A S P E C T S 0 F T HE

R A D I A T I O N C H E M I S T R Y

A, N D

P R O T E C T I O N O F P E P T I D E S

A thesis submitted in partial fulfillment of the requirements for the degree of

M A S T E R 0 F S C I E N C E

in the University of New South Wales

by

J.A. HOURIGAN, B.Sc.

July, 1971. (11)

The work contained in this thesis has not been submitted for a degree or similar award to any other University or Institution.

J.A. HOURIGAN (111)

Aspects of the radiation chellliatry and protection of the polypeptide chain of catalase were investigated, employing the soluble enz,111le and also insoluble, poly(diazostyrene)-bound catalase. Prior investigation of the structure of the inaolubilized catalase by ion-exchange chromatography of its hydrolysate showed that approximately 140 residues (histidine, lysine, cysteine and tyrosine, i.e. roughly half the total number of these residues per catalase aolecule) were covalently linked to poly(diazostyrene) molecules. The conjugate was pictured as a catalase molecule to which a large number (20-100) of poly(diazostyrene) molecules were covalently linked. The results indicate further, and in agreement with the results of irradiation experiments, that the tyrosine and arginine residues are mainly located in the interior of the active conformer of the catalase molecule. A novel pyrolysis-gas chromatographic technique was developed for the estimation of the protein content of poly(diazostyrene)-bound catalase. The method was calibrated against the results of quantitative ion­ exchange chromatography of the hydrolysates of the bound enzymes. It was then extended to allow the prediction (iv)

of the protein content of poly(diazostyrene)-bound catalase prepared by a known procedure from a given ratio of protein to polymeric carrier. The radiation sensitivity of catalase was investigated, first, free in dilute, oxygenated aqueous solution and secondly, bound to poly(diazostyrene) as a dilute, oxygenated, aqueous dispersion. Hydroxyl radicals, resulting from {-irradiated water molecules, inactivated the enzyme and degraded cysteine and/or cystine in particular, amongst the amino acid residues. Although the radical scavenging and repair agent, diglycylglycine, offered some protection against the effects of (-radiation on catalase, protection of similar molar concentrations of catalase by the insolubilization was much more effective, probably due to a combination of conventional radical scavenging and repair processes with proximity, conformational and other steric factors. lV)

TABLE o:r CONTENTS

TITLE -PAGE 1 • Introduction. 1

1.1 Radiation Effects OD Peptides and Related Compounds. 1 1.2 Radiation Chemistry of Water. 4 1. 3 Radiation Chemistry of Aqueous Solutions. 7 1.4 Mathematical Treatment of Protection. 11 1.5 Catalase. 22 1.6 Insolubilized Enzymes. 24 1.7 The System to be Investigated. 29

2. Experimental Methods 0 30 2.1 Materials. 30 2.2 Apparatus. 33 2.3 Preparation of Insolubilized Catalase. 37 2.4 Properties of Insolubilized Catalase and Constituent Polymers. 42 2.5 Estimation of Catalatic Activity. 46 2.6 The Effects off-Radiation. 49

3. Discussion of Results• 54 3.1 The Radiolytic Inactivation of Catalase in Solution. 54 (vi)

Table of Contents (Continued) TITLE -PAGE 3.2 Species Responsible for the Radiolytic Inactivation of Catalase in Solution. 72 3.3 The Properties of Polymer-Bound Catalase. 85 3.4 The Radiolytic Inactivation of Insolubilized Cata.lase. 121

