein, may be prepared by affinity chromatography on columns of agarose to which Gly-Gly-Tyr(Bzl)-Arg (Blumberg et al., 1970), p-aminophenylmercuriacetate (Sluyterman and Widjenes, 1970), or glutathione-2-pyridyl disulfide were attached (Brocklehurst and Little, 1970). 2. Physicochemical properties and structure Various physical properties of papain and three other sulfhydryl proteases, are listed in Table 12. Generally speaking, there does not appear to be any major difference in the size and charge distributions among the various enzymes. One of the most characteristic differences is the absence of carbohydrate in papain and chymopapain (Arnon, 1970). -115- Table 12. Physicochemical properties of some cysteine proteases. Stem Property Papain Ficin Bromelain* Chymopapain B Molecular weight 23,000 25,500 20,000 34,500 33,200 17 2- * B 25.00 21.00 19 .00 18 .40 Isoelectric point 8.75 9.00 9 . 55 10.40 No. of amino acids 212 248 179-285 318 % Carbohydrate 0 3.4 1.4-2.1 0 * Stem bromelain consists of various enzymes B at 280 nm Source: Brocklehurst et al. (1981) Liener (1974) -116- The papain molecule is composed of a single polypeptide chain of 212 amino acid residues, the sequence of which has been determined. Six of the seven cysteine residues are engaged in disulfide bond formation, two being in the first half of the chain (Cys-22-Cys-63 and Cys-56-Cys-95) and one in the second half (Cys-153-Cys-200). The seventh cysteine residue (Cys-25) forms part of the catalytic site (Brocklehurst et al., 1981). The structural conformation is stabilized by the three disulfide bridges. Their rupture results in loss of biological activity, catalytic as well as immunological (Shapira and Arnon, 1969). The high isolectric point (pl= 8.1) of papain suggests that a high proportion of the charged amino acids are basic. Around 18% of the total amino acid residues in papain have charged groups, 7.1% acidic ( Asp and Glu) and 11.3% basic (Lys, His and Arg), which gives a ratio of acidic to basic amino acids of 0.63 (Yada, 1984). The average Bigelow hydrophobicity for papain is 1,159 cal residue-1, which is similar to other proteases such as chymosin, pepsin and trypsin (Yada, 1984). Hydrophobicity values similar to other proteases were also found when this parameter was measured with fluorescent probes (Yada, 1984). In its secondary structure, papain has 28% helix, 14% beta sheet, 17% beta turn and 41% random structure (Chang et al., 1978). The three dimensional structure of papain has been determined -117- by X-ray crystallography to a resolution of 0.28 nm. For a review of X-ray studies of papain see Drenth et al. (1971). The papain molecule is ellipsoidal with approximate dimensions of 5.0 x 3.7 x 3.7 nm. It is a binuclear protein, constructed around two hydrophobic cores. The two lobes contains the same number of amino acid residues and are separated by a cleft in which the active center region lies (Brocklehurst et al., 1981). 3. Stability Crystalline papain shows a high degree of stability. As crystals in suspension in NaCl solutions, it can be kept at 4°C for months without detectable loss in activity (Arnon, 1970; Brocklehurst et al., 1981). Papain is an enzyme with high thermostability; the papain powder resists dry heat at 100<>C for 3 hours (Arnon, 1970). Papain in solution also shows a remarkable temperature stability, which is pH dependent. The enzyme is unstable under acidic conditions. Below pH 2.8 the enzyme suffers a drastic decrease in activity (Arnon, 1970). Papain is less heat stable than chymopapain (Skelton, 1968). Papain is unaffected by high concentrations of some denaturing agents such as methanol (up to 70%), dimethyl sulfoxide (up to 20%), and urea (8 M) (Arnon, 1970). However exposure to 10% trichloroacetic acid or 6 M guanidine hydrochloride causes irreversible changes in the structure and activity of papain (Arnon, 1970). Since papain activity depends on a free -SH group it is -118- expected that all thiol reagents should act as inhibitors. Papain is inactivated in the presence of air and low concentrations of cysteine (Brocklehurst et al., 1981; Arnon, 1970). 4. Activity Papain is a protease with broad side chain specificity, and will degrade most protein substrates more extensively than trypsin, pepsin or chymotrypsin, in many cases giving rise to free amino acids (Arnon, 1970). Studies on papain-catalyzed cleavage of alpha and beta chains of haemoglobin, oxidized chains of insulin and of glucagon indicate that peptide bonds of the type A-X-Y, in which X-Y is the scissile bond and A is Phe, Val, lie or Leu are cleaved preferentially (Brocklehurst et al., 1981). In addition to the hydrolysis of peptide bonds, papain is very effective as an esterase or thiol esterase, and also possesses transferase activity. Papain is capable of catalyzing not only transamidation and transpeptidation, but transesterification reactions as well (Arnon, 1970). E. CHEMICAL MODIFICATION OF PAPAIN Exploration of the active site of an enzyme by chemical modification frequently provides important evidence about its properties (Lowe, 1976). Since papain is a sulfhydryl enzyme, -119- it is not surprising that most of the chemical modification studies on this protein have concerned the modification of the essential cysteine residue (Cys-25). Many of the studies may be considered more as kinetic than chemical modification studies, since more emphasis was placed on the kinetics of the inactivation or inhibition. A discussion on the kinetics of irreversible inhibition (i.e. irreversible modification) is also given in this literature review (see section "Irreversible inhibitors"). 1. Modification of Cys-25 Modification of the Cys-25 group of papain has been carried out with a variety of alkylating agents (e.g. iodoacetate, iodoacetamide, chloroacetate). Irreversible inactivation of the enzymes occurs by alkylation but in most cases no observable changes in structure occur (Lowe, 1976; Brocklehurst et al., 1981). Papain alkylated with iodoacetic acid has been reported to have the same immunological interactions with antipapain serum as native papain. These results suggest that very little, if any, structural change occurred during this modification (Shapira and Arnon, 1969). Oxidation of the Cys-25 residue of papain has been performed with a variety of reagents; however, in many cases only the effect on the papain activity was reported. Since oxidation per se causes losses in the activity, it is difficult to indicate -120- if changes in structure occur upon chemical modification of papa in. Reaction of papain with 2-bromo-21,4'dimethoxy acetophenone produced a derivative that was used to prepare modified papain molecules in which cysteine-25 was changed into: (1) dehydro-serine, (2) serine and (3) glycine (Clark and Lowe, 1978). Serine- and dehydro-serine papain lacked esterase activity, but possessed binding properties to affinity chromatography columns similar to those of native papain. These results indicate that the tertiary structure of the native enzyme has been preserved (Clark and Lowe, 1978). 2. Modification of other amino acid residues Few studies aimed at modifying other residues in papain have been reported. In general, chemical modification of amino acid residues, other than the essential cysteine residues, in sulfhydryl enzymes presents a difficult problem. Since the cysteine group located at the active site is so reactive, most reagents react with it in preference to any other residue which might also be a functional component of the active site (Gleisner and Liener, 1973). In order to solve this problem, the technique of reversible blocking of the cysteine residue with tetrathionate has been used with success (Liu, 1967; Gleisner and Liener, 1973). Gleisner and Leiner (1973) modified the histidine residue located at the active site of ficin. A fully active derivative of ficin was prepared by -121- reversibly blocking its active thiol group with sodium tetrathionate and by oxidation of a methionine residue with sodium metaperiodate. Treatment of this active derivative with bromoacetone in the presence of 2 M urea at pH 6.5 resulted in its complete inactivation. The sole change in its composition was the loss of one of its two histidine residues. Modification of histidine residues by photooxidation with methylene blue caused loss of the caseinolytic activity of papain and bromelain. Discrepancies between the rate of the loss of proteolytic activity and that of histidine residues suggests that factors other than oxidation of histidine residues were involved in the mechanism of inactivation (Murachi and Okumura, 1974). Tetrathionate was also used in this experiment as a blocking agent for the cysteine groups of papain and ficin. Succinylation of papain caused, as expected, a dramatic change in the isolectric point (pi), from 8.2 to 4.3. In spite of the change in pi, an activity assay with benzoyl-glycine ethyl ester demonstrated a loss of activity of only 10% (Sluyterman and DeGraff, 1972). One of the most widely used reagents for the modification of tryptophan residues is N-bromosuccinimide, which with an intact enzyme generally oxidizes the indole to an oxyindole ring and may subsequently brominate it. It is known, however, that this reagent is capable of modifying cysteine, methionine, tyrosine and histidine residues (Lowe and Whitworth, 1974). In -122- a detailed investigation, Lowe and Whitworth (1974) studied the modification of tryptophan residues of papain by N- bromosuccinimide. The cysteine group of papain was protected first, as a disulfide with mercaptoethanol. The authors reported that a 2 molar equivalent of the reagent N- bromosuccinimide modified tryptophan-69, and a 4 molar equivalent modified tryptophan-69 and -177. The enzymic activity, measured with N-benzyloxycarbonylglycine p-nitrophenyl ester, was not seriously impaired. By contrast, photooxidation of tryptophan -177 and additional conversion into formylkynuremine, led to complete loss of enzymic activity toward N-a-benzoyl-L-arginine ethyl ester with apparently only minor changes in papain conformation (Jori and Galiazzo, 1971). As mentioned before, reduction of all the disulfide bonds of papain resulted in total loss of activity in papain. It should be pointed out that in order to obtain a complete reduction of the three disulfide bonds of papain, it was necessary to incubate the enzyme with 0.34 M (3-mercaptoethanol plus 6 M guanidine hydrochloride for three hours (Shapira and Arnon, 1969). It was also possible to selectively reduce only one disulfide bond of papain. In this case, it was necessary to incubate the protein with the reducing agent plus 8 M urea; 60% of the original activity was retained (Shapira and Arnon, 1969). Reacting this reduced papain with mercury caused the formation of an intramolecular mercaptide bond -S-Hg-S-, instead of the -123- original disulfide bond -S-S-. This enzyme derivative was enzymatically active on a range of substrates. The internal mercury atom, in contrast to the one found in the active site sulfhydryl, was retained in the molecule under the conditions required for activation of papain (5 mM cysteine and 2 mM EDTA). The immunological reactivity of this derivative with antipapain serum was essentially identical with that of native papain (Shapira and Arnon, 1969). Mercury papain, papain with Cys-25 blocked with mercury, is a commonly used derivative of the enzyme. It has been shown to have the same structure as papain, and since it is reversibly inactive, it is more stable during storage than the native papain molecule (Arnon, 1970). -124- F. ENZYME KINETICS 1. Reaction rates The mathematical equations that describe the changes in concentration of the product or reactant during a chemical or enzymatic reaction at any time are called reaction rates. The reaction rates most frequently found in enzymatic reactions are: zero, first and second order reaction rates (Whitaker, 1972). (a) Zero order reactions In a zero order reaction the rate of disappearance of reactant or rate of appearance of product is independent of the concentration of reactant. A monomolecular, irreversible zero order reaction is represented as: A- * ¥P (1) where A is the reactant and P is the product. The rate of conversion is expressed mathematically for a zero order reaction as: -dA/dt = k = dP/dt (2) By integrating Eq.(2) between the limits of substrate concentration at zero time (A0) and at time t (A), the following equation is obtained: Ao-A = kt = P (3) The plot of lAol-lA) or IP] against time gives a straight line with intercept of zero and slope k. Zero order reactions with respect to substrate concentration in enzymatic reactions are encountered whenever the enzymes are saturated with substrate (Whitaker, 1972). (b) First order reactions In a first order reaction the rate of the reaction depends on the reactant concentration and is expressed mathematically as: -dA/dt = kA = dP/dt (4) Integrating Eg.(4) between the limits of reactant concentration of Ao at time 0, and A at time t, gives the following equation: In A = In A0 - kt (5) In enzyme-catalyzed reactions, the rate of reaction is first order with respect to substrate concentration when the enzyme is less than 5% saturated with the substrate (Whitaker, 1972). (c) Second order reactions This type of reaction is characterized as being dependent upon two molecules, either of the same compound or of two different compounds. A second order reaction can be represented as: A + B "s *P (6) Mathematically the rate of reaction of a second order reaction is expressed as: -dA/dt = -dB/dt = dP/dt = kAB (7) Depending on the initial concentration of A and B, three types of second order reactions can be found (Whitaker, 1972): -126- I. Type I When the concentration of A is equal to the concentration of B, the reaction rate can be expressed as: -dA/dt = kA* (8) By integrating Eq.(8) under the condition that at t=0, [A]=[AoJ or [B]=[B0] one obtains: 1/tA] - 1/[A0] = kt or 1/[B] - l/[Bo] = kt (9) Eq.(9) is widely used to determine the second order rate constant of the reaction of an enzyme with an irreversible inhibitor compound. The second order rate constant, k (M_I s"1), is obtained by plotting the data according to Eq.(9). II. Type II When the concentrations of the reactant A and B are similar, the reaction rate is expressed as Eq.(7). By integrating Eq.(7) and expressing the equation in terms of initial reactant concentration we obtain (Whitaker, 1972): kt = 2.3/( [Bol-IAol ) log{ ( [Ao]/[B0] ) * ( [Ba]-[P] )/( [AA]-[P] ) } (10) Eq.(lO) is also commonly used to determined the second order rate constant of inactivation of an enzyme with an irreversible inhibitor (Tonomura et al., 1985). III. Type III When the concentration of one of the reactants is relatively high in relation to the other reactant, it can be assumed that the concentration of the reactant present in high concentration is constant over the course of the reaction and the second order rate equation can be simplified to the first order reaction rate equation (Eq.(5)). 2. States of an enzymatic reaction Enzyme catalyzed reactions follow the progress curve shown in Fig. 21. Generally three states for the overall time course of an enzyme reaction can be recognized: Phase I : The pre-steady state or transient initial phase, a few seconds in duration, Phase II : A steady-state phase, Phase III: A nonlinear phase up to completion. (a) The pre-steady state The initial part of the curve where the velocity, or rate of the reaction, expressed as dP/dt, is increasing with time is called the pre-steady state. According to the Michaelis-Menten theory (see section "Effect of substrate concentration on the initial velocity"), it is the time required to establish the equilibrium between the formation and breakdown of the enzyme- substrate complex. Since the formation of the enzyme-substrate complex is an adsorptive phenomena that is controlled by diffusion, the pre-steady state can only be studied by special techniques such as stopped-flow or perturbation methods (Engel, 1984). -128- [Product] phase Reaction time Figure 21. Progress reaction curve for an ideal enzyme reaction. -129- (b) The steady state In this part of the reaction all the reactants are in dynamic equilibrium and therefore operate at maximum efficiency. If the initial substrate concentration is high enough to saturate the enzyme, there will be a period in which the reaction rate with respect to time is zero order, i.e. dP/dt = k. The length of this period will depend, among other things, on the initial substrate concentration (Fullbrook, 1983). (c) The nonlinear state As the reaction proceeds the initial reaction velocity decreases, even if complete enzyme activity is retained. This is due to the reaction approaching its equilibrium point and the reverse reaction beginning to operate (Fullbrook, 1983). The integrated form of the Michaelis-Menten equation can be used to describe the three states, assuming that the enzyme maintains its full activity during the whole course of the reaction (Fullbrook, 1983). 