4. Conclusion. 132 5. Acknowledgements. 134 6. References. 136

APPENDIX

1. Results• 148 1.1 Properties of Insolubilized Catalase. 148 1.2 The Effects of '6'-Radiation. 160

2. Published Reaction Rate Constants. 177 2.1 Reactions of the Hydroxyl Radical. 177 2.2 Miscellaneous Reactions. 181 1 •

l IftRODUC!IOB l.l IU.D~IOB BPlBCTS OB PEPTIDES ilD BELATED COIIPOUlf.DS

!he large volW118 of litera'turel-4 published in the last decade on the radiation cheaistry of peptides and such related substances a■ amino acid■, proteins and enzyaes, is a reflection of the iaportance of this area of investigation. The irradiated materials were aost commonly in the solid state or either in oqgenated or deoqgenated aqueous solutions14• As one facet of this work, many investigators have attempted to protect the irradiated aaterials froa the effects of irradiation5• 6• Protection, in thia sense, occurs when the destniotiv• effect of the irradiation on the compound under study 1• reduced by an added substance. The converse is sensitizat­ ion. The aeohanisa of protection usually falls into on• or other of the categories shown below5- 7• a) Transfer of energy or charge by physical processes can protect an irradiated molecule by removing excess energy before the molecule can decompose. The protector aay then decoapose. This mechanism haa been coaaonly observed during irradiation of organic liquida, particul­ arly when the protector is an aromatic coapound. b) Scavenging of radiation-produced free radicals (See Section 1.2) by protector molecules will sometimes reduce the destructive effect of irradiation. In these instances, the protector is usually a highly reactin coapoUDd towards the radiation-produced free radicals so that it oompetea successfully forth• with the sub­ atrate, even when the former•a concentration is au.eh lower than the latter's• c) A similar effect is obtained if the protector interacts chemically with a substrate radical to restore it to ita original state. This process is usually known as repair. d) A fourth approach is to form a •complex• between the protector and the moeities in the target compound which are particularly sensitive to irradiation. The complex may be unreacj1ve towards radicals or it may facilitate energy or charge transfer away from the target compound. Protection will be treated mathematically in Section 1.4. In order to reduce anomalous effects from the term­ inal residues during the experimental irradiation of peptides, it is advisable to work with polypeptides. In this project, radiation-induced alteration in the activity of an enzyae waa used aa a sensitive measure of the effects of radiation on a polypeptide. Kost preTious work on the irradiation of enz111es has been perfol"lled on their dilute aqueous solutiona2• How­ ever enzyaea in Nature, exist both in solution (often oxygenated) and bound to insoluble cell components8• Therefore, in the present study the radiation chemistry and protection of an enzyae both in oxygenated aqueous solution and suspension was investigated. The enzyae, oatalase (H2o2 : H2o2 oxidoreductase 1.11.1.6), was chosen aa it is very widely distributed biologically and because an appreciable amount is known about its general properties. (The aias of the project are des­ cribed more completely in Section 1.7). 1.2 RilIATI0I CHllfISTRY 0~ WATER

The radiation chemistry of water baa been Tery

intensively studied since the mid 1940 1 s and baa been 9-12 reviewed several tiaea • When pure water is irradiated with 60co y-rays (aean photon energy 1.25 KeV.) the energy is primarily absorbed by Compton scattering and as a result the water is exposed to a flux of very fast recoil electrons13: + •- These initial Compton recoil electrons, as well as second­ ary electrons produced by ionization of other water molecules, lose energy and undergo thermalization within 10-13 second of the initial ionization. Following ther­ malization of an electron, adjacent water molecules become polarized and the electron is finally hydrated after about 10-10 second. In accordance with the uncertainty prin­ ciple, the electron is "smeared out• over a large number of water molecules. The formation of the hydrated electron can be represented as e-

According to the generally accepted diffusion :model14, the overall effect of this process will be the formation of a track of excited and ionized species 5. along the path ot the Compton recoil electron, with short side-tracks or •spurs•, formed by secondary electrons, being spaced at intervals along the main track. When absorbed in water, sparsely ionizing radiation such as 60co (-rays, give spurs ot about 201 initial diameter and about 104 1 apart15 • An appreciable traction ot the ettects ot such radiation can be attributed to reactions between intact solvent molecules and active species which have diffused from the spur into the bulk ot the solution.

The species initially formed on radiolysis, H2o: and e;q initiate a complex series ot reactions, the most important of which are shown below:

Ho+ + 2 + H2o ~H30.

•aq- + OH• ~011

Ho+l . + 011 ~ 2H20 Ho+ 3 ' + •;q ~H•

a· + oH· ~H2o

H• + - 8 aq ~H2 + 011

H• + H• ~H2

•aq + e;q ~H2 + 2011

oa· + oa· ~H2o2 6.