3. Measurement of velocity of enzyme catalyzed reactions Since the reaction velocity of an enzyme-catalyzed reaction is not constant over the entire reaction time, the rate most frequently determined is the initial rate, or initial velocity of the reaction (Ainsworth, 1977). This method involves determination of rate of the reaction as close to time zero as possible. This method has the following advantages: (a) it is -130- unaffected by instability of the enzyme, (b) it is unaffected by the product concentration since this is zero, and (c) substrate concentration can be taken as that initially added to the reaction (Whitaker, 1972). (a) Effect of substrate concentration on the initial velocity Many factors affect the initial velocity of an enzyme catalyzed reaction: enzyme and substrate concentration, pH, temperature, ionic strength and the presence of activators or inhibitors. From a kinetics point of view and at a constant enzyme concentration, substrate concentration is one of the most important factors which determine the velocity of an enzymatic reaction (Dixon and Webb, 1964). In most cases when the initial velocity is plotted against substrate concentration a section of a rectangular hyperbola is obtained, as shown in Fig. 22. The shape of the curve is typical of a process that depends on a simple dissociation (Fullbrook, 1983). A theory involving dissociation of this type was put forward in 1913 by Michaelis and Menten and has been the foundation of the greater part of enzyme kinetics. This theory assumes that the enzyme (E) forms a complex (ES) with the substrate (S) and that the complex (ES) dissociates into the free enzyme (E) and the end product (P) of the enzyme reaction (Dixon and Webb, 1964). E + S— »ES (11) ES^ »E + P (12) -131- Vmax Figure 22. Typical graph o£ the initial velocity of an enzymatic reaction as a function of initial substrate concentration. -132- The derivatization of the Michaelis-Menten equation can be found in many biochemical textbooks (e.g. Whitaker, 1972). The Michaelis-Menten equation is the equation of a right hyperbola and describes the data as plotted in Fig. 22: v = Vmax [S]/ (Km +[S]) (13) v = initial velocity [S ] = initial substrate concentration Vmax = maximum velocity Km = Michaelis constant The Michaelis constant (Km) can be defined as the value of the substrate concentration that gives an initial velocity equal to half the maximum velocity at that enzyme concentration. Thus it is a measure of the affinity of the enzyme for the substrate; the smaller the value of Km the higher the affinity that the enzyme shows for the substrate (Fullbrook, 1983). The Km of enzymes range widely but for most industrially used enzymes it lies in the range of 10"1 to 10"9 M (Fullbrook, 1983). Km is independent of enzyme concentration and is a true characteristic of the enzyme for a specific substrate under defined conditions of temperature, pH and ionic strength (Dixon and Webb, 1964). (b) Determination of Km and Vmax According to Eq.(13) at a given initial substrate concentration, two parameters, Km and Vmax, define the initial velocity of an enzymatic reaction. In order to calculate Km and Vmax, initial velocities at different substrate concentrations -133- must be found. G. DETERMINATION OP INITIAL VELOCITIES Determination of accurate initial velocities is a prerequisite for the calculation of accurate Michaelis-Menten parameters of an enzymatic reaction (Durance et al., 1986). The most common method to estimate initial velocities is the fixed-time assay. In this method the reaction is started and at a predetermined time, a sample is analyzed for product or substrate concentration. Since it is assumed that the initial velocity is constant over the predefined initial time, meaning that the reaction is zero order with respect to time, the initial velocity, Vo is calculated by: (So-S)/(t-t0) = Vo (14) where So is the initial substrate concentration and S is the substrate concentration at time t. The main advantage of this method is its simplicity. However, estimates of the initial velocities tend to be inaccurate even when the traces are almost straight (Atkins and Nimmo, 1980). When two variables are mutually correlated (e.g. reaction time and product concentration) linearization through data transformation is quite useful (Pujii and Nakai, 1980). Pujii and Nakai (1980) developed a procedure for data transformation for linearization and suggested that this transformation could be used to find the reaction order which best fitted the reactant- -134- product relationship during the course of the reaction. Durance et al. (1986) applied this technique, with excellent results, to determine reaction orders for hypothetical models and kinetic parameters with reduced standard errors for an enzymatic hydrolysis. This method has also been applied in shelf life studies of corn products (Arteaga, 1988). The linearization method is as follows: for a monomolecular, irreversible reaction the rate of disappearance of a substrate can be expressed as: dS/dt = kS" (15) where S is the substrate concentration, t is reaction time, n is the order of the reaction, and k is the rate constant. By integrating Eq.(15) the following equations are obtained (Durance et al., 1986): S1"" = So1-" + (n-l)kt for n f 1 (16) InS = InSo - kt for n = 1 (17) The dependent variables of these equations (S1-" and InS) are transformed according to Durance et al. (1986) to: YB = So1"" + (n-l)kt for n ? 1 (18) lnY = InSo - kt for n = 1 (19) B values are selected, using the computer program reported by Durance et al. (1986) such that the coefficient of determination of the line YB against time is maximized. The reaction order (n) is then calculated from: n = 1-B (20) The value of the rate constant k is taken as the slope of the -135- equation for the linearized data calculated by linear regression. By obtaining the derivative of this linearized equation, and equalizing it to zero, the value of the initial velocity is obtained. After initial velocities have been calculated at different initial substrate concentrations, different methods can be used to calculate Km and Vmax. For best results, initial velocities should be obtained at 6 to 10 initial substrate concentrations ranging from 0.1 to 10 Km. Atkins and Nimmo (1980) reviewed the present methods for estimation of Michaelis-Menten parameters. The Michaelis-Menten equation (Eq.(13)) may be plotted in several different ways for the determination of Vmax and Km from a set of measurements of velocity at different substrate concentrations. The most common graphical methods are the Lineweaver-Burk method and the Hofstee method. A detailed discussion of these methods can be found in many biochemical textbooks. More recently, non-linear regression analysis (Oestreicher and Pinto, 1983; Lutz et al., 1986) has been used to calculate Km and Vmax more accurately. H. NITROPHENYL ESTERS AS SUBSTRATES FOR PAPAIN Nitrophenyl ester derivatives of specific compounds are commonly used substrates in enzyme kinetics experiments (Whitaker, 1972). Since the nitrophenyl ester is a good leaving -136- group and the concentration of nitrophenol liberated can be easily determined spectrophotometrically at different pH's, the rate of hydrolysis can be followed continuosly. Recently the construction of a p-nitrophenolate sensitive electrode and its application to an enzyme assay was reported (Katsu et al., 1987). Some of the most common artificial substrates used in kinetic studies of papain are the nitrophenyl esters of carbobenzoxyglycine. Both the pre-steady and steady state kinetics for the papain catalyzed hydrolysis of this nitrophenyl are well documented (Ascenzi et al., 1987; Kirsch and Ingelstrom, 1966). 1. Steady state kinetics for the papain catalyzed hydrolysis of carbobenzoxyglycine Kirsch and Ingelstrom (1966) studied in detail the steady state kinetics of the papain-catalyzed hydrolysis of esters of carbobenzoxyglycine. They concluded that the reaction of papain with the p-nitrophenyl ester provides a very sensitive and convenient assay for enzyme activity. The reaction is fast, taking 1 min to complete, is easily followed spectrophotometrically, requires very little enzyme (about 20Ug per assay), and the high solubility of the substrate makes it possible to work at saturating conditions. Moreover, the compound is commercially available at a reasonable price. However, since nitrophenyl ester assays are inherently unspecific, the use of this substrate is limited to fairly pure enzyme preparations (Kirsch and Ingelstrom, 1966). -137- Kirsch and Ingelstrom (1966) determined values of Km and Vmax for the hydrolysis of p-nitrophenyl, m-nitrophenyl, o-nitrophenyl and ethyl esters of carbobenzoxyglycine. Initial velocities were estimated using fixed time assays. According to the kinetic data reported, those authors proposed that enzyme and substrate formed a noncovalent complex in the first stage of the reaction, which was followed by an acylation step with a rate constant dependent upon the reactivity of the substrate. The final reaction was the deacylation of the enzyme. Similar results have also been reported by other researchers (Hubbard and Kirsch, 1968; Hollaway and Hardman, 1973). Values obtained by different researchers for the kinetic parameters Km, Vmax and Kcat (= Vmax/enzyme concentration) for the papain-catalyzed hydrolysis of p- nitrophenyl ester of carbobenzoxyglycine catalyzed are shown in Table 13. 2. Pre-steady state kinetics for the papain catalyzed hydrolysis of carbobenzoxyglycine In a recent article, Ascenzi et al. (1987) reported the steady and pre-steady state of the papain catalyzed hydrolysis of a p-nitrophenyl ester of carbobenzoxyglycine over the pH range of 3 to 9.5 at 21°C. Their result suggested a minimum three-step reaction mechanism, involving an acyl enzyme intermediate. The pH profile of the kinetic parameters reflected the ionization of two groups with pK values of 4.5+0.1 and 8.8±0.15 in the free enzyme. -138- Table 13. Michaelis-Menten parameters for the papain catalyzed hydrolysis of carbobenzoxyglycine p-nitrophenyl ester*-. Km Kc«B Reference (UM) ,(s-1) Kirsch and Ingelstrom (1966) 13.0 2.73 Alecio et al. (1974) 8.3 8.00 Lin et al. (1975) 16.0 14.00 Ascenzi et al. (1987) 16.0 3.16 12.0 13.0 *-The reported range of conditions used during the kinetics experiments were: temperature: 21-25°C buffer: 0.02-0.1 M phosphate pH: 6-7 BKC»T = Vmax/[E] -139- I. ENZYME INHIBITION 1. Definition and classification Any substance which reduces the velocity of an enzyme-catalyzed reaction, by whatever mechanism, is an inhibitor (Whitaker, 1972). Inhibitors influence the rate of the enzyme-catalyzed reaction either by blocking the active site, or by bringing about a change in the structure affecting the efficiency of the active site (Engel, 1984). Based on kinetic considerations inhibitors can be classified into two groups: irreversible and reversible inhibitors. The interest in enzyme inhibitors for cysteine proteases has grown significantly in recent years, due to the fact that some enzymes in this family are degradative agents in certain disease states, such as muscular dystrophy, some forms of heart disease and cancer. Inhibitors to these enzymes are therefore of pharmaceutical interest (NRC Canada, 1988; 1987). (a) Irreversible inhibitors This group includes all compounds that react with an enzyme to form stable covalent bonds (Whitaker, 1972). Reagents such as iodoacetic acid and fluorodinitrobenzene, whose action is to combine chemically with specific amino acid residues of the enzyme, are classical examples of irreversible inhibitors (Dixon and Webb, 1964). Even though the covalent bond formed between enzyme and inhibitor may in some cases be reversed by chemical -140- treatment, from a strictly kinetic point of view the inhibition is considered irreversible. In some cases the inhibitors bind so tightly to the enzyme that irreversible inhibition kinetics can be used in order to characterize the mode of inhibition (Ainsworth, 1977). I. Kinetics of irreversible inhibition As mentioned by Tsou et al. (1985), in most textbooks of enzyme kinetics, attention is always focused on the effect of reversible inhibitors on the steady-state kinetics of enzyme catalysis. The kinetics of the binding of irreversible inhibitors with the enzyme molecule usually receive little more than passing mention. Irreversible inhibition can be seen as a second order irreversible reaction, where the rate of association or binding is the second order rate constant (Whitaker, 1972). For a simple one-to-one binding irreversible reaction: E + I S ».EI (21) where E is an enzyme, I is the inhibitor, and EI is a binary complex, the rate of the inhibition may be written as: -dE/dt = -dl/dt = dEI/dt = k[E][I] (22) By integrating Eg.(22) under the conditions that at t=0, [I]=[Iol or [E]=[E0] one obtains (Tonomura et al., 1985; Chung and Folk, 1970): kt=l/( [Io)-[E0] ) ln{( [E0]/[Io] )*( ( [Io]-[Eo] ) + ( [Et=]/[Eo] ) )} (23) -141- In the case of tEol=tIol Eq.(23) is simplified to: l/[Et] - l/[Eo] = kt or l/[It] - l/[Iol = kt (24) Usually the amount of free enzyme at different time intervals [Et], is determined by enzyme activity (Tonomura et al., 1985). Since, in enzyme kinetic studies, activity (i.e. velocity) is normally proportional to the concentration of free enzyme. Eq.(24) can also by expressed in terms of activities (Tonomura et al., 1985; Barrett et al., 1982): l/at - l/a0 = kt (25) where at is activity at t time, and a0 is activity at zero time. If the concentration of inhibitor is much greater than the enzyme concentration ([I0] >> [E0]), the reaction can be assumed to be first order and the pseudo-first order rate constant, koBa, can be calculated according to the following equation: — K.OB3 t= ln([Et]/[E0] ) (26) or in terms of activities: In (at/a0) (27) The apparent second order rate constant for inactivation, k, is calculated as (Barrett et al., 1982): K — NOBS /[Io] (28) where [Io] is the initial inhibitor concentration. When reaction of an irreversible inhibitor with an enzyme involves a group near the active site of the enzyme, the presence of substrate (or a competitive inhibitor) may change the rate of reaction of inhibitor with the enzyme. In most cases, the rate of reaction will decrease as substrate concentration is increased to -142- saturation (Whitaker, 1972). This concept of inhibition competition can equally be applied to both reversible and irreversible inhibitors (Tsou et al., 1985). Reversible competitive, noncompetitive and uncompetitive inhibitors are distinguished by their effects on Km and Vmax (see section Reversible inhibition). Similar criteria can also be derived to distinguish three types of irreversible inhibitors from the effect of substrate concentration on the apparent rate constant of binding of irreversible inhibitors to the enzyme (Tsou et al., 1985) . Conditions to distinguish three types of irreversible inhibitors are of the same type used for reversible inhibitors. Whereas equilibrium constants are used to distinguish the three types of reversible inhibition, rate constants are used in irreversible inhibition (Tsou et al., 1985). According to Tsou et al. (1985) plots of substrate concentration against the inverse of the second order rate constant can distinguish these three types of inhibition. For competitive inhibition, the plot of 1/k against substrate concentration will be a straight line. For noncompetitive inhibition, the apparent rate constant is the true rate constant and is independent of substrate concentration. For uncompetitive inhibition, a plot of 1/k as a function of 1/tS] will be a straight line. -143- II. Irreversible inhibitors o£ sulfhydryl enzymes All reagents for chemical modification of sulfhydryl groups (e.g. DTNB, 2PDS, tetrathionate, alkylating agents) are, from a kinetic point of view, irreversible inhibitors of sulfhydryl enzymes. They have already been discussed (see the section "Modification of sulfhydryl groups in proteins"). An interesting irreversible inhibitor of sulfhydryl enzymes is the compound E-64, L-(trans)-epoxysuccinyl-leucylamido(4- guanidino) butane. This inhibitor was first isolated from the extract of a solid culture of Aspergillus iaponicus TPR-64 (Hanada et al., 1978). Since this compound combines in an eguimolecular and irreversible form with the essential -SH of papain and other sulfhydryl proteases (Barrett et al., 1982; Hanada et al., 1978), it has been used for active-site titration, to determine the operational molarity of enzyme solutions, thus calibrating rate assays (Ascenzi et al., 1987; Zucker et al., 1985; Barrett et al., 1982). (b) Reversible inhibition Since many biochemical textbooks discuss in detail the kinetics of reversible inhibition, only a brief discussion will be presented here. Reversible inhibitors form noncovalent complexes with the enzyme. They can be removed from an enzyme by dialysis, chromatography, etc., with complete restoration of the uninhibited reaction rate (Engel, 1984). -144- I. Kinetics of reversible inhibition Three basic types of reversible inhibitors are recognized. They are called competitive, noncompetitive and uncompetitive inhibitors and they are distinguished from one another by their effects on the velocity of the enzyme catalyzed reaction when measured as a function of a particular substrate concentration (Ainsworth, 1977). For the schematic reaction: E + S* ES I-EH. + P (29) K-i It has been found that: a) Any inhibitor that displaces the equilibrium E + S« *ES modifies the value of Km. b) Any inhibitor that changes the maximum concentration of ES modifies Vmax. II. Competitive inhibition This is the simplest case of reversible inhibition. The assumption here is that binding of the inhibitors and binding of the substrate are mutually exclusive (Engel, 1984). The basis for this is that the inhibitor has structural features sufficiently similar to those of the substrate to enable it to occupy all or part of the substrate binding site. Since substrate at infinite concentration must be able to compete successfully with inhibitors at finite concentration, the maximum rate (Vmax) is unaltered by a fixed concentration of inhibitor. -145- III. Uncompetitive inhibition This is the opposite of competitive inhibition, since the assumption is that the inhibitor can bind only to the enzyme-substrate complex (Engel, 1984). The inhibitor promotes binding between the enzyme and substrate, thus decreasing Km. Once the enzyme-substrate complex is formed, it may be either productive (form product) or non-productive (binds inhibitor). (Engel, 1984). IV. Noncompetitive inhibition In this case, the inhibitor can bind with equal ease to either the free enzyme or to the enzyme-substrate complex, but the enzyme-substrate-inhibitor complex is inactive. Since substrate and inhibitor do not compete with each other for the same binding site on the enzyme, inhibition cannot be eliminated by adding more substrate, as is the case with a competitive inhibitor (Whitaker, 1972) V. Reversible inhibitors of sulfhydryl enzymes Many reversible inhibitors of cysteine proteases have been reported. Urea and guanidine hydrochloride, at low concentrations, inactivate papain in a noncompetitive way, while cyanate ions react with papain in a mixed type form (Nakamura and Soejina, 1970). Urea at high concentrations (2 M), methanol, acetonitrile and dimethylsulphoxide exhibit competitive inhibition (Sluyterman, 1967b). -146- ,. Egg-white cystatin is a tightly-binding inhibitor of ficin, papain, cathepsin H and L and also dipeptidyl peptidase (Nicklin and Barrett, 1984). It has been reported that this cystatin shows a competitive reversible inhibition of cathepsin B. With papain it was shown to form equimolar complexes (Nicklin and Barrett, 1984) . Other cystatins have been found in mammalian tissues and body fluids. Their inhibitory properties are very similar to those of egg white cystatin, and amino acid sequences show evolutionary homology (Nicklin and Barrett et al., 1984). These inhibitors can be grouped into three families, on the basis of structural similarities. Family 1 consists of low- molecular weight (all,000) inhibitors, without a disulfide bridge, and are found mainly intracellularly. Family 2 consists of low molecular weight (»13,000) inhibitors with two disulfide bridges, mainly found in body fluids. Recently three new cystatin type protease inhibitors which belong to family 2 have been isolated from human saliva (Isemura et al., 1987). Family 3 consists of high molecular weight inhibitors (Isemura et al., 1987). Although E-64 is an irreversible inhibitor (see Irreversible inhibitors of sulfhydryl proteases) of sulfhydryl proteases, Hanada et al. (1978) applied reversible inhibition kinetics to study its inhibition effect on papain, and reported that E-64 was a noncompetitive inhibitor. This result is surprising since E-64 has been found to react with the active site of the sulfhydryl -147- proteases, thus, its inhibitory action should be competitive. More detailed studies carried out by Barrett et al. (1982) confirmed the active-site-directed nature of the reaction of papain with E-64. Leupeptin, acetyl-L-leucyl-L-leucyl-L-arginyl, is another tightly- binding reversible inhibitor of cysteine proteases. This inhibitor has exhibited a protective effect against mouse muscular dystrophy, and its possible usefulness in the treatment of muscular dystrophy in man has been studied (Umezawa and Aoyagi, 1983). Two other reversible inhibitors, antipapain and chymostatin, together with leupeptin, have an a-amino aldehyde group in the C-terminal part of their peptide molecule. The terminal aldehyde group is involved in the specific binding to enzymes, including the hydroxyl or thiol group of serine or cysteine proteases, respectively (Umezawa and Aoyagi, 1983). -148- MATERIALS AND METHODS A. MATERIALS Papain (twice crystallized), cysteine-HCl, ethylene diamine- tetraacetic disodium salt (EDTA), 5,51-dithiobis 2-nitrobenzoic acid (DTNB) and carbobenzoxyglycine p-nitrophenyl ester were from Sigma Chemical Company (Saint Louis, MO). Sephadex G-25 superfine and DEAE-Sephadex A-25 were from Pharmacia Fine Chemicals (Uppsala 1, Sweden). Sodium tetrathionate was obtained from ICN Pharmaceuticals, Inc. (Plainview, NY). Acetonitrile (HPLC grade, wavelength cutoff 190 nm), was from Caledon Laboratories Ltd. (Georgetown, ON). All reagents were of analytical grade or better. Glass distilled water was used in the preparation of all solutions and buffers. Weighing in the milligram range was done on an UM3 ultrabalance (Mettler, Greifensee, Switzerland). B. DETERMINATION OF PROTEIN CONCENTRATION Protein concentration of papain solutions was determined from absorbance readings at 280 nm with a E^d cm) of 25.0 (Brocklehurst et al., 1981). A molecular weight of 23,800 daltons (Brocklehurst et al., 1981) was used for all calculations. In some experiments, protein was measured at least in -149- duplicate either by using the dye-binding assay as described in the Bio-Rad protein standard assay bulletin (Bio-Rad Instruction Manual 82-0275-1282, Bio-Rad Laboratories, Richmond, CA) developed by Bradford (1976), or by digesting the samples using the rapid Micro-Kjeldahl method of Concon and Soltess (1973). Digested samples were analyzed for total nitrogen content using an Auto Analyzer II (Technicon Instruments Co., Tarrytown, NY). The crude protein content was then calculated by multiplying the total nitrogen content by a factor of 6.25. C. DETERMINATION OF PROTEOLYTIC ACTIVITY 1. Optimization of the conditions to measure proteolytic activity of papain Initially, the ability of papain to hydrolyze casein was determined using the method of Hanada et al. (1978). However, it was found that this method had poor repeatability. As mentioned by West (1988), enzyme assays are generally not as accurate nor reliable as good chemical analysis. In order to increase Its precision, an optimization of three parameters of this method: enzyme concentration, incubation time and incubation temperature, was carried out. The objective of this optimization was to minimize the standard deviation of the proteolyic activity readings within replicates. The optimization method used was a new optimization approach which involved a central composite rotatable design (CCRD) for -150- three variables, followed by multiple regression and computational simplex optimization. According to the CCRD the complete experimental plan consisted of a total of 16 experiments. Table 14 shows the levels for each of the three factors used. Four replications were done for each of the 16 experimental conditions. Proteolytic activity was measured as described below, with the modification that papain concentration, incubation time and incubation temperature were varied according to the CCRD. Using the standard deviation (four replicates) of the absorbance reading at 280 nm of the filtrates of each experimental condition as the dependent variable, a quadratic model was obtained through multiple regression. The equation obtained was entered as part of the computational simplex optimization program. Minimization of this function was performed in order to find the conditions associated with the smallest standard deviation. 2. Proteolytic activity determination The method of Hanada et al. (1978) was used to measure the proteolytic activity of native and tetrathionate modified papain, with the modifications in papain concentration, incubation time and incubation temperature, which gave the smallest standard deviation according to the results obtained with the optimization method described above. -151- Table 14. Upper and lower limits for the three factors used for optimization of the proteolytic activity determination of papain. Limits Factor Lower Upper Papain concentration (mg/mL) 0.03 0.10 Incubation time (min) 5.0 10.0 Incubation temperature(°C) 35 45 -152- The substrate solution was 1% (w/v) Hammersten casein (BDH Chemicals, Vancouver, B.C.) in 0.01 M Tris-HCl buffer pH 8, which was prepared according to the Food Chemical Codex (FCC III, 1981). The method was as follows: in a stoppered test tube a half mL of a solution of papain (100 Hg/mL in 0.05 M phosphate buffer, pH 6.8) was mixed with 0.25 mL of 40 mM cysteine and 20 mM EDTA in 0.05 M phosphate buffer pH 6.8, and 0.25 mL of 0.05 M phosphate buffer pH 6.8. The mixture was incubated at 35°C for 15 min. After this period of time, 5 mL of the 1% casein solution (pre-equilibrated at 35°C) was added and the contents of the test tube were mixed immediately using a Vortex mixer. The mixture was subjected to a further incubation for 5 min at 35<>C. Then 5 mL of a 0.44 M trichloroacetic acid (TCA) solution was added to stop the enzyme reaction and precipitate the unhydrolysed substrate. The contents of the test tubes were completely mixed. In order to separate the precipitated protein, centrifugation (10,000 xg, 15 min, 4<>C) followed by filtration under vacuum through Whatman No.42 filter paper was carried out. All filtrates were completely clear. The absorbance of the filtrate was measured at 280 nm with a Cary 210 spectrophotometer (Varian Associates Inc., Palo Alto, CA). Proteolytic activities were expressed as International Units i.e. flmoles of tyrosine liberated min_lmg"x of sample, under assay conditions. A standard curve prepared with L-tyrosine plus casein solution under the conditions for assay was used to convert absorbance to Hmoles of tyrosine. -153- D. DETERMINATION OF THE INFLUENTIAL FACTORS FOR MAXIMUM INHIBITION AND REACTIVATION OF THE PROTEOLYTIC ACTIVITY OF PAPAIN BY TETRATHIONATE The fractional factorial design Lis (21B) of Taguchi (1957) was used to determine the factors which may significantly affect the inactivation of the proteolytic activity of papain by tetrathionate and the subsequent reactivation of the tetrathionate inactivated papain by cysteine. In the inactivation experiments the factors evaluated were: molar ratio of tetrathionate to papain, pH, temperature and time of the inactivation reaction. For the reactivation reaction the same factors were considered, plus the additional factor of cysteine concentration during the activation reaction. The factors together with their assigned levels are shown in Table 15. The scheme used is presented in Fig. 23. For assay of enzyme inhibitory activity, the method reported above for the determination of proteolytic activity was used, with the modification that papain was first activated with the cysteine-EDTA buffer for 15 min at 350C, and then the solution was freed from cysteine by passage through a Sephadex G-25 superfine column, previously equilibrated with nitrogen saturated 0.05 M phosphate buffer pH 6.8, containing 20 mM EDTA. In order to prevent reoxidation of papain, the elution was also carried out under nitrogen saturated conditions. This activated papain solution was diluted with 0.05 M phosphate buffer pH 6.8 or with 0.05 M Tris-HCl pH 10.0, to a proper concentration (0.1 -154- Table 15. Factors and the assigned levels investigated for their possible influence on the inhibition of the proteolytic activity of papain, and on the subsequent reactivation of activity by cysteine. Levels Factor Lower Upper [Tetrathionate]/[papain] 10 100 pH 6.8 10 Reaction time (min) 5 10 Cysteine (mM)*- 20 40 Reaction temperature (°C) 22 40 "•Factor considered only in the reactivation experiments -155- [TT]/[Papain] Temperature Time [Cysteine] Figure 23. Scheme used in the Taguchi Lit fractional factorial experiment. The factor of cysteine concentration was only evaluated in the reactivation experiments. [TTl/[Papain] = molar ratio tetrathionate to papain in the inhibition reaction. -156- mg/mL) for the assay. To 0.25 mL of the diluted activated papain solution was added either 0.25 mL of nitrogen saturated phosphate buffer or a fresh solution of tetrathionate in the same buffer. The concentration of tetrathionate in the solution was such that it gave, upon dilution, a molar ratio of tetrathionate to papain of either 10 or 100. The mixture was incubated under the conditions of time and temperature according to the fractional factorial design. After the appropriate incubation time addition of casein and TCA, centrifugation, filtration and absorbance reading were performed as previously described. The percentage inhibition was calculated as follows (Hanada et al., 1978): % inhibition = 100(A-B)/A (34) where A stands for the absorbance without tetrathionate and B for the absorbance with tetrathionate. For the reactivation experiments the method was as follows: after the activated papain solution was incubated with or without tetrathionate, 0.25 mL of 0.05 M phosphate buffer pH 6.8 with or without cysteine was added at a concentration according to the fractional factorial experiment (20 mM or 40 mM). The mixture was further incubated at 40°C for 10 min. After this, 5 mL of the 1% casein solution was added and the other steps were carried out as described before. -157- The percentage reactivation was calculated as follows: % reactivation = 100(C/A) (35) where A is the absorbance of the sample without tetrathionate plus cysteine, and C is the absorbance of the sample with tetrathionate and cysteine. The values of % inhibition and % reactivation (in duplicates) were analyzed using a Taguchi's fractional factorial analysis of variance computer program written in IBM-BASIC (Arteaga, 1986). E. PREPARATION OF TETRATHIONATE-MODIFIED PAPAIN The tetrathionate-modified papain (TT-papain) was prepared as follows: papain (25 mg) was dissolved in 10 mL of 0.05 M phosphate buffer pH 6.8, containing 20 mM EDTA and either 40 mM cysteine or 20 mM |3-mercaptoethanol. The mixture was incubated for 10 min at 40°C. After this period the mixture was placed on a column of Sephadex G-25 superfine (2 cm x 40 cm) equilibrated with nitrogen saturated 0.05 M phosphate buffer pH 6.8 containing 10 mM EDTA. Protein was eluted with the same nitrogen saturated buffer at a flow rate of 40 mL/hr and 4 mL fractions were collected. The absorbance at 280 nm of the eluent was continually monitored with a LKB 2138 UVICORD UV-detector (LKB-Produkter AB, Stockholm-Bromma 1, Sweden) connected to a SP- H6V plotter (Riken Denshi Co., Denmark). Protein emerged between 32-40 mL of the effluent, and these fractions were pooled. The protein concentration of the pooled fractions was immediately -158- determined by measuring absorbance at 280 nm. A predetermined amount of sodium tetrathionate was added to this protein solution based on the protein concentration. The ratio of moles of tetrathionate to moles papain for the chemical modification varied in the range of 10 to 500. The reaction mixture was incubated at room temperature for 15 min. After this period of time, the excess tetrathionate was removed by passing the mixture through a column of DEAE-Sephadex A-25 anion exchanger, which was equilibrated with 0.05 M phosphate buffer pH 6.8 containing 0.2 M NaCl. Since at this pH papain has a net positive charge, it did not bind to the anion exchange resin, and it was eluted immediately from the column. The protein fractions were pooled, freeze-dried and stored with dessicant at -20°C until further analysis. F. CIRCULAR DICHROISM Circular dichroism spectra were measured using a JASCO J-500A spectropolarimeter (Japan Spectroscopic Co., Ltd., Tokyo, Japan) under a constant nitrogen flush at 20°C. The instrument was calibrated by the two-point calibration technique at wavelengths of 290.5 and 192.5 nm using d-10-camphorsulfonic acid in distilled water as reported by Chen and Yang (1977). -159- 1. Optimization of the conditions for measuring the CD spectra of papain Preliminary experiments shoved that the determination of CD spectra of papain had the problem of high noise levels. Although the CD signal is inherently noisy (Wollmer et al., 1983) and it is well known that this problem can affect the prediction of secondary structure of proteins (Hennessey and Johnson, 1982), the selection of the "best" experimental conditions to measure CD is usually done by trial and error based on previous experience. It was thought that by using an objective optimization method, it could be possible to increase the quality of the CD spectrum of papain. Based on preliminary experiments and previously published information, the following four factors, which affect the determination of the CD spectra of proteins, were selected for optimization: (1) papain concentration, (2) bandwidth, (3) time constant, and (4) the value of the product of scan rate times the time constant. By entering the limits to the simplex centroid optimization program (SCO) (Nakai and Arteaga, 1988) different sets of experimental conditions were obtained. Papain was dissolved in 0.05 M phosphate buffer pH 6.8 and filtered through a 0.44 um Millex-HA filter (Millipore Corporation, Bedford, MA) and diluted with the same buffer to the proper concentration (i.e. absorbance at 280 nm) according to the SCO. Five scans over the range 280-190 nm were recorded for each -160- experimental condition and the standard deviations o£ the ellipticity values (00) at three wavelengths (222, 209, and 200 nm) were calculated. Since the photomultiplier voltage (PM) is a measure of the noise level (JASCO, 1979), the PM at those wavelengths was recorded. The objective function to be minimized was a combination (product) of three nonconfllcting parameters: (1) the total scanning time, (2) the mean of the standard deviation of ellipticity values at the three wavelengths, and (3) the mean of the photomultiplier voltages at those wavelengths. In order to estimate the accuracy of each CD scan, the values of the mean residue ellipticity ([6]HR«) at 222, 209 and 200 nm were calculated using the formula reported below and the values obtained were compared to published data. The coefficients of variation of (IOIMRW) at 222, 209 and 200 nm were also calculated for each experimental condition. 