OH- eaq- + H2o 2 -+OH• +

OH• Ho• + H2O2 >H2o + 2

oa· + H2 ► H2o + H• Kost of these reactions are extremely fast (See Appendix 2), the rate constants being of the order of 109 - 1011 •-l seo-1 • In a f-irradiated closed system containing pure water these •products• will destroy each other in such a way that no net chemical change will be detected12• 1.3 BADIA.TIO! CHEMISTRY 01 ~QUEOUS SOLUTIONS

Published work in this field demonstrates two main lines of research16•17• Some workers have attempted to explain radiation effects by working baok from the G values (i.e. yields per 100 ev. of energy absorbed) of the final producta17• Others, relying usually on pulse radiolytic experiments, have investigated the initial attack of radicals on various substratea16• Relatively little has yet been achieved in the study of the many secondary reactions which occur after initial attack and ultimately lead to the final products. An attempt has been made to interpret the results of the present study in terms of initial attack by radiation-produced oH• radicals. Consequently, the reactivity of e;q, a• and H02 will not be reviewed incittail. The oa• radical readily oxidizes inorganic anions and cations18 and abstracts hydrogen at the '(,-carbon in moat saturated aliphatic compounds*16•19• Since the OH• radical behaves as an electrophile in the latter reaction one would expect electron-releasing groups ad­ jacent to the ~-carbon to enhance the reactivity of the substrate and electron-withdrawing groups to decrease it.

* A selection of rate constants is given in Appendix 2. 8.

10 The order of reactivity found by Anbar and Coworkers J amongst various functional groups roughly substantiates this expectation. In the reaction + OH• _. R CH• X the order of reactivity for various X substituents is:

With thiols the His abstracted from the sulfhydryl group rathP.r than the 0(-carbon 20 ,

+ +

Aromatic compouncis are susceptible to electrophilic attack by oH• and as a result yield hydroxy-cyclohexa­ dienyl radicals. the latter radicals rapidly decompose to complex products, the identity of which depends on the nature of the aromatic compound and the presence or ~bsence of im~urities or oY..ygen. The initial reactions with benzene are thought to be21- 23:

--+ ~OH~ (XOH OH V· 02 ~ . O· Oz .. ~. 1.rc-•J• ~•Jru,3-,eroK,Y• .:!j'Clv:~,:ov.: ,J.;.-~n,11 c,rcl<>nexahe11¥l r'"l.ll:."l .. riol:1ic:1l l 1

* The unex~ected reactivity of RCH2No 2 is possibly due to 20 additional reactions of CH 0 witn the N0 2 group • 9.

The above considerations are reflected in the order of reactivities of oH• with amino acids. The important feature of these results is the reactivity associated with the side chains of the various amino acids, because the terminal amino and carboxyl groups are comm.on. More­ over, when relating these reactivities to amino acid residues in peptides and proteins, the terminal groups will, in the main, be converted to amide groups and since these will be common, the relative reactivity of different residues should be reflection of the reactivity of the side chains they contain. The rate constants for reactions between OH• and amino acids are summarized in Appendix 2. It is apparent that a num.ber of amino acids are quite reactive, having rate constants in excess of 109 M-1 sec-1 • These acids, in order of decreasing reactivity are cysteine,* tryptophan,tyrosin£ ~ * The rate constants for these acids shown in Appendix 2 are for acidic solutions only, while the remaining acids have been compared in neutral solutions. In the case of cysteine, RSH (pXa 8.33; ref. 24) a change to neutral solutions will increase the concentration of the conjugate base, RS-, and probably lead to greater reactivity with OH• (ref.20) while with tyrosine (pXa 10.l; ref. 24), the value is so high that an increase in pH from£!• 2 to £,!•7 will have little practical effect on its degree of ionization or reactivity with OH•. 10.