2. Ffls-yy CP Spectra (19Q-24Q nm) CD spectra for native and tetrathionate-modified papain were scanned from 240 to 190 nm. A cell of 0.1 cm pathlength was used. Sample preparation was as described above. The conditions used were those obtained through the optimization technique described above, which were: 0.023 mg/mL papain concentration, 1.6 nm bandwidth, 1.6 time constant, and 0.88 for the product of time constant times the scan rate. -161- The results of the CD analysis were expressed in terms of the mean residue ellipticity (181M»W) in the standard units of deq cm*/dmol. Without correcting the Lorenz refractive index factor, a mean residue weight of 99 (Chang et al., 1978) was used for all calculations. Each protein solution was measured a minimum of three times. The baseline spectrum for each protein sample was obtained by running the buffer under conditions identical to those used for the sample. The mean residue ellipticity (9]MRW was calculated using the following formula: [9]M»W = 9Q»MRW (36) 10»L»c where 8° is the observed ellipticity in degrees, MRW is the mean residue weight (taken as 99), L is the pathlength in cm, and c is the protein concentration in mg/mL. 3. Near-UV Spectra (250 -350 nm) In this spectral region the noise level of the CD signal was small, thus, there was no need to optimize the conditions for near-UV CD measurements. A 1 cm pathlength cell was used with a protein concentration of 1.0-2.0 mg/mL. In this case, the CD results were expressed in terms of the difference in molar absorptivity between left and right circularly polarized light, 5e. The fle was calculated according to the following equation: -162- <5e = [9]MBW N (37) 3300 where N= total residues in the protein. In the case of papain, N=212 (Brocklehurst et al., 1981). G. SECONDARY STRUCTURE PREDICTION Two methods were used to predict the secondary structure fractions of papain based on its CD spectra. The constrained regularization procedure of Provencher and Glockner (1981) was used by entering the [8]MRW at 1 nm intervals from 240 to 190 nm. The second method used was the procedure reported by Siegel et al. (1980). In spite of its simplicity this method has been shown to be highly accurate and versatile (Yada, 1984). In this method, CD data in the form of [8]MRW at wavelengths between 210 and 240 nm provide an accurate prediction of the fraction of helical structure of a protein. The IBM-BASIC computer program used is reported in Appendix I. H. FLUORESCENCE AND DIFFERENTIAL ABSORPTION SPECTROSCOPY The effect of pH on the fluorescence emission spectra of native and tetrathionate modified papain was investigated over the pH range of 4 to 10. Fluorescence measurements were performed with a Shimadzu spectrofluorophotometer Model RF-540 (Shimadzu Co., Kyoto, Japan). -163- The emission spectra, at room temperature (approximately 22<>C), over the range 460-300 nm at an excitation wavelength of 282 nm, were measured in the following 0.02 M buffers: sodium acetate, pH 4.0 to 5.5; sodium phosphate, pH 5.5 to 8.0; Tris-chloride, pH 8.0 to 9.0; glycine NaOH, pH 9.0 to 10.0. No influence of buffer ions on the emission spectra was observed. In all cases a protein concentration of 0.01 mg/mL was used. The wavelength of 282 nm has been reported to be the maximum excitation wavelength of papain. (Barel and Glazer, 1969). Activation of both native and tetrathionate-modified papain was carried out by incubating the enzyme with 20 mM cysteine and 10 mM EDTA in 0.02 M phosphate buffer pH 6.8 for 20 min at 40<>C. After this incubation, activator-free enzyme solution was obtained by passage through a Sephadex G-25 superfine column under nitrogen saturated conditions. Solutions of activated papain were immediately diluted to the appropriate concentration with the corresponding buffer, which had been previously saturated with nitrogen. Absorption spectra measurements were made with a Cary 210 spectrophotometer (Varian Associates Inc., Palo Alto, CA) in the region 350-260 nm. To get a baseline, a sample cuvette containing native papain in 0.02 M phosphate buffer pH 6.8, was scanned against a reference cuvette containing the same enzyme solution. The solution in the sample cuvette was replaced with a solution of tetrathionate-modified papain of the same concentration, and the differential absorption spectrum was recorded. -164- I. DETERMINATION OF TOTAL -SH GROUPS OF PAPAIN The thiol group content of papain was measured according to the method of Habeeb (1972) using 5,51-dithiobis 2- nitrobenzoic acid (DTNB). Five to ten mg of native or tetrathionate modified papain was dissolved in 6.0 mL of a 10% solution of sodium dodecyl sulfate (SDS) in 0.02 M sodium phosphate pH 8, containing 20 mM EDTA. The sample was incubated at room temperature for 10 min. In some experiments, before the addition of SDS, papain (native or TT-papain) was activated with cysteine (40 mM) for 10 min followed by removal of cysteine by gel filtration through a Sephadex G-25 superfine column, under nitrogen saturated conditions as described before. After addition of SDS and incubation, 0.1 mL of a freshly prepared solution of DTNB (40 mg DTNB in 10 mL of 0.1 M sodium phosphate buffer pH 8) was added to 3 mL of the SDS-protein solution, and the mixture was further incubated at room temperature for 10 min. The absorbance of the solution was read at 410 nm against a protein solution in SDS to give apparent absorbance. A reagent blank was subtracted from the apparent absorbance to give the net absorbance. An extinction coefficient of 13,600 M"1 cm"1 (Hanada et al., 1978) for the nitromercaptobenzoate anion was used for all calculations. -165- J. INSOLUBILIZATION OF PAPAIN WITH TETRATHIONATE Preliminary experiments showed that under certain conditions tetrathionate caused precipitation of papain. Tetrathionate was reported to cause precipitation of pig muscle glyceraldehyde-3- phosphate dehydrogenase (Parker and Allison, 1969). In order to give more insight on this observation the experiments described below were carried out. 1. Effect of concentration of tetrathionate and 0-mercaptoethanol on the precipitation of papain The effect of tetrathionate and 0-mercaptoethanol concentration on the precipitation of papain was studied using response surface methodology. A central composite rotatable design (CCRD) of two variables was used. The levels of tetrathionate and 0-mercaptoethanol evaluated are presented in Table 16. Papain was dissolved in 0.05 M sodium phosphate pH 6.8 to a concentration of 10 mg/mL. The required 0-mercaptoethanol was added to give a final concentration according to the CCRD and the mixture was incubated at 40°C for 10 min. Then the required amount of tetrathionate was added and the mixture incubated at 60°C for 10 min, followed by immersion in an ice bath for 5 min. After centrifugation (10000 xg, 20 min, 4°C) protein content of an aliquot of the supernatant was measured using the Bio-Rad method. Absorbance readings were converted to protein concentration values using a standard curve prepared with native papain. -166- Table 16. Levels of the factors used in the RSM experiment of the precipitation of papain by tetrathionate. Coded levels Factor -1.4 -1 0 +1 +1.4 [Tetrathionate] (mM)*- 0 15 50 85 100 [0-mercaptoethanol] (mM)*- 0 15 50 85 100 xFinal concentration -167- 2. Effect of PH and temperature on the precipitation of papain by tetrathionate Papain was dissolved in distilled water and after no pH adjustment (pH 5.5) or pH adjustment to 8.5 with NaOH, 0-mercaptoethanol (80 mM) and tetrathionate ( 100 mM) were added. The mixture was Incubated for 10 min at 22, 40, or 60<>C, followed by immersion in an ice bath for 10 min. CentrifugatIon and protein determination of an aliquot of the supernatant were carried out as described above. 3. Resolubilization of the precipitated protein The effect of different chemical reagents (urea, SDS, and 0-mercaptoethanol) on resolubilization of the precipitated protein was studied. The following procedure was used: the precipitated protein (50 mg), which was insoluble in water, was dispersed in 5 mL of 0.02 M phosphate buffer pH 6.8. Then the corresponding chemical reagent was added to obtain the desired final concentration. The mixture was incubated for 60 min at 45<>C, followed by immersion in an ice bath for 10 min. The mixture was then centrifuged (10000 xg; 4°C; 15 min). Samples that contained urea were dialysed for 48 hr against three changes of distilled water (5 L) at 4°C (Spectrapor membrane tubing No.l, Spectrum Medical Industries, Inc., Los Angeles, CA) before protein determination. An aliquot of the supernatant as well as the insoluble fraction remaining after centrifugatlon were transferred to 30 mL Micro-Kjeldahl flasks and evaporated until dryness in a -168- forced-air convection oven at 80°C (6 hr). Digestion, total nitrogen determination, and calculation of crude protein concentration were carried out as described before. 4. Analysis of the precipitated protein Protein content of the precipitate was measured using the Micro-Kjeldahl method as stated before. Proteolytic activity of the solid was measured using casein as substrate as described previously. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the native and tetrathionate-modified papain (soluble and insoluble), was carried out using a PhastSystem*M (Pharmacia, Uppsala 1, Sweden). PhastGel gradient 10-15% gels and PhastGel SDS buffer strips (Pharmacia, Uppsala, Sweden) were used. The gels had a 13 mm stacking gel zone and a 32 mm continuous 10 to 15% gradient gel zone with 2% crosslinking. The buffer system in the gels was 0.112 M acetate (leading ion) and 0.112 M Tris, pH 6.4. The buffer system in PhastGel SDS buffer strips was 0.02 M tricine (trailing ion), 0.02 M Tris and 0.55% SDS, pH 7.5. The buffer strips were made of 2% agarose. The preparation of the samples was as follows: to 0.5 mL of the protein solution (3-7 mg protein/mL) in 0.01 M phosphate buffer pH 7.0 were added 20 UL of p-mercaptoethanol and 0.2 mL of 10% SDS in 0.01 M phosphate buffer pH 7.0, and 0.3 mL of 0.01 M phosphate buffer pH 7.0. The mixture was heated at 100°C for 5 min, the samples were cooled to room temperature, and 50 UL -169- of the tracking dye (bromophenol blue) was added. Application and separation of the samples were performed as recommended in the PhastSystem manual (Pharmacia, 1986). After electrophoresis, the gel was immediately stained and destained using the recommended fast coomassie staining procedure (Pharmacia, 1986). -170- K. DETERMINATION OF Vmax AND Km FOR THE PAPAIN-CATALYZED HYDROLYSIS OF CARBOBENZOXYGLYCINE P-NITROPHENYL ESTER The method used was essentially the one reported by Kirsch and Ingelstrom (1966). Papain was activated in freshly prepared 0.02 M phosphate buffer pH 6.8, 1 mM EDTA and 0.35 mM cysteine for at least 1 hr at 40°C. It has been reported that, under the conditions mentioned above, activation is complete after 45 min and constant activity is maintained for at least 4 hr (Kirsch and Ingelstrom, 1966). The papain-catalyzed hydrolysis of carbobenzoxyglycine p-nitrophenyl ester was followed by first transferring 0.2 mL of the solution of the nitrophenyl ester in acetonitrile into a 1 cm quartz cuvette placed in the cell compartment of a Cary 210 spectrophotometer (Varian Associates Inc., Palo Alto, CA). The reaction was initiated by adding 2.8 mL of the activated enzyme solution. During the course of the reaction, the change in absorbance at 400 nm was continuously measured. The initial velocity for the enzymatic reaction under each experimental condition was corrected for the reaction in the absence of enzyme. Six initial substrate concentrations were used and at least three complete reaction curves were taken for each substrate concentration. All measurements were performed at room temperature (20-22°C). In order to obtain the initial velocity values (V0) necessary -171- for the calculation of Km and Vmax, the linearization method reported by Durance et al. (1986) was compared to the conventional fixed time assay method. Using the computer program reported by Durance et al. (1986) modified for an IBM-PC, the optimum value of B was found; the reaction order with respect to time was derived (Eg. 20) and according to this value, data was linearized using Eq. 18 or Eq. 19 (see section "Determination of initial velocities" in the LITERATURE REVIEW of this chapter). The equation for the linearized data was obtained by linear regression. Initial velocity at each initial substrate concentration was taken as the first derivative of the curve at t=0. For the conventional fixed time method, the initial velocity (Vo) was assumed to be constant over an initial predefined period of time (Eq. 14). Since the reaction time in most cases was relatively short (30-100 sec) the initial time period was taken at 15 to 30 sec depending on the substrate concentration. The initial velocities calculated by the linearization procedure and by the conventional fixed time method were fitted to the Michaelis-Menten equation by the computer program of Oestreicher and Pinto (1983), modified for running on an IBM-PC computer. This program also calculated the standard error and the residual standard error of Km and Vmax. -172- L. INHIBITION EXPERIMENTS In order to characterize the inhibitory effect of sodium tetrathionate on the activity of papain, a set of inhibition experiments was carried out. Since tetrathionate reacts with cysteine (Inglis and Liu, 1970) present in the activation buffer, it was necessary to incorporate a gel filtration step in order to separate cysteine from papain prior to the inhibition experiments. Papain was activated in 0.02 M phosphate pH 6.8 containing 10 mM of EDTA and 40 mM of cysteine for 15 min at 40<>c. This solution was freed from cysteine by passage through a Sephadex G-25 superfine column previously equilibrated with nitrogen saturated 0.02 M phosphate buffer pH 6.8 and 20 mM of EDTA. The elution was carried out under nitrogen saturated conditions in order to prevent reoxidation of papain. 1. Reversible inhibition experiments Tetrathionate was assumed to act as a reversible inhibitor, and the kinetic parameters for reversible inhibition were calculated. The setup of this experiment was as follows: three different concentrations of the inhibitor (sodium tetrathionate) were evaluated at five substrate concentrations. An aliquot of 0.2 mL of the solution of the p-nitrophenyl ester in acetonitrile was transferred into a 1 cm quartz cuvette placed in the Cary 210 spectrophotometer, followed by 0.2 mL of sodium tetrathionate in 0.02 M phosphate buffer pH 6.8. The reaction was -173- initiated by adding 2.8 iL of the gel filtered activated papain in nitrogen saturated 0.02 M phosphate buffer pH 6.8 containing 20 mM of EDTA. The reaction product was analyzed by measuring the change in absorbance at 400 nm. Appropriate controls were run for all experiments. Initial velocities were calculated as mentioned before and the type of reversible inhibition was determined using the computer program ENZYME (Lutz et al., 1986). This program is a weighted nonlinear least squares curve fitting computer program, implemented in compiler BASIC for the IBM-PC, used to estimate the parameters of enzyme kinetics obeying Michaelis-Menten kinetics and seven inhibition models. 2. Irreversible inhibition experiments (a) In the absence of substrate The inactivation study in the absence of substrate was as follows: 1 mL of a sodium tetrathionate solution was added to 50 mL of a gel filtered activated papain solution to start the inactivation reaction. The mixture was kept at room temperature and the residual enzyme activity (i.e. initial velocity) was assayed every 15 sec as mentioned above, using 1.5 mM p-nitrophenyl ester in acetonitrile as substrate. A control mixture containing all of the components of the reaction mixture except for the inhibitor was included. The result of the inactivation is presented as a second order rate constant. -174- (b) In the presence of substrate In order to characterize the type of irreversible inhibition by tetrathionate, the effect of different substrate concentrations on the second order rate constant of inhibition was evaluated. The experimental setup was as follows. A 0.2 mL aliquot of the solution of the p-nitrophenyl ester in acetonitrile was transferred into a 1 cm quartz cuvette placed in the Cary 210 spectrophotometer, followed by 0.2 mL of sodium tetrathionate in 0.02 M phosphate buffer pH 6.8. The reaction was initiated by adding 2.8 mL of the solution of the gel filtered, activated papain in nitrogen saturated 0.02 M phosphate buffer pH 6.8 containing 20 mM of EDTA. The reaction product was analyzed as mentioned before. Appropriate controls were run for all exper iments. Since the tetrathionate concentration used was much higher than that of papain, the reaction was assumed to be first order. To obtain the second order rate constants at each substrate concentration, linearization of the data (substrate concentration vs. time) was carried out and derivatization of the linearized equation obtained by linear regression was performed for the inhibited reactions. From the derivatized equations, the velocity of the reaction at different time intervals was calculated. -175- The reaction rates o£ the control reactions were practically constant over the time period analyzed (zero order with respect to time) and were calculated using the Michaelis-Menten equation (Eq. 13). The difference between the velocities of the control reaction and the inhibited reaction, at the same initial substrate concentration, at different time intervals was calculated. Taking the velocity as a measure of the activity of papain, semilogarithmic curves of activity at each substrate concentration as a function of time (Eq. 13) were found to be linear and the observed (pseudo first-order) rate of inactivation, k0b«, was taken as the value of the slope of these lines. The apparent second order rate constant for inactivation, k, was taken as (Barrett et al., 1982): k = kob-/[I] (38) where [I]= initial concentration of inhibitor. M. STATISTICAL ANALYSIS Calculations were carried out on an IBM-PC. Data handling and mathematical operations were performed with LOTUS 1-2-3" (Lotus Development Co., Cambridge, MA). The STATGRAPH program (STSC, Inc., 1985) was used for all statistical evaluations. -176- RESULTS AND DISCUSSION A. OPTIMIZATION OF THE CONDITIONS TO MEASURE PROTEOLYTIC ACTIVITY OF PAPAIN A central composite rotatable design (CCRD) for three variables was used in designing this experiment. The independent variables papain concentration, temperature and time of incubation were coded as Xi, X* and X3, respectively. Altogether 16 combinations (Including a replicate of the central point) formed the CCRD. The levels of the three independent variables together with the values of the response are shown in Table 17. The response was taken as the standard deviation (SD) of four replicates of the absorbance at 280 nm of the TCA-filtrate at each experimental condition. The data in Table 17 was fitted to a second order model commonly used in response surface methodology (RSM) (Nakai and Arteaga, 1988): Y= 0o + E 0iXi + E 0nXi* + E E PijXiX, (39) where 0i are regression coefficients and Xi are the independent variables related to Y. The adequacy and fitness of the model were tested by analysis of variance (Table 18). The results showed that this model did not explain the data variation adequately. A logarithmic transformation of the response, Ln(SD), as recommended by Draper (1985), also did not produce a statistically acceptable model. -177- Table 17. Central compositive rotatable design matrix used for the optimization of conditions to measure proteolytic activity of papain, and results for each experiment. Factors [Papain] Temp Time Experiment (mg/mL) (°C) (min) Mean*- S.D1 1 0.050 37.0 6.5 0.090 0.008 2 0.050 37.0 9.0 0.050 0.007 3 0.050 43.0 6.5 0.112 0.009 4 0.050 43.0 9.0 0.117 0.021 5 0.090 43 . 