'aethionine, phenylalanine, histidine, cystine (these last three having comparable reactivities) arginine and isoleucine. The order of these reactivities with the electrophilic oa• are explained by the presence of groups such as the thiol in cysteine, activated aromatic or heterocylic rings in tyrosine and tryptophan, sulphide in methionine, aromatic or heterocyclic rings in phenylalanine and histidine, disulphide in cystine, the protonated guandino group (pKa 12.48; ref. 24) in arginine and the relatively long carbon chains in arginine and isoleucine. In irradiated dilute aqueous solutions of proteins, free radical reactions such as those discussed above can lead at least, to conformational changes3• 25 • 26 and, it is reasonable to expect, enzymic inactivation. Other reactions accompanying free radical attack on dilute aqueous solutions of proteins are cross-linking to produce aggregates and chain scission to produce fragments27 • 1 1 •

1.4 IUTHEMATICil TREA.TMENT OP PROTECTION

It is common28 to refer to radiation effects in dilute aqueous solutions as "indirect effects" and to those in the solid state or bulk liquids as "direct effects•. This classification arises because the effect• in aqueous solutions are usually ascribed to free radicals produced from water by the radiation (See Section 1.2) while those in bulk liquids or the solid state are as­ cribed to "direct hits" by photons on sensitive sites in the target compound. The direct effect has been treated mathematically by Lea29 • The mathematical treataent of the indirect effect is more applicable in the present study and will be considered in detail.

1.4.1 Kinetic Treatment of the "Indirect Effect" The kinetic treatment of the indirect effect in dilute aqueous solutions of enzymes was originally de­ veloped by Dale and Coworkers3O and has been well sum­ marized by Allen31 • Briefly, these investigators showed that when the logarithm of the residual enzymic activity decreases linearly with dose, one can assume that the radiation-produced radicals react as readily with de­ activated enzyme molecules as with active enzyme molecules. This conclusion was shown to hold for pure aqueous enzylllic solutions and for those containing a concen­ tration of protector so large that irradiation did not materially alter its concentration. For a solution containing a protector, the equation had the form.31 E ln I D (1) 0 - where a is a constant dependent on the ratio of the rate constant for reaction of radicals with protector molecules to the rate constant for reaction of radicals with enzyme molecules, D is the dose absorbed by the solution,

1 0 is the initial concentration of active enzyme, I is the concentration of active enzy'Jlle after absorption of dose, D, G is the inactiTation yield, i.e. the number of molecules of enz,YJlle inactivated per 100 ev. of energy absorbed, P is the concentration of protector. It is often convenient to refer to the 37~ dose, D37, i.e. the dose required to reduce the enzyme activity to 37~ of its initial value; at this point - -1 ••• - (2) 13.

But in a pure solution, P = O and equation (2) reduces to (3) • E0 T or, by rearrangement G (4)

It follows from the definition of G that its reciprocal is the dose abaorbe4 per unit concentration of the enzyme. Similarly, the term a;G ia a measure of the nwaber of radicals reacting with unit concentration of protector, or, in other words, the dose absorbed per unit concen­ tration of protector. Other investigators have extended equation (2) to allow for the reaction of radiation-produced radicals with buffer components or iapuritiea32•33. Apart from an exponential variation of residual activity with dose, this derivation additionally asswaea that the concentrat­ ion of radicals reaches a steady state during the irrad­ iation and that radical recombination is negligible. (This ia usually tru.e34 when the total concentration of solutes is above 10-6 Kand can be asauaed to operate in these experiaents). Equation (2) then takes the fora - + C (5) where A ia ! i.e. the dose absorbed per unit con­ centration of enzyae, B ia a i.e. the dose absorbed per unit con- ~ oentration of protector, i.e. a measure of the amount of radiation diverted from the enzyme by the protector, or a measure of the protective ability of the protector, or the reciprocal of the protector's G va1ue,* C ia the dose absorbed by the buffer. In the derivation it is assumed that this is a constant. Several other groups have derived similar expressions for the kinetics of the radiation-produced inactivation of dilute aqueous solutions of enzyaea35. Three particular cases of equation (5) will be of interest in the present study; these oases are illust­ rated in Pigure (1) (a), (b) and (c). Figure (1) (a) describes the case where protection is due to simple radical competition between the enzyme and the protector. Thus the slope of the parallel lines obtained either in the presence or absence of protector will give the value of A and hence of the inactivation yield for the enzyae.