0 6.5 0.072 0.002 6 0.090 37.0 9.0 0.133 0.030 7 0.090 37.0 6.5 0.166 0.022 8 0.090 43.0 9.0 0.232 0.009 9 0.100 40.0 7 . 5 0.100 0.016 10 0.030 40.0 7.5 0.063 0.012 11 0.070 45.0 7.5 0.137 0.021 12 0.070 35.0 7.5 0.080 0.011 13 0.070 40.0 10.0 0.037 0.017 14 0.070 40.0 5.0 0.073 0.012 15 0.070 40.0 7 . 5 0.012 0.003 16 0.070 40.0 7.5 0 . 013 0.005 *-Mean of the absorbance reading at 280 nm (n=4) BStandard deviation (n=4) -178- Table 18. Analysis of variance of the second order model*'* Source of variation DF Mean Square F-value Model 9 0.0000517 0.70 n.s Error 6 0.0000752 Total 15 "Model Eq. (39) BR*(adjusted for DF) = 0.00 n.s not significant at p= 0.05 -179- Inclusion of a three term interaction (X1X2XJ) into the second order model gave a highly significant model (Table 19). The multiple coefficient of determination, R», for this modified second order model was 0.95 (p<0.01), which indicates that this model accounted for 95% of the total variation based on the regression. A plot of the predicted against the observed values for the dependent variable (i.e. SD) is shown in Fig. 24. Included in this plot is a line with slope equal to 1. This type of plot is useful to detect cases when the variance is not constant or a transformation of the dependent variable is needed. This plot shows that the points are distributed fairly uniformly about the diagonal line, suggesting that the model is reasonable. A plot of residuals versus predicted values (Fig.25) shows that the residuals are randomly scattered, suggesting again the adequacy of the fitted model. 1. Localization of optimum condition The common method of finding the optimum in RSM is by derivatizing the fitted model in relation to each independent variable, then equalizing the derivatives to zero in order to find a stationary point, and lastly, solving the system by simultaneous equations (Nakai and Arteaga, 1988). Canonical analysis can also be performed in order to characterize the shape of the fitted response (Nakai and Arteaga, 1988). -180- Table 19. Analysis of variance for the modified second order models- Source of variation DF Mean Square F-value Model 10 0.000083 29.30*- Error 5 0.0000028 Total 15 *-R* (adjusted for DF) = 0.953 ** significant at p<0.01 Variable* Coefficient T-value Prob(> jTj) Xi -27.8002 -11.0895 Xj 0.0004 -0.0783 -10.1408 0.0005 X, -0.2414 -10.2126 0.0005 Xx* 8.7341 5.6554 0.0048 X,* 0.0005 6.3621 0.0031 X,* 0.0018 5.8326 0.0043 XxX* 0.6642 10.6593 0.0004 XxX3 3.6177 11.3634 0.0003 X2X3 0.0054 9.2726 0.0008 XxX2X3 -0.0900 -11.339 0.0003 CONSTANT 2.4751 10.7857 0.0004 "•Code: Xx = [Papain] (mg/ml) X» = temperature (<>C) X3 = time (min) -181- 0.030 -i 0.025 0.020 - > q5 0.015 - CO o 0.010 0.005 - 0.000 I i i i i i i i i i | i i i i i i i i i i i i i i i i i i i i i III T i i i i i 0.000 0.010 0.020 0.030 0.040 Predicted Figure 24. Plot of predicted values according to the modified second order model of the SD of the determination of the proteolytic activity of papain against the corresponding experimental ones. A line with slope 1 is included in the plot. -182- 20.00 -i 15.00 o o oo 10.00 H I O 5.00 H x 0.00 o -5.00 H Or: -10.00 -15.00 -20.00 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I » I » I » I » I I I 0.000 0.010 0.020 0.030 0.040 Predicted Figure 25. Plot of residuals for the modified second order model. -183- However with the inclusion o£ the three term interaction (XiXzXs), the above mathematical approach is not possible since the number of variables is greater than the number of equations. Although other mathematical procedures could be used, they are somewhat complex. Another method of localizing the optimum condition is by means of the graphical method suggested by Floros and Chinnan (1988); however, the necessity of plotting different response surfaces makes this method time consuming. A more efficient approach to find the optimum condition is by using computational simplex optimization (CSO) as described by Nakai and Arteaga (1988). By entering the equation, obtained using multiple regression, as a function subroutine in the Morgan and Deming simplex program (1974), it is possible to minimize the equation, thus finding the conditions that produce the highest repeatability (i.e. lowest standard deviation). The computer program used is given in Appendix II. Minimization of the fitted model by CSO gave the following optimum conditions (Fig. 26): papain concentration, 0.1 mg/mL; incubation time, 5 min and temperature, 35<>C. To confirm the optimum values obtained by the CSO, response surfaces were generated (Fig. 27-29). In all response surfaces, one factor was maintained constant at its optimum level as defined by the CSO. This "graphic optimization" confirmed the optimum values obtained by CSO. It is worth noting that even when the responses are relatively complex, CSO is able to obtain -184- Terminating difference value- 0.0000 Lover and upper limits LL: 0.070 3S.000 5.000 UL: 0.100 4S.000 10.000 Initial simplex XI X2 X3 RESPONSE Vertex 1 0.070 35.000 S.000 0.007 Vertex 2 0.098 37.357 6.179 0.004 Vertex 3 0.077 44.428 6.179 0.028 Vertex 4 0.077 37.357 9.714 0.026 Vertex 5 (Reflection) 0.066 28.715 7.750 Response Vertex 6 (Contraction-W) 0.079 40.500 6.571 Response 0.006 Vertex 7 (Reflection) 0.088 37.881 5.000 Response 0.002 Vertex 8 (Expansion) 0.094 38.143 5.000 Response 0.004 Vertex 9 (Reflection) 0.100 42.158 6.833 Response 0.022 Vertex 10 (Contractlon-W) 0.079 36.790 5.458 Response 0.000 Vertex 11 (Reflection) 0.098 35.000 5.000 Response -0.014 Vertex 12 (Expansion) 0.100 35.000 S.000 Response -0.015 Vertex 13 (Reflection) 0.080 35.757 5.000 Response -0.002 Vertex 14 (Reflection) 0.08S 35.000 5.306 Response -0.005 Vertex 15 (Reflection) 0.097 35.000 5.000 Response -0.014 Vertex 16 (Reflection) 0.100 35.000 5.204 Response -0.013 Vertex 17 (Reflection) 0.100 35.000 5.000 Response -0.015 Vertex 18 (Reflection) 0.098 3S.OO0 5.000 Response -0.014 Vertex 19 (Contractlon-R) 0.099 35.000 5.000 Response -0.014 Vertex 20 (Reflection) 0.100 35.000 5.000 Response -0.015 Vertex 21 (Reflection) 0.100 35.000 5.000 Response -0.015 Vertex "22 (Contraction-R) 0.100 35.000 5.000 Response -0.015 Vertex 23 (Massive contractlon-R) 0.100 35.000 5.000 Response -0.01S Pinal average values 0.100 35.000 Figure 26. Computational optimization used to obtain the best experimental conditions for proteolytic activity determination of papain. The response is the standard deviation calculated using the modified second order model. -185- Figure 27. Response surface of standard deviation against incubation time and enzyme concentration, at a constant incubation temperature of 35<>C. Standard deviation values are in the range of 0.000 to 0.030 -186- Figure 28. Response surface of standard deviation against incubation time and temperature, at a constant papain concentration of 0.1 mg/ml. Standard deviation values are in the range of 0.000 to 0.030 -187- Figure 29. Response surface of standard deviation against incubation temperature and enzyme concentration, at a constant incubation time of 5 min. Standard deviation values are in the range of 0.000 to 0.030 -188- an optimum response. The optimum conditions found by the CSO were verified by carrying out a set of proteolytic activity determination using the optimum computed experimental conditions of papain concentration, temperature and incubation time. The results of this verification confirmed the validity of the optimization technique. By using the optimum experimental conditions, the standard deviation of four replicates of absorbance at 280 nm in the proteolytic activity determination was 0.003. In summary it can be concluded that this new optimization approach of combining CCRD with CSO is an efficient method of optimization. B. DETERMINATION OF THE INFLUENTIAL FACTORS FOR MAXIMUM INHIBITION AND REACTIVATION OF THE PROTEOLYTIC ACTIVITY OF PAPAIN BY TETRATHIONATE The % inhibition and % reactivation data for the 1G experiments following a fractional factorial experimental design Lis (21S) of Taguchi (1957) were analyzed by analysis of variance. Significance of the factors was evaluated and possible interactions were determined for the inhibition of the proteolytic activity of papain by tetrathionate, and for the subsequent reactivation of the proteolytic activity of papain by cysteine. The results of the analysis of variance on the inactivation experiments are presented in Table 20. All factors and -189- Table 20. Analysis of variance (Taguchi's Lis 2XB) for the inhibition of the proteolytic activity of papain by tetrathionate (TT). Source of variation DF Mean Square F -value Molar ratio TTtPapain (MR) 1 175.62 0 .28 n.s. Reaction pH (pH) 1 602.00 0 .97 n.s. Reaction time (t) 1 6.08 0 .01 n.s. Reaction temp. (T) 1 116.10 0 .19 n.s. MR x pH 1 14.68 0 .02 n.s. MR x t 1 617.38 0 .99 n.s. MR x T 1 989 .93 1 . 59 n.s. pH x t 1 220.88 0 .36 n.s. pH x T 1 42.93 0 . 07 n.s. t X T 1 116.10 0 .19 n.s. Error"2 5 621.06 Total 15 n.s. not significant at p= 0.05 eThe sums of squares values for the factors and interactions that do not appear in the ANOVA table were very low and, therefore, incorporated into the error sums of squares. -190- interactions were found to be nonsignificant (p>0.05). This result indicates that under the conditions tested, the inhibition of the proteolytic activity of papain by tetrathionate was not affected by changes in the levels of the factors. In most cases the % inhibition was over 90%, meaning that 90% of the original proteolytic activity of papain was inhibited by reaction with either a 10 or a 100 molar excess of tetrathionate. Neither changes in pH (6.8 or 10.0), incubation time (5 or 10 min), nor temperature (22 or 40<>C) significantly affected the % inhibition. These results agree with the fact that tetrathionate reacts in a very specific manner with sulfhydryl enzymes. Fig. 30 shows the effect of the two levels of molar ratio of tetrathionate to papain on the % inactivation. Both levels caused more than 90% inhibition of the proteolytic activity. The results of the analysis of variance for the reactivation experiments are presented in Table 21. The pH during the inactivation reaction, and cysteine concentration during the reactivation reaction were computed to be highly significant (p<0.01) sources of variation in the reactivation reaction by cysteine. The molar ratio of tetrathionate to papain and reaction time of the inactivation reaction were also found to be significant sources of variation (p<0.05). Factors in the inactivation reaction (molar ratio of tetrathionate to papain, pH, temperature and time) had an effect on the reversibility, as measured by % reactivation, of the -191- 110-j 100- 40- 30- 1 1 1 i i 1 1 1 1 1 r 1 0 50 100 [Tetrathionate]/[Papain] (mol/mol) Figure 30. Effect of two levels of the molar ratio of tetrathionate to papain on the inactivation of papain. (Mean±SEM n=8) -192- Table 21. Analysis of variance (Taguchi's Lis 21B) for the reactivation by cysteine of the proteolytic activity of tetrathionate-inactivated papain. Source of variation DF Mean Square F-value Molar ratio TT: papa in (MR)*- 1 64.56 9.44* Reaction pH (pH)*- 1 327.81 47.92" Reaction time (t)*- 1 72.36 10.58* Cysteine (Cys) 1 1134.78 165.89" MR x pH 1 344.09 50.30" MR x t 1 116.95 17.10** Cys x pH 1 508.50 74.34" Cys x t 1 189.86 27.75" Cys x TB 1 349.39 51.08" pH x T 1 175.56 25.66" Error0 5 6.84 Total 15 "significant at p < 0.05 "significant at p < 0.01 ^-Factors of the inactivation reaction. B T = incubation temperature GThe sums of squares values for the factors and interactions that do not appear in the ANOVA table were very low and, therefore, incorporated into the error sums of squares. -193- reaction between papain and tetrathionate. Since % reactivation is a measure of the reversibility of the inactivation process, it can be expected that if tetrathionate, upon reacting with papain, does not cause irreversible conformational changes in the enzyme molecules, the original proteolytic activity of papain will be observed (i.e 100% reactivation) after treatment of the inactivated enzyme with the reducing agent (i.e. cysteine). In general, values of % reactivation higher than 85 were obtained, which indicates that in most cases the inactivation was reversible. This suggests that no major conformational changes in papain took place due to the reaction of papain with tetrathionate. The effect curve, in Fig. 31, shows the interaction between the molar ratio of tetrathionate to papain and reaction time on the % reactivation. At any level of these factors the % reactivation was higher than 85%, indicating that 85% of the original proteolytic activity of papain was regained after treatment with cysteine. At the lower level of the molar ratio of tetrathionate to papain (10) a significantly higher reactivation was obtained when the inactivation reaction time was only five minutes than when it was 10 min. In both cases the % reactivation was greater than 90%. A significant interaction was also found between the molar ratio of tetrathionate to papain and pH of the inactivation reaction. A significantly higher reactivation was obtained when the pH of the inactivation reaction was 6.8 and the molar ratio -194- 10CH Rx. time= 5 min I c 90- •o —~ o Rx. time= 10 min > I o D 0) 80- 70 i 100 [Tetrathionate]/[Papain50 ] (mol/mol) Figure 31. Effect curve for the interaction between the molar ratio of tetrathionate to papain and reaction time of inactivation reaction on the % reactivation of the proteolytic activity of papain. (Meantconfidence limits calculated at p<0.05) -195- of tetrathionate to papain was 10 (Fig. 32). As expected, the concentration of cysteine during the reactivation reaction had a significant effect on the % reactivation value. Contrary to what was expected, a lower % reactivation was obtained when a higher level of cysteine (40 mM) was used for reactivation. Since the reducing agent (i.e. cysteine) was essential for reactivation, it was thought that a higher % reactivation should be obtained at the higher cysteine concentration. A possible explanation of this phenomenon is that disulfide interchange reactions may have occurred at the higher cysteine concentration. This could cause irreversible inactivation of some of the papain molecules, producing a lower value of % reactivation. When the reactivation reaction was carried out with 20 mM cysteine a 100% reactivation value was obtained, which indicates that the inhibition of the proteolytic activity of papain was totally reversible. When the reactivation was carried out with 40 mM of cysteine, the factors involved in the inhibition reaction (time, pH, and temperature) had a significant effect on the % reactivation. Fig. 33-35 show that at a cysteine concentration of 40 mM, the higher the pH or temperature and the longer the reaction time, the lower the value of % reactivation. The overall results of the fractional factorial experiments indicate that the reaction of papain with tetrathionate, under the conditions studied, caused a complete inhibition of the -196- 110-, 80- 70 H 1 1 1 1 1 1 i 1 1 1 1 0 50 100 [Tetrathionate]/[Papain] (mol/mol) Figure 32. Effect curve for the interaction between the molar ratio of tetrathionate to papain and pH of the inactivation reaction on the % reactivation of the proteolytic activity of papain. (Meantconfidence limits calculated at p<0.05) -197- 110-i 100- 90 80- pH 10.0 70- 60- 50 I i —r~ 15 20 25 30 35 40 45 [Cysteine] (mM) ure 33. Effect curve for the interaction between the cysteine concentration during the reactivation and pH of the inactivation on the % reactivation of the proteolytic activity of papain. (Mean+confidence limits calculated at p<0.05) -198- Figure 34. Effect curve for the interaction between the cysteine concentration during reactivation and temperature of inactivation on the % reactivation of the proteolytic activity of papain. (Mean±Confidence limits calculated at p<0.05) -199- 110-. 