* It is assumed that radical-protector reactions lead to inactivation of both participants. rrotector vresent (a)

..t'rotector absent

+ B.P E 0

D.37

~rotector vresent in concentrations (b) r' l'' an-i P"; P">P' Frotector absent

Protector present ( C)

.Protector absent

E 0

Fit.:ure 1: I<'o!':t:ci of_Vari~tion of_th~37 Dos~_!~~h th~_lEit.!.~~ £££~~Etr~!~£E_Of En~tme 1 ~o• 16.

!furthermore, in the presence of known amounts ot pro­ tector, the values of Band C can be calculated troa the intercepts produced by extrapolation of the lines to zero enzyme concentration. The value for B obviously leads to an estimate for the yield of inactivation of the protector.* In figure (l)(b), the slope of the straight line varies with the concentration of the protector and can even decrease to negative values (P • P•). It is evident that this graph does not describe a simple case of radical competition between the enzyme and the pro­ tector. Nakken36 encountered this situation during the protection of pyrido:xal-5-phosphate by R-aminobenzoio acid. He fitted the following empirical equation to the graph: + C (6) where 1 is a function of protector concentration; the remaini~g terms have been defined previously.

Nakken concluded that 1E0 was the "dose•, which after being absorbed by the protector, was transferred back to pyridoxal-5-phosphate. The incidence of this reaction of protector radicals with intact pyridoxal-5- phosphate molecules increased with increasing protector

* See footnote page 14. 17. concentration. Terms such aa U rather than A-1 could not be used to explain the negative slope which Nakken found at relatively high concentrations of a-amino­ benzoic acid. The case illustrated by Figure (l)(c) is relevant to the discussion given in Section 3.1.1. It is pro­ posed that this situation has certain similarities to Nakken•s results and is consistent with the empirical equation:

• (A+ F)E0 + BP + C (7)

Thus, it is possible that the protector obeying this expression protects the enzyme by radical competition but also increasingly repairs the inactivated enzyme molecules as the protector concentration is increased.

1.4.2 The Protection Quotient Probably the most useful of empirical measures of the protective ability of compounds is the protection quotient,Q. This was defined by Dale and Coworkers as the ratio of the dose taken up by the protector to that taken by the indicator (the enzyme, in the present study) for equa1 weights of eaoh37 • Thus, Q - Eo (Di - De ) (8) r ( De ) 18.

where is the dose giving a specified degree of enzymic inactivation in the absence of protector, DP is the dose giving the same degree of inactivation in the presence of protector,

E0 is the weight of enzyme per ml9 P is the weight of protector per ml. Since it is empirical by definition, Q can be used to assess protectors regardless of the shape of the inactivation - dose curves; it will be recalled that the kinetic treatment considered in Section 1.4.1 requires that these curves be exponential. Nevertheless, invest­ igations of Q will be more fruitful if the experimental results do fit exponential inactivation-dose curves. Thus by deriving expressions for DP and D• at 37~ residual enzymic activity and substituting these into equation (8) Dale --et a1.37tound that Q - (9) where d is a measure of the relative rate of uptake of radicals by protector and enzyme and is constant, m is a constant related to the lifetime 19.

of the radicals and their rate of destruction in collision with the enzyme. Rearrangement of equation (9) gives:

l i (10) + •Q a - Q And so, a plot of l against l should give a straight a 10 line if protection is due to si~ple free-radical compet­ ition between the enzyme and the protector and if the irradiated system obeys the other conditions associated with the derivation of equations (2) and (5). This treat­ ment will be applied to the results of the present study. Dale and Coworkers37 also devised a method for ex­ perimentally recognising the situation where the protect­ or not only competes for the radicals with the enzyme but also after reaction with the radicals interacts with intact enzyme molecules causing the latter's inactivation*. They identified the interaction by plotting Q against P on a double logarithmic scale, and referred to the