70- 60-| -i 1 1 1 1 1 15 20 25 30 35 40 45 [Cysteine] (mM) Figure 35. Effect curve for the interaction between the cysteine concentration during reactivation and time of inactivation on the % reactivation of the proteolytic activity of papain. (Meantconfidence limits calculated at p<0.05) -200- not significantly affected by changes in the molar ratio of papain to tetrathionate, pH, temperature or time of the inhibition reaction. In order to obtain total reversibility of the inhibition, the inhibition reaction must be carried out at pH 6.8, and at a temperature of 22°C (Fig. 36), and the reactivation reaction must be carried out with 20 mM cysteine. C. OPTIMIZATION OF CONDITIONS FOR MEASURING THE CD SPECTRA OF PAPAIN Experiments were performed according to the SCO as previously described. SCO with the four factors was initiated by performing five experiments called "initial simplex". After obtaining the response values, which were in this case the combination of three nonconflicting parameters, the response values were reported back to the computer to obtain new experimental conditions. Table 22 shows the series of the optimization experiments for achieving the best conditions for measuring the CD spectra of papain. A total of 18 experiments were carried out, with vertex number 15 giving the lowest response (i.e. best scan). Since the sizes of scales of the three parameters that were included in the evaluated response were different, it was necessary to transform two of the responses; the natural logarithm of the total scan time was used and the mean of the photomultiplier voltage at 222, 209, and 200 nm was divided by 100 (Table 22). These transformations were arbitrarily selected. Two cycles of centroid, reflection and curve fitting plus an additional centroid were done. Mapping was performed by plotting -201- A f ,) not significantly affected by changes in the molar ratio of papain to tetrathionate, pH, temperature or time of the inhibition reaction. In order to obtain total reversibility of the inhibition, the inhibition reaction must be carried out at pH 6.8, and at a temperature of 22<>C (Fig. 36), and the reactivation reaction must be carried out with 20 mM cysteine. C. OPTIMIZATION OF CONDITIONS FOR MEASURING THE CD SPECTRA OF PAPAIN Experiments were performed according to the SCO as previously described. SCO with the four factors was initiated by performing five experiments called "initial simplex". After obtaining the response values, which were in this case the combination of three nonconflicting parameters, the response values were reported back to the computer to obtain new experimental conditions. Table 22 shows the series of the optimization experiments for achieving the best conditions for measuring the CD spectra of papain. A total of 18 experiments were carried out, with vertex number 15 giving the lowest response (i.e. best scan). Since the sizes of scales of the three parameters that were included in the evaluated response were different, it was necessary to transform two of the responses; the natural logarithm of the total scan time was used and the mean of the photomultiplier voltage at 222, 209, and 200 nm was divided by 100 (Table 22). These transformations were arbitrarily selected. Two cycles of centroid, reflection and curve fitting plus an additional centroid were done. Mapping was performed by plotting -202- Table 22. Simplex-centroid optimization of the conditions for CD spectrophotometry of papain. Factors Papain Bandwidth TC*- Product3 Technique Vertex (Aieo) (nm) (sec) (nm) RESC Super Simplex Initial Simplex 1 0..50 0 1..00 0 1,.00 0 0,.10 0 26..0 0 2 1.,88 8 1,,21 9 2,,53 0 0,.29 7 123. ,00 3 0,.82 8 1,.92 6 2,.53 0 0,.29 7 16..0 0 4 0.,82 8 1,,21 9 2., 530 0,,93 3 16.,8 0 5 0..82 8 1,.21 9 7,.47 9 0,. 297 32..0 0 Centroid 6 0.,74 6 1,.34 1 3.,38 5 0,.40 7 40.,2 4 Reflection 7 0.. 500 1,.46 3 4 .24, 0 0 .51, 7 8..4 0 Curve fitted 8 0.,50 0 1., 478 4 ,. 347 0..53 0 8., 39 Centroid 9 0,.66 4 1,.40 6 2,. 601 0,.46 5 37.. 22 Reflection 10 0 .. 500 1.,59 3 1.,00 0 0 ,.63 3 6.,8 0 Curve fitted 11 0..50 0 1,.78 0 1,. 000 0 ., 802 7..1 9 Centroid Search Initial Simplex 12 0. 450 1. 500 1. 000 0 . 500 29 . 60 13 0. 959 1. 609 1. 656 0 .609 32. 70 14 0. 570 1. 963 1. 656 0 .609 12. 24 15 0. 570 1. 609 1. 656 0 .963 6. 00 Centroid Search 16 0. 570 1. 609 3. 777 0 .609 25. 60 17 0. 540 1. 670 2 .02 2 0 .670 17. 30 18 0. 563 1. 713 2. 277 0 .713 17. 00 *• TC = time constant B Product = TC (min) x Scan rate (nm/min) e RE = Response = Ln(total scan time (min) x A x _B 100 where A = E S.D. (n=4) of [6] at 200f 209 and 22 nm 3 B = E PV at 200, 209 and 222 nm 3 S.D = standard deviation [9] = ellipticity (m<>) PV = photomultiplier voltage (V) -203- the response values obtained against each factor. The response surfaces appeared to direct the search for a lower response toward lower protein concentration, longer band width and higher values of the product of time constant times the scan rate. After the centroid search, the optimum conditions were found to be as follows: papain concentration, 0.23 mg/mL; band width, 1.6 nm; time constant, 2 sec, and the value of the product of time constant times the scan rate 0.8 nm. Fig. 37 shows the actual traces of the CD scan at vertices 1 and 15. It is evident that a significant improvement in the quality of the CD scan was achieved by using SCO. Johnson (1985) recommended that for optimum CD measurements the protein concentration should be such that the absorbance at 280 nm(l cm pathlength) is less than 1. In the present study the optimum concentration gave an absorbance at 280 nm of 0.575. Hennessey and Johnson (1982) recommended that the product of time constant times the scan rate should be not more than 0.33 nm. The operation manual for the spectropolarimeter (Jasco, 1979), however,indicated that values in the range of 3.33 to 13.33 nm were adequate. Su and Jirgensons (1977) used a value of 0.42 nm for the above parameter when measuring the CD spectra of many proteins, including papain. The noise level of the scan decreased as the time constant increased. However at high time constant, it was necessary to decrease the scan rate in order to compensate for the reading delay of the instrument. -204- Figure 37. Trace of the far-UV CD spectrum of papain measured under different conditions. (A) CD spectrum measured under the conditions of vertex 1. (B) CD spectrum measured under the optimum conditions (Vertex 15). Band width is related to the amount of light that reaches the photomultiplier, and in many cases longer band widths give lower noise levels. Increasing the band width to more than 2 nm is not recommended (Johnson, 1985) since artifacts in the signal are produced. It should be pointed out that the optimum conditions found are only for measuring the CD of papain. The same methodology can be applied to find the optimum conditions for the CD determination of other proteins. Not all proteins produce a CD scan with high noise levels, and therefore an optimization of the experimental conditions to measure the CD may not always be required. The high noise levels in the CD scan of papain are due to the high absorption coefficient [E3-* (1 cm) =25.0) and relatively weak CD bands of this enzyme. In proteins where the absorption coefficient is low or the ellipticity values high, the noise levels of the CD scan are lower. Table 23 shows the numerical values of [8]MRW at 222, 209 and 200 nm, together with the corresponding coefficient of variation for each vertex of the SCO. Reference values for [0]MRW at those wavelengths for papain are also included. As expected, higher coefficients of variation were obtained at the shorter wavelength (200 nm) and in some cases coefficients of variation of more than 100% occurred. This table demonstrates the importance of selecting the optimum conditions for measuring the CD spectrum of papain; changes in protein concentration, band width, time constant and the product of time constant times scan rate had an -206- Table 23. Values of mean residue ellipticity*- (181M**) and corresponding coefficient of variation (C.V)» at three wavelengths for the different vertices of the slmplex-centroid optimization. Wavelength (nm) 222 209 200 Vertex [6]„. C.V(%) [6)«. C.V(%) [9]*.. C.V(%) 1 -7 .27 8. 51 -8. 74 14 .16 -5. 41 68 . 57 2 -8 . 89 17. 78 -15. 47 8 . 33 -10. 83 95. 24 3 -7 .28 1. 31 -13. 77 1 .69 -6 . 40 30. 60 4 -7 .04 0 . 68 -13. 77 1 .69 -7 . 89 56 . 88 5 -7 .38 0. 65 -14 . 15 1 .64 -7 . 29 45. 95 6 -8 .70 0 . 4 4 -14 . 85 1 .04 -11. 34 38 . 18 7 -9 . 50 0. 6 0 -10 . 83 0 .48 -7 . 27 25. 53 8 -9 . 50 0. 30 -10. 83 0 .48 -7 . 27 25. 53 9 -8 .78 5. 07 -12. 68 4 . 07 . -7 . 22 78 . 57 10 -9 .40 3. 03 -11. 24 3 .21 -5. 8 3 79 . 65 11 -9 .40 1. 82 -11. 34 9 .09 -6 . 70 65. 38 12 -8 .25 4 . 00 -9 . 24 14 . 29 -6. 77 113. 82 13 -8 .52 2. 42 -9 . 55 8 • 65 -3. 10 250 . 00 14 -8 .68 1. 50 -9 . 12 4 .76 -6 . 51 40. 00 15 -8 .96 0. 50 -10. 40 1 .70 -7 . 70 20. 30 16 -9 . 00 3. 00 -11. 30 4 .10 -8 . 00 37. 75 17 -9 .17 0. 98 -11. 00 2 .40 -8 . 20 45. 00 L8 -9 .17 0. 98 -10 . 60 2 .00 -8 . 76 45. 00 Reference values1 -11 .70 - -12. 60 - -8. 88 *-Units: (10*") deg cm* drool-1 •Coefficient of variation (%) = Standard deviation x Mean °Source: Yang et al. (1986) -207- important effect on the reproducibility. As Hennessey and Johnson (1982) have indicated, "If CD spectroscopy is to go beyond empirical correlations so that the spectra themselves can be used to determine secondary structure, a higher degree of accuracy is required." The results of this study show the usefulness of SCO in finding the best conditions to carry out the determination of the CD of papain. A small number of experiments were needed and a significant improvement in the quality of the CD spectrum of papain was obtained. Furthermore, this same approach can be used for other proteinsand also for optimization of other analytical techniques. D. CD OF NATIVE AND TETRATHIONATE MODIFIED PAPAIN Typical far-UV CD spectra for native and tetrathionate-modified papain (TT-papain) (molar ratio of tetrathionate to papain for the modification =200) are shown in Fig. 38. The two spectra are superimposable, which suggests that no change in the secondary structure of papain occurred upon the modification. Similar results were obtained when the modification was carried out with other molar ratios of tetrathionate to papain. The CD spectra of both papains showed negative peaks at about 222 and 209 nm, and a small shoulder at about 205 nm, with [8]HR« of -9.2x10', -10.3x10' and -7x10' deg cm* dmol, respectively. The -208- Figure 38. Far-UV CD spectra of native and TT-papain. (molar ratio of tetrathionate to papain during reaction = 200). Note: both proteins gave the same spectra. -209- position of the peaks and the overall shape of the CD spectra are very similar to the one previously reported by Su and Jirgenson (1977); however, somewhat smaller [9JM»W values were obtained in the present study. Since tetrathionate is thought to react specifically with the only cysteine residue of papain, Cys-25, it is very unlikely that major conformational changes would occur upon this modification. The fact that natural, reversibly and irreversibly inactivated papain have the same CD spectra as fully activated papain (Sluyterman, 1967c) supports this observation. In the near ultraviolet region the three aromatic residues, phenylalanine, tyrosine and tryptophan give rise to CD signals when their side chains are in asymmetric surrounding or involved in interactions in the proteins. Furthermore, disulfide bridges may contribute significantly between 250 and 300 nm (Heindl et al., 1980; Strickland, 1974). Typical near-UV CD spectra for native and tetrathionate- modified papain (molar ratio tetrathionate to papain during the modification =200) are shown in Fig. 39. The two proteins had nearly identical spectra. Since the near-UV CD spectrum is related to the tertiary structure of proteins (Strickland, 1974), this result suggests that little, if any, change occurred in the tertiary structure of papain due to modification with tetrathionate. Similar results were obtained when modification was carried out with other molar ratios of tetrathionate to papain. -210- Wavelength (nm) Figure 39. Near-UV CD spectra of native ( .) and TT-papain (- - -) (molar ratio of tetrathionate to papain during reaction = 200). -211- Following the approach o£ Strickland (1974), the peaks at 262 and 268 nm may be assigned to phenylalanine residues, while the one at 277 nm is due to tyrosine residues. Assignment of the negative peak at 290 nm is more difficult since it could be due to either tryptophan or disulfide residues. Usually the disulfide CD begins at longer wavelengths (320 to 350 nm) and gradually intensifies to give one or two bands located above 240 nm. Disulfide bands are much broader than the CD bands observed for the aromatic amino acid side chains, and the longest wavelength of disulfide CD band can be expected to peak below 290 nm (Strickland, 1974). For many proteins the peak of the disulfide CD band at the longest wavelength is negative. The near-UV CD spectrum obtained in this experiment is very similar to the one reported by Su and Jirgenson (1977). E. SECONDARY STRUCTURE OF NATIVE AND TETRATHIONATE-MODIFIED PAPAIN Table 24 shows the predicted values obtained through the use of two published algorithms, for the different secondary structure fractions of native papain and tetrathionate-modified papain (molar ratio of tetrathionate to papain for the modification = 200). The results obtained confirm the fact that no change in the secondary structure of papain occurred upon modification with tetrathionate. As reported by Yada (1984), the algorithm of Provencher and Glockner (1981) when applied to the CD data predicted more closely than the algorithm of Siegel et -212- Table 24. Predicted secondary structure fractions of native and tetrathionate-modified papain* (TT-papain) based on CD data, using two algorithms, and X-ray determined secondary structure fractions. Secondary structure fraction Helix 0-sheet 0-turn Random Protein 1B 23 12313 13 Native .28 .40 .22 .14 .31 .10 .17 .28 .47 .39 papain TT-papain - .40 .22 - .31 .10 - .28 - .39 *Molar ratio of tetrathionate to papain during modification = 200. Similar results results were found at all ratios tested (10-500). BMethods: 1. X-ray (Chang et al., 1978). 2. Siegel et al. (1980) . 3. Provencher and Glockner (1981). Note: The method of Siegel et al. (1980) does not estimate 0-turn or random fractions. -213- al. (1980) the secondary structure fractions obtained with X-ray crystallography. F. FLUORESCENCE AND UV-ABSORPTION OF NATIVE AND TETRATHIONATE MODIFIED PAPAIN Native, activated papain (i.e. papain that was incubated with 20 mM of cysteine and 4 mM of EDTA prior to analysis) showed an inflection point at about pH 8.0 in the pH-fluorescence profile, and an increase in the fluorescence intensity with further increase in pH, reaching a maximum at pH 9.5 (Fig. 40). In contrast, the pH-fluorescence profile of unactivated, native papain and tetrathionate-modified papain did not show any inflection point at pH 8.0 (Fig. 40-41). The pH-fluorescence profile of native activated papain is in excellent agreement with the one previously reported by Barel and Glazer (1969). According to Barel and Glazer (1969), the presence of an inflection point at pH 8.0 in the pH-fluorescence profile of activated papain is due to ionization of the sulfhydryl group at the active site of the enzyme. Since unactivated, native papain has the sulfhydryl group partly blocked in the form of a mixed disulfide with half-cystine or partly existing at the oxidation state level of sulfenic acid (Brocklehurst et al., 1981), no free sulfhydryl group is present and hence no inflection point at pH 8.0 is observed. The observation that tetrathionate modified papain (molar ratio of tetrathionate to papain for the chemical modification reaction = 10 to 500) did not show an inflection point at pH 8.0 (Fig. 41) was a clear indication that the -214- 120-1 Figure 40. Effect of pH on the relative fluorescence intensity at 22°C of native papain. Unactivated (• ), and active papain (o). -2] 5- 100-1 1 1 1 1 1 1 1 1 1 3456789 10 11 pH Figure 41. Effect of pH on the relative fluorescence intensity at 22°C of TT-papain (molar ratio of tetrathionate to papain during reaction - 200). Unactivated (O), and active papain (o). -216- sulfhydryl group of papain was blocked upon reaction with tetrathionate, as expected. Alkylation of papain with either iodoacetate or iodoacetamide was reported (Barel and Glazer, 1969) to produce a similar effect. When the tetrathionate-modified papain was incubated (i.e. activated) with cysteine plus EDTA, a very similar pH-fluorescence profile to the one of activated native papain was obtained (Fig. 41). Figure 42 shows the effect of the molar ratio of tetrathionate to papain for the modification, on the fluorescence intensity at 352 nm. A quenching effect was observed when the chemical modification was carried out with more than a 50 molar excess of tetrathionate over papain. Since tryptophan residues contribute a major proportion of the fluorescence intensity of papain, this observation suggests that some tryptophan residues were modified by tetrathionate. The fact that tetrathionate has been reported to react with tryptophan residues in proteins (Inglis and Liu, 1970) gives support to this hypothesis. However, since amino acid analysis of the modified papain was not carried out, no definitive conclusion can be drawn. Similar to the CD results, no differences in the absorbance spectra was obtained between native and tetrathionate-modified papain (molar ratio of tetrathionate to papain for the chemical modification^ 10 to 500). This data also suggests that no conformational change occurred in papain upon chemical modification with tetrathionate. Activation with cysteine did not affect the ultraviolet -217- JH 100-0 CD 20-j 1 1 1 1 1 1 1 1 1 1 1 0 100 200 300 400 500 [Tetrathionate]/[Papain] (mol/mol) Figure 42. Effect of the molar ratio of tetrathionate to papain during the chemical modification on the relative fluorescence intensity at 352 nm (pH 6.8 and 22°C). -218- spectrum of tetrathionate modified papain. As reported by Barel and Glaser (1969) the same was also true for native papain. G. DETERMINATION OF SH GROUPS IN NATIVE AND TETRATHIONATE-MODIFIED PAPAIN Table 25 summarizes the results of SH determination of native and tetrathionate-modified papain. Native, unactivated papain (i.e. native papain that was not activated with cysteine and EDTA) showed a content of 0.1+0.02 mole of SH/mole of papain. As expected, upon activation with cysteine, the content of SH groups increased significantly to 0.7+0.06 mole of SH/mole of papain. Since no affinity chromatography technique was used to separate active from inactive papain, it was not possible to prepare papain with 1 mole of SH/mole of protein. The reaction of activated papain (freed from activators by gel filtration) with tetrathionate (molar ratio of tetrathionate to papain = 10 to 500) caused the complete disappearance of SH. Incubation of this modified papain with 40 mM cysteine and 10 mM EDTA restored the content of SH group of the modified papain to a level very similar to that found in native, activated papain. These results confirm the fact that tetrathionate reacts in a reversible manner with the sulfhydryl group of papain. -219- Table 25. Sulfhydryl content of native and tetrathionate-modified papain (TT-papain)*-. SH content*3'0 Protein (moles SH/ mol protein) Non-activated0 Native papain 0.1 ±0.02 TT-papain 0.0 ±0.00 Activated™ Native papain 0.7 ±0.06 TT-papain 0.6 ±0.10 *-Molar ratio tetrathionate papain during inactivation = 200. BA molecular weight of 23,800 was used for all calculations. °Mean ±S.D (n=4) DNon-activated indicates samples that were not incubated with cysteine and EDTA prior to the assay. "Activated indicates samples that were incubated with cysteine and EDTA, and then freed from cysteine, under nitrogen saturated conditions, by gel filtration, prior to the assay. -220- H. INSOLUBILIZATION OF PAPAIN WITH TETRATHIONATE Preliminary experiments showed that when a papain solution was heated to 60°C and 0-mercaptoethanol and tetrathionate were added, precipitation of the protein occurred. It is important to point out that this insolubilization of papain with tetrathionate was not an objective of this thesis research; moreover, it's occurrence was not expected. Since this precipitation was an effect of the chemical modification of papain by tetrathionate, a series of experiments were conducted in order to give more insight into this phenomenon. The effect of concentration of tetrathionate and 0-mercaptoethanol on the precipitation of papain is depicted in Fig. 43. This response surface was generated from the quadratic model fitted to the data obtained using RSM (Table 26). The fitted model was shown to be highly significant (Table 27). Up to 90% of the original papain was precipitated with the combined addition of 100 mM 0-mercaptoethanol and 50 mM tetrathionate. Addition of 0-mercaptoethanol alone did not produce high levels of precipitation (Fig. 43). Regarding the effect of pH and temperature on the precipitation of papain with tetrathionate, it was found that practically no precipitation occurred at a basic pH (pH >8) at all the incubation temperatures tested. At a neutral pH (pH 6.8), very slight precipitation occurred at room temperature. The highest level of protein precipitation occurred when the pH -221- -222- Table 26. Experimental data for the two-factor, five-level response surface analysis of the effect of tetrathionate (TT) and 6-mercaptoethanol (13-ME) concentration on the precipitation of papain. Factor, mM TT 13-ME % Protein Treatment precipitated*"B 1 15 15 47. 21 2 85 15 77.28 3 15 85 67.43 4 85 85 88.63 5 50 100 90.20 6 50 100 62.00 7 100 50 81.90 8 0 50 17.39 9 50 50 81.39 10 50 50 83.81 ^Protein precipitated was calculated as follows: (1 - mg protein in supernatant after treatment ) x 100 initial mg protein in solution sProtein was determined using the Bio-Rad protein assay -223- Table 27. Analysis of variance for the second order model*-for the precipitation of papain by tetrathionate, obtained using backward stepwise multiple regression. Source of variation DF Mean Square F-value Model 5 13219.308 217.695* Error 4 60.724 Total 9 AR*(adjusted for DF) = 0.990 "significant at p<0.001 Var iableJ Coefficient T-value ProbO |Tj ) 0.481 9.574 0.0004 X* 1.956 4.783 0.0031 X** -0.012 •4.981 0.0025 XiX2 -0.004 -1.870 0.1107 *Code: Xi (0-mercaptoethanol] (mM) X2 [Tetrathionate] (mM) -224- of the papain solution was not adjusted with NaOH (pH 5.0) (Table 28). It was also observed that precipitation occurred almost instantaneously at 60°C. Upon addition of tetrathionate to the solution of papain containing 0-mercaptoethanol, a drop in pH occurred (Fig. 44), possibly due to the formation of some H2S via reduction of the tetrathionate with 0-mercaptoethanol. Experiments in which the pH of a papain solution with 0-mercaptoethanol was decreased by addition of 0.1 N HC1, showed that the drop of pH per se was not responsible for the precipitation. Addition of thioacetamide, a compound which yields H2S upon solubilization in water, to a papain solution with 0-mercaptoethanol did not produce any precipitation. Thus, the possibility that the drop in pH or the formation of H2S was responsible for the precipitation of the enzyme was ruled out. Protein analysis of the precipitated material by micro-Kjeldahl indicated that it consisted of almost pure protein (70-90% protein d.b.). Neither urea nor SDS at a final concentration of 8 M and 10% respectively, were able to resolubilize the precipitated protein. Addition of 0-mercaptoethanol to a final concentration of 100 mM solubilized approximately 70% of the insoluble protein. Addition of SDS (10% final concentration) together with 100 mM of 0-mercaptoethanol was the condition in which the highest level of solubilization was achieved (Table 29). -225- Table 28. Effect of temperature and pH on the precipitation of papain by tetrathionate*. Conditions of incubation % Protein pH Temperature (<>C) precipitated**'0'0 5.0 22 10.5 ± 1.9 5.0 40 30.3 ± 2.0 5.0 60 82.8 ± 2.9 6.8 22 4.7 ± 1.0 6.8 40 13.9 ±- 3.6 6.8 60 23.5 + 2.2 8.0 22 3.0 ± 0.7 8.0 40 2.2 + 0.3 8.0 60 3.3 + 0.1 10.0 60 2.0 + 1.0 *A11 samples were incubated with 100 mM of 3-mercaptoethanol and 100 mM of tetrathionate. (See MATERIALS AND METHODS for details) BProtein precipitated was calculated as follows: (1 - mg protein in supernatant after treatment ) x 100 initial mg protein in solution °Protein was determined using the Bio-Rad Protein assay. "Values are Mean±S.D (n=3) -226- 2.00 | i i i i i i i i i | i i i i i i i i i i i i i i i i i i i i i i i i i i i i I 0 100 200 300 400 Reaction time (sec) Figure 44. Change of pH during the reaction between tetrathionate, 0-mercaptoethanol and papain. Final [tetrathionate] = [0-mercaptoethanol] = 100 mM, Final [papain] = 10 mg/mL. -227- Table 29. Effect of different reagents on the resolubilization of the precipitated papain. Reagent Final concentration % Resolubilization*"B'a Urea 8 M 3.0 SDS 10 % 4.9 0-ME** 100 mM 75.3 Urea - SDS 8 M, 10% 5.3 Urea - 0-ME 8 M, 100 mM 72.9 0-ME - SDS 100 mM, 10% 80.3 *•% Resolubilization was calculated as follows: ( mg protein in supernatant after treatment ) x 100 initial mg protein in the precipitate BProtein was determined using Micro-Kjeldahl. cMean of two replicates. -228- The Insoluble protein was found to have no proteolytic activity (Table 30). However, incubation of the insoluble protein with 0-mercaptoethanol, in addition to resolubilization, caused a partial reactivation of the proteolytic activity. But on a protein basis, the proteolytic activity of the insoluble papain after incubation with 0-mercaptoethanol was much less (approximately 20 times) when compared to native papain (Table 30). SDS-Gel electrophoresis of the insoluble papain produced a similar pattern to that of native papain (Fig. 45). As indicated in the MATERIALS AND METHODS section, preparation of the samples for the electrophoresis included addition of 0-mercaptoethanol together with SDS and heating in boiling water. This treatment caused complete resolubilization of the insoluble protein. This result gives support to the hypothesis that disulfide bond formation caused precipitation of papain. At this moment it can not be said if intra- or inter- molecular bond formation was involved. Taking into consideration that papain has only one free sulfhydryl group per molecule, together with the resistance of its disulfide bonds to reduction, it is more likely that inter- molecular disulfide bonds were formed. Electrophoresis was also tried without reducing the insoluble protein (i.e. without incubation with 0-mercaptoethanol), however, since no resolubilization was achieved in the presence of SDS alone, it was impossible to carry out the elecrophoresis. -229- Table 30. Proteolytic activity of native and insoluble papain. Protein % Proteolytic activity' Native non-activated° 2.0 activated*5 100.0 Insoluble non-activated 0.0 activated 6.5 activated3 12.5 *•% Proteolytic activity (P.A) was calculated as follows: P.A of sample x 100 P.A of native, activated papain P.A was expressed in )ig tyrosine min"1 mg protein"1 BMean of two replicates. °Non-activated indicates samples that were not incubated with 0-mercaptoethanol and EDTA prior to the assay. DActivated indicates samples that were incubated with 0-mercaptoethanol (10 mM) and EDTA (10 mM) prior to to the assay. "Sample that was activated with 100 mM of 0-mercaptoethanol and 10 mM EDTA prior to the asay. -230- I 1. 4 * - . • -t. . '-. 1 2 Figure 45. Electrophoretogram of soluble and insoluble papaya -231- Parallel experiments showed that tetrathionate also caused the precipitation of the protein components of papaya latex (prepared as described in MATERIALS AND METHODS of CHAPTER 1) and of a sample of commercial papain (PANOL, Enzyme Technology Corp. New York, NY) under the same conditions that caused precipitation of pure papain. SDS-gel electrophoresis of the insoluble material resulting from the reaction of tetrathionate with papaya latex and the commercial papain sample also gave patterns similar to the corresponding soluble materials (Fig. 46). SDS-gel electrophoresis under non-reducing conditions of soluble chemically modified papain (molar ratio of tetrathionate to papain = 200) showed the formation of a high molecular weight fraction not originally present in native papain (Fig. 47). It appears that even under conditions where precipitation did not occur tetrathionate caused formation of high molecular weight aggregates. These results suggest that insolubilization or precipitation of papain with tetrathionate was mainly due to the formation of aggregates via disulfide interchange since only (3-mercaptoethanol produced a significant resolubilization of the precipitated papain. Disulfide interchange usually occurs at higher rates when the pH of the reaction is basic (pH>8). In this case, no precipitation occurred when the pH of the reaction was basic. Moreover, greater precipitation occurred at pH 5.0. A possible -232- 2 3 4 5 6 7 8 Figure 46. Electrophoretogram of native (1) and insoluble (2) papain. (1) soluble commercial papain (Calbiochem papain). (2) insoluble commercial papain (Calbiochem papain). (3) insoluble commercial papain (PANOL). (4) soluble commercial papain (PANOL). (5) soluble pure papain. (6) and (7) insoluble papaya latex. (8) soluble papaya latex. -233- 12 3 4 5 6 7 8 Figure 47. Electrophoretogram under non-reducing conditions of soluble tetrathionate-modified pure papain and of tetrathionate-treated commercial papain and papaya latex. (1) tetrathionate-modified pure papain (molar ratio of tetrathionate to papain = 200). (2) tetrathionate-modified pure papain (molar ratio of tetrathionate to papain = 100). (3) control pure papain. (4) and (5) tetrathionate-treated (1%, w/w) commercial papain (PANOL) (6) tetrathionate-treated (1%, w/w) papaya latex. (7) protein standars. (8) control papaya latex. -234- explanation of this pH effect can be related to the stability of papain at different pH's. It is well known that papain is unstable at acidic pH (Arnon, 1970). Therefore it is possible that the acidic pH caused destabilization of the papain molecule which facilitated the formation of aggregates. Reduction of papain with 0-mercaptoethanol prior to the addition of tetrathionate was essential for precipitation, indicating that either the presence of 0-mercaptoethanol and/or the reduced papain molecule were involved in the precipitation mechanism. Although resolubilization of the precipitated papain was possible, the lower proteolytic activity of the resolubilized papain compared to native papain suggested that irreversible denaturation had occurred. Taking into consideration the high stability of papain to temperature and denaturing agents such as urea, that tetrathionate induced precipitation of papain under relatively mild conditions was not expected. Further research is needed in order to have a better understanding of this phenomenon. -235- K. KINETIC PARAMETERS FOR THE PAPAIN CATALYZED HYDROLYSIS OF CARBOBENZOXYGLYCINE P-NITROPHENYL ESTER The reactions of the enzyme with the nitrophenyl ester of carbobenzoxyglycine at several different initial ester concentrations are shown in Fig. 48. Since the reaction rate for this enzymatic reaction was relatively high, it was necessary to design a system that permitted rapid addition of the enzyme solution into the cuvette placed in the cell compartment of the spectrophotometer, in order to obtain accurate results. The system developed is shown in Fig. 49. Addition of the enzyme solution into the cuvette containing the substrate solution placed in the cell compartment of the spectrophotometer was performed with an automatic pipettor (Dispensette, Brinkmann Brand, W. Germany), calibrated to dispense exactly 2.8 mL of the papain solution. As depicted in Fig. 50 this system permitted an accurate determination of the zero time of the reaction. As reported by Kirsch and Ingelstrom (1966) the initial rates of the reactions were not very sensitive to substrate concentration, indicating that the Km was very low. As expected, the rate of appearance of nitrophenol in the absence of enzyme was appreciable and was dependent upon the concentration of the substrate. Values of B and reaction order for each initial substrate concentration calculated using the linearization procedure are shown in Table 31. For the calculation of B values, only the data of substrate concentration obtained every 2 sec up to 20-30 sec were used. The reactions were 80-95% completed -236- 0.40 -, Time (sec) Figure 48. Papain-catalyzed hydrolysis of carbobenzoxyglycine p-nitrophenyl ester at pH 6.8 and 22°C. The reaction mixture contained 0.02 M sodium phosphate buffer, 1 mM EDTA, 0.35 mM cysteine, 6.7% (v/v) acetonitrile and 3.3 x 10-"7 papain. Initial substrate concentration as shown. -237- Automatic pipette Cell compartment of the Spectophotometer Tygon tubing r~ \ Papain solution Nitrogen Cuvette et with substrate v • ^ J Figure 49. Schematic diagram of the system used for the enzyme kinetics experiments. -238- O time zero Reaction Time Figure 50. Idealized progress reaction curve (A40o vs. time) for the papain-catalyzed hydrolysis of carbobenzoxyglycine p-nitrophenyl ester. -239- Table 31. Values of B and corresponding empirical reaction order with respect to time (nT) at different initial substrate concentrations for the reaction of papain-carbobenzoxyglycine p-nitrophenyl ester. [Initial] Best estimate 5 10" M of B nT 19 .0 -0.19 1.19 9 . 52 -0.05 1.05 6.66 -0.20 1.20 4.76 1.00 0. 00 2.76 1.00 0.00 0.92 1.00 0.00 -240- within this period. This agrees with the recommendation o£ Durance et al. (1986) who indicated that, for accurate determination of the reaction rates using this linearization approach, the substrate depletion data used for calculations should include data when at least 80% of the initial substrate has been transformed to the corresponding product. Regression equations using only data for the initial 20-30 sec of the reaction and their r* values are shown in Table 32, together with the initial velocities derived from those equations. The derived V0 values and V0 values estimated by fixed time assay were used to compute Km and Vmax (Table 33). Both methods gave similar results. No reduction in the standard error was obtained using the linearization procedure. The Km and Vmax values obtained in this experiment were within the range of previously reported values (Table 13). Although the linearization procedure of Durance et al. (1986) did not improve the accuracy of the determination of the kinetic parameters Km and Vmax in this case, through the use of this methodology it was possible to obtain the equation that relates the change of velocity of the reaction to time. This relationship was used to determine the rate constant of inactivation. This approach can only be used when product inhibition does not occur and the substrate concentration is high enough to remain at saturating conditions during the course of all of the reaction, so that decrease in velocity (i.e. activity) is due to the reaction of the inhibitor with the enzyme and not to the -241- Table 32. Results of curve fitting the kinetic data for the reaction of papain-carbobenzoxyglycine p-nitrophenyl ester at various initial substrate concentrations. Initial velocities (V0) determined by the fixed time assay method are included for comparative purposes. [Initial] Derived Fixed time (10-»M) Regression Equation*' r * 19 .00 y-o.it - 0.0092X + 5 .105 0.99 1.80 1.49 9.50 y-o. so— 0.0016X + 1 .590 0.99 1.80 1.38 yo 6.60 .io = -7.0 x 10"«X + 0.1457 0.99 1.58 1.38 4.76 Y= -1 .2 x 10"SX + 4.5 x 10"s 0.96 1.20 1.24 2.86 Y= -1 .1 x 10-«X + 3.3 x 10-9 0.96 1.10 1.04 0.92 Y= -7 .2 x 10-TX + 2.6 x 10"s 0.93 0.72 0.61 xUnits: Y, 10-SM residual substrate; X, sec BUnits: (10-8M)/sec -242- Table 33. Kinetic parameters of the papain-carbo• benzoxyglycine p-nitrophenyl ester reaction (± standard error), computed with the program of Oestreicher and Pinto (1983) using initial velocities estimated by fixed time assays or derived from experimentally determined curves. Fixed time Differential Parameter method method Km* 1.53 ± 0.5 1.70 ± 0.6 VmaxB 1.63 ± 0.2 1.95 ± 0.2 Kcat° 8.15 ± 0.2 9.75 ± 0.3 * 10-'M B (10-6M)/sec c sec -x -243- decrease in substrate concentration. L. CHARACTERIZATION OF THE INHIBITORY EFFECT OF TETRATHIONATE 1. Reversible inhibition Since linearization did not improve the accuracy of the determination of Km and Vmax, the fixed-time method was used to estimate the initial velocities in these experiments. Assuming that tetrathionate acted as a reversible inhibitor of papain, it was found that the rate of inhibition or inactivation of papain by tetrathionate decreased as the substrate concentration increased. Using the computer program ENZYME (Lutz et al., 1986) to analyze the experimental data it was found that tetrathionate significantly affected the Km (p<0.005) but not Vmax, indicating that tetrathionate acted as a competitive inhibitor of papain. This result is consistent with the nature of the reaction of tetrathionate with Cys-25, which is part of the active-site of papain. Most references indicate that if an irreversible inhibitor reacts with a group near or in the active site, such as tetrathionate with the only cysteine residue of papain, its mode of inhibition should be competitive. The results in this study support this suggested mechanism. -244- A Lineweaver-Burk plot of the enzyme activity of papain at three different concentrations of tetrathionate is shown in Fig. 51. It clearly shows the competitive inhibition nature of tetrathionate on the activity of papain. It was necessary to use relatively high concentrations of tetrathionate in order to measure changes in the initial velocities at the different substrate concentrations evaluated. A inhibition or dissociation constant (Ki) of 6.6 X 10~4 was obtained by means of the computer program ENZYME. This value indicates that tetrathionate did not bind to papain as tightly as other inhibitors such as E-64 (Ki=1.8 X 10"«) (Hanada et al,, 1978) or cystatin (Ki < 5"") (Nicklin and Barrett, 1984). Since tetrathionate is an inorganic compound, It may not have as high affinity for the active site of papain as more complex molecules which act as substrate analogs of papain. 2. Irreversible inhibition (a) In the presence of substrate Figure 52 shows the inhibitory effect of a constant concentration of tetrathionate at three different concentrations of substrate. It is evident that a high concentration of substrate protected papain from inhibition. Since a relatively high concentration of the inhibitor was used, the reaction was assumed to be first order (Eq. 5) and the pseudo-first order rate constant of inhibition at each substrate concentration was -245- Figure 51. Lineveaver-Burk plot for the papain activity at different concentrations of tetrathionate. 10.6 x IO'4 M (•) 20.6 x 10"* M («A) 31.8 x IO"4 M (O) Without tetrathionate (O) -246- Figure 52. Inhibitory effect of tetrathionate (TT) in the presence of three different initial substrate concentration at a constant TT concentration (1.06 x 10-3 M. The three top lines are the reactions without TT. The initial substrates concentration were: 1.90 x 10-" ( O ,e ) 0.95 x 10-" (Q ,0 ) 0.48 x 10-4 (A ,if ) -247- calculated. The second order rate constant was obtained in the usual way by dividing the pseudo-first order constant by the inhibitor concentration. In order to characterize the irreversible mode of inhibition of papain by tetrathionate, a plot of the inverse of the second order rate constant (k) against substrate concentration as suggested by Tsou et al. (1985) was evaluated. Fig. 53 shows a linear relationship (r*=0.98) which indicates that tetrathionate is an irreversible competitive inhibitor of papain. (b) In the absence of substrate Fig. 54-56 summarize the changes in the catalytic activity of papain which occurred upon treatment of this enzyme with tetrathionate in the absence of substrate. A period of about 10 min was required for 90-95% inactivation at levels of 1 mole of tetrathionate per mole of papain (Fig. 54). At levels of less than 3 moles of tetrathionate per mole of enzyme, the degree of inactivation obtained after 30 sec of incubation appeared to be linearly related to the ratio of reagent to enzyme (Fig. 55). The kinetics of papain inactivation by tetrathionate at levels of 1-3 moles of tetrathionate per mole of enzyme at 22<>C was second order. Using Eq. 11 ( when mole tetrathionate/mole papain =1) or Eq. 12 (when mole tetrathionate/mole papain >1) the value of k (second order rate constant for tetrathionate inactivation) was 16,919 M-Xsec'1 (Fig. 56). -248- Figure 53. Effect of carbobenzoxyglycine p-nitrophenyl on the second order rate constant of inactivation of papain by tetrathionate. (Mean±S.D., n=3). The regression line is shown. -249- 100.00 H. Molar ratio Tetrathionate:Papain 0 1:1 2:1 3:1 80.00 - -o 0) c 60.00 LY > 40.00 - o < 20.00 0.00 100 200 i—i—i—i—i Time (sec) 300 400 Figure 54. Change in the catalytic activity of papain as as a result of the reaction with tetrathionate Activity was measured with carbobenzoxyglycine p-nitrophenyl ester as the substrate. -250- Figure 55. Effect of the molar ratio of tetrathionate to papain on the catalytic activity of papain at two reaction times. Activity was measured with carbobenzoxyglycine p-nitrophenyl ester as the substrate. -251- 0.12 -i Molar ratio Enzyme : Tetrathionate 0 1:1 0.10 i A 1:2 1:3 .0.08 O °-06 0.04 - 0.02 z 1 0.00 i i i i I i i 1 I I I I I I I I I I I I I i i i i i i i i * * * i 500 600 700 0 100 200 300 400 Time (sec) Figure 56. Progress of inactivation of papain, by different levels of tetrathionate (TT). The value of Y was calculated based on Eq.ll when the molar ratio of TT to papain was 1:1. For the other molar ratios Eq.12 was used. Activity was measured with carbobenzoxyglycine p-nitrophenyl ester as the substrate. -252 Table 34 presents values o£ second order rate constants for different irreversible inactivators of papain and also for the inactivation of guinea pig liver transglutaminase and glyceraldehyde-3-dehydrogenase with tetrathionate. With the exception of E-64 (k= 638,000 M^sec-1) and cystatin (k= 1.0 X 107 M_1sec_x), papain reacts faster with tetrathionate than with other inactivators such as iodoacetamide or hydrogen peroxide. According to the values of k reported for the inactivation of other enzymes with tetrathionate, it would appear that tetrathionate reacts faster with papain than with transglutaminase and dehydrogenase. In a recent article, Prasad and Horowitz (1987) reported that inactivation of bovine liver rhodanase (EC 2.8.1.1) with tetrathionate was instantaneous and a linear function of tetrathionate concentration. Although no k was reported, the term "instantaneous" could indicate that the k for the reaction of rhodanase with tetrathionate was significantly higher than the reaction of tetrathionate with papain. Since the reactivity of cysteine residues in proteins is affected by neighboring groups (Means and Feeney, 1971) it is well known that proteins react at different rates with reagents that modify these residues. This could account for the different values of k for the inactivation of sulfhydryl enzymes with tetrathionate. -253- Table 34. Second order rate constants of inactivation for some papain inhibitors and for tetrathionate with two other enzymes. k Enzyme Inhibitor (M*1 s"x) Reference Papain N-ethylmaleimide 166.00 (1) Papain N-heptylmaleimide 3.05 x 10' (1) Papain N-decylmaleimide 21.45 x 101 (1) Papain Chloroacetyl 0.38-5.24 (2) amino acid Papain Cyanate 156.70 (3) Papain Cystatin 1.00 x 10' (4) Papain 2-PDS 942.00 (5) Papain E-64 63.80 x 10« (6) Papain H,0, 61.00 (7) Papain Iodoacetic 2.3 x 10* (6) Papain o-methylsourea 1.33 x IO"4 (8) G-3P-D* Tetrathionate 80.80 (9) TGLU" Tetrathionate 8.33 (10) Reference: (1) Anderson and Vasini, 1970 (2) Oka and Morihara, 1986 (3) Sluyterman, 1967a (4) Nicklin and Barrett, 1984 (5) Brocklehurst et al., 1981 (6) Barrett et al., 1982 (7) Lin et al., 1975 (8) Banks and Shafer, 1972 (9) Pihl and Lange, 1962 (10) Chung and Folk, 1970 *D-glyceraldehyde 3-phosphate dehydrogenase •Transglutaminase -254- CONCLUSIONS The major objectives of the present work were to investigate the effects of chemical modification with tetrathionate on the activity of papain and to characterize the inhibition effect of tetrathionate on the activity of papain in terms of enzyme kinetics. Due to the fact that two methods that were used in this study, i.e., circular dichroism and proteolytic activity determination, had problems of high noise levels and lack of repeatability, respectively, optimization of the operating conditions of the methods , in order to improve their precision was carried out by means of simplex optimization. A new optimization approach, consisting of the combined use of a central composite rotatable design and computational simplex optimization, was applied to find the experimental conditions which yielded the highest precision for the proteolytic activity determination of papain by using casein as a substrate. The optimum conditions were as follows: papain concentration, 0.1 mg/mL; incubation time, 5.3 min and incubation temperature 35<>C. Simplex centroid optimization was used for optimization of the conditions to measure the CD spectra of papain. After a total of 18 experiments (vertices), the following optimum conditions were obtained: papain concentration, 0.23 mg/mL; band width, 1.6 nm; time constant, 2 sec, and for the value of the product of time -255- constant times the scan rate, 0.8 nm. A significant improvement in the repeatability and signal to noise ratio of the CD scan of papain was achieved. The results of the two fractional factorial experiments indicated that the reaction of papain with tetrathionate caused a complete inhibition of the proteolytic activity of the enzyme. This inhibition or inactivation was not affected by changes in the molar ratio of papain to tetrathionate, pH, temperature and time of the inactivation reaction. In order to obtain complete reactivation of the proteolytic activity, the inhibition reaction should be carried out at pH 6.8, 22<>C and the reactivation reaction with 20 mM cysteine. The results of the UV-absorbance, near-UV CD and far-UV CD spectral analysis indicated that no major changes in secondary or tertiary structure occurred in papain upon chemical modification with tetrathionate. The reversibility of blocking the only cysteine residue of papain by tetrathionate was confirmed by determination of sulfhydryl groups and pH-fluorescence profiles of the modified papain. Decreases in fluorescence intensity of the modified papain from that of the native papain suggests that at high molar ratios of tetrathionate to papain for chemical modification, some tryptophan residues were also modified. Preliminary experiments showed that when a papain solution was heated to 60°C in the presence of 0-mercaptoethanol and tetrathionate, the enzyme precipitated. Using response surface methodology, experiments were carried out to find the conditions -256- producing the largest quantity of precipitate. Addition of 0-mercaptoethanol and tetrathionate at levels of 100 and 50 mM, respectively, precipitated 90% of the originally soluble protein. Solubility measurements and electrophoresis of the insoluble protein demonstrated the possibility of inter-molecular disulfide bond formation. The last part of this thesis included a series of enzyme kinetics experiments. The substrate used throughout these experiments was carbobenzoxyglycine p-nitrophenyl ester. The standard errors of the Km and Vmax values for the papain- catalysed hydrolysis of the p-nitrophenyl derivative were not reduced when initial velocities were calculated using the linearization approach of Durance et al. (1986), as compared to values calculated using a fixed time method. However, the linearization method gave equations that related the change in velocity with time for each initial substrate concentration. 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Acta 828:196. -274- APPENDIX Appendix 1. Listing of the IBM-BASIC version of the computer program used by Siegel et al. (1980) to determine secondary structure fractions from CD spectra. -275- 10 REM CD ANALYSIS SIEGEL METHOD 20 DIM A$(72),D$(72),I$(72),S$(72),D(13,10) 30 FOR 1=1 TO 13:FOR J=l TO 10 :READ D(I,J): NEXT J:NEXT I 4 0 REM CONVERT 50 FOR 1=1 TO 13 60 D(I,8)=D(I,8)~2 70 NEXT I 80 INPUT "NAME OF PROTEIN";1$ 9 0 INPUT "CODE";D$ 100 N=13 110 FOR 1=1 TO 13 120 PRINT D(I,1);"NM [THETHA] = " 130 INPUT T 140 D(I,4)=T 150 NEXT I 160 REM CALCULATIONS 163 LPRINT "PROTEIN ";I$ 16 4 LPRINT "CODE= ";D$ 170 FOR 1=1 TO 13 180 D(I/5)=(D(I/4)-D(I/2))/D(I/3) 18 5 PRINT "WAVELENGTH ";D(J,1);" [THETA] "; D(I,4);" FRACTION"; D (I , 5 ) 186 LPRINT "WAVELENGTH " ; D ( 1,1) ; " [THETA] "; 0(1,4);" FRACTION";D(I,5) 190 D(I,6)=(D(I/7)+D(I/8))/(D(I/3)"2)+ ((D(I,4)-D(I/2))~2)*D(I,9)/(D(I,3)"4) 200 D(I,6)=D(I,6)+2*(D(I,4)-D(I,2))*D(I,10)/(D(I,3)~3) 210 NEXT I 230 S1=S2=S3=0 240 FOR 1=1 TO 13 250 S1=S1+ (0(1,5)70(1,6)) 260 S2=S2+(1/D(I,6)) 270 NEXT I 280 FOR 1=1 TO 13 290 S3=S3+((D(I,5)-S1/S2)~2) 300 NEXT I 310 X=S1/S2 320 S4=l/S2 330 S5=S3/((N-1)*N) 340 REM PRINT OUTPUT 350 PRINT 360 PRINT "PROTEIN: ";I$ 370 PRINT "AVERAGE VALUE OF HELIX (X-BAR); ";X 375 LPRINT "AVERAGE VALUE OF HELIX (X-BAR); ";X:LPRINT 380 PRINT "CORRESPONDING TO "; X*100; "% HELICAL STRUCTURE" -276- Appendix 2. Listing of an IBM-BASIC computer program which utilizes the simplex algorithm of Morgan and Deming (1974) used for computational optimization of the conditions to measure the proteolytic activity of papain. -277- 1 CLEAR:CLS: DIM X(10,100)/Y(200),L(10),U(10)/M(11,10)/S{10),B(10): INPUT "Max imization?(Y/N) ",X$:PRINT:INPUT "No. of factors? ",NN: INPUT "Maximum vertices to compute? ",r1V: INPUT "Terminating difference value? ",TERM 2 INPUT "How many vertices without prohibit-trespassing? ",ZR:PRINT: FOR 1=1 TO NN:PRINT "Factor No. ";I: INPUT " Enter lower then upper limits ",L(I),U(I):PR I NT:NEXT I 3 LPR1NT "Terminating difference value=" ; US I NG "ft ft ft . ft ftft ft " ; TERM : LPRINT:LPRINT "Lower and upper limits":LPRINT " LL:";: FOR J = l TO NN : LPRINT USING "ft ft ft » ft ft . ft ft ft " ; L (J ) ; : NEXT J.-LPRINT: LPRINT " UL:";:FOR K=l TO NN 4 LPRINT USING "ft ft ft ft ft ft . ft ft ft " ; U (K ) ; : NEXT K : LPRINT : LPRI NT : LPRI NT : P=(1/(NN*SQR(2)))*(NN-1+SQR(NN+1)):Q=(1/(NN*SQR(2) ) ) *(SQR(NN +1)-1) : FOR J=l TO NN:M(1,J)=L(J):NEXT J:FOR 1=2 TO NN+l:FOR J = l TO NN 5 IF I-1=J THEN M(I,J)=L(J)+P*(U(J)-L(J)) ELSE M(I,J ) =L(J)+Q*(U(J)-L(J) ) 6 NEXT J:NEXT I:FOR K=l TO NN+l:FOR J=l TO NN:S(J)=M(K,J):NEXT J: GOSUB 46:B(K)=R:FOR L=l TO NN:M(K,L)=S(L):NEXT L:NEXT K: FOR XX = 1 TO NN + l:FOR 1=1 TO NN:X(I , XX)=M(XX,I):NEXT I:NEXT XX: FOR Y = l TO NN + 1:Y(Y)=B(Y):NEXT Y 7 LPRINT TABOO) "Initial s implex": LPRINT TAB(16) "XI";: LPRINT TAB(25) MX2";:LPRINT TAB(34) "X3";:LPRINT TAB(43) "X4";: LPRINT TAB(52) "X5";:LPRINT TAB(60) "RESPONSE":XX=XX-1:Y=Y-1: FOR 1=1 TO XX:E(I)=100-(X(1,I)+X(2,I)+X(3,I)+X(4,I ) ) 8 NEXT I:FOR J=l TO XX:LPRINT "Vertex ";USING "ftftft";J;: LPRINT USING "ftftftftft.ftftft";X(l,J);X(2,J);X(3,J);X(4,J);E(J);: LPRINT TAB(56) USING "ft ft ftftft.#ftft";Y(J):NEXT J:LPRINT: QO=0:WHILE Q0=0:WORST=B(1):WL=1:FOR 1=2 TO NN+1 9 IF(X$="Y")=0 THEN 11 ELSE IF B(I) -278- 24 C=-.5:C$="(Contraction-W)":GOSUB 41: IF(R>WORST)=0 THEN 25 ELSE FOR J = l TO NN : Q(J)=S(J) :NEXT J: GOSUB 45:GOTO 26 25 C=-.25:C$="(Massive contraction-W)":GOSUB 41: FOR K=l TO NN:Q(K)=S(K):NEXT K:GOSUB 45 26 GOTO 37 27 IF(REFL -279- Appendix 3. Listing of the IBM-BASIC computer program used to generate the data (grid) for plotting the response surface of the effect of |3-mercaptoethanol and tetrathionate on the precipitation of papain. Note: The actual plotting was done with the commercial software Golden Graphic System (Golden Software, Inc., Golden, Colorado). -280- 5 REM PROGRAM FOR GENERATION OF DATA POINTS FOR RS 7 REM MER=[MERCAPTOETHANOL] TETRA=[TETRATHI ONATE] 8 REM Y= % PAPAIN PRECIPITATED 10 OPEN "prers .daf'FOR OUTPUT AS ff 1 20 FOR MER=0 TO 100 STEP 10 30 FOR TETRA=0 TO 100 STEP 10 35 REM EQUATION OBTAINED BY BACKWARD REGRESSION ANALYSIS 40 Y=TETRA*1.956241+MER*.481129+ (TETRA"2)*-.012221+TETRA*MER*-.00 4 0 0 9 4 5 NORMALIZATION OF DATA 5 0 MERN = ((MER-10 0)*10)/10 0: TETRAN=((TETRA-100)*10)/100 60 LPRINT USING "###.######";MER,MERN,TETRA,TETRAN,Y 65 REM SAVING THE DATA IN THE FILE 70 PRINT #1,MERN;TETRAN;Y 8 0 NEXT TETRA 9 0 NEXT MER 100 CLOSE -281-