* The original publication refers to this change as the conversion of the enzyme to a "meta-stable state•37 • During the intervening years the free-radical nature of the indirect effects of ionizing radiation on dilute aqueous solutions has been elucidated (see Section 1.2) and in this study the interaction referred to above will be regarded as a free radical reaction. 20.

variation obtained as the phenoaenon of the "changing quotient•37 • In this project the "changing quotient•

was recognised from a graph of D37 versus E0 as described above in ligure (l)(b) and equation (6) and originally proposed by Nakken36• 1.4.3 The Dilution Effect

Dale and Coworkers38 have shown that irradiation inactivation of dilute aqueous solutions of enzymes is characterised by the "dilution effect". This means that dilution of the solution gives an apparent increase in the radiation sensitivity of the enzyme or conversely that concentration of the solution gives an apparent decrease in the radiation sensitivity of the enzyme. The arguments in support of this phenomenon have been ably summarized by Bacq and ilexander28 • If in­ activation is indirect, then it will be due to reaction between radiation-produced solvent radicals and the enzyme. ilthough one would expect the extent of reaction to be dependent on the concentration of both radicals and enzyae, the concentration of the former is usually so low in aqueous solution(,!:!• 10-6 Mor less39 ) that in practice the enzyme is often at least in incipient excess. Bearing 21. in mind that the concentration of radicals is dependent on the dose, these circwastances lead to a constant in­ activation yield and a decreasing percentage inactivation at increasing enzyme concentration, i.e. the solution appears increasingly resistant to the radiation as the enzyme concentration is increased. It is convenient to demonstrate the dilution effect by plotting the inactivation yield at D37 against the initial concentration of the enzyme. After allowing for the dose absorbed by the buffer (if any) the yield should increase to a maximum value and then remain constant with increasing concentration of enzyme. 22.

1.5 CATALASE

This enzyme is widely distributed throughout Nature40 (e.g. in vertebrate liver and blood as well as various llicro-organisms) where it presumably promotes the so-called "catalatic" reaction:

Catalase is a chromoprotein, containing the ferri-proto­ porphyrin IX prosthetic group in which 4 of the co­ ordination positions to the iron are taken by the pyrrolic nitrogens. Of the remaining two positions (above and below the plane of the porphyrin group), one is evidently taken by an amino acid residue of a protein group and the other by a water molecule40• Each catalase molecule contains 4 such prosthetic groups distributed evenly amongst 4 presumably identical subunits41• Catalase used in the present study was from bovine liver; it had a molecular weight of 244,000 (ref. 42~ an iso-electric point of 5.8 (ref. 43) and optimua activity between pH6 and 8 (ref. 44). The •chanism of the catalatic reaction (equation (10)) has been extensively investigated and the reaction kinetics are consistent with the following second order 23.

reaction equation45 :

- ndx - k e x where X is the concentration of hydrogen peroxide, e is the concentration of catalase, k is the rate constant found from the slope of a semi-logarithmic plot of residual peroxide concentration versus time. Chance and Coworkers45 developed a rapid titration with permanganate for the estimation of the residual concen­ tration of hydrogen peroxide and used it to estimate catalatic activity. This method is advantageous as it estimates the initial activity before the catalase protein can be inactivated by hydrogen peroxide45 • A number of other methods e.g. slow titrimetric methods and gasometric assays, used for the estimation of catalatic activity do not meet this criterion. Con­ sequently the catalatic activity was estimated in the present study by rapid permanganate titration. Spectrophotometric methods45 were not employed because of the physical state of the insolubilized catalase. The recently introduced methods using the Clark electrode (specific for o:xygen)46 were not available when the present project was begun. 24-

1.6 INSOLUBILIZED ENZYMES

ilthough pure enzymes are invariably soluble in aqueous solution47 , interest has grown recently in methods for rendering enzymes water-insoluble. The resulting insolubilized enzyme (immobilized enzyme, poly­ mer- bound enzyme, polymer-enzyme conjugate) often differs in properties and application from the original free enzyme48-50. Several methods of preparation are available, namely49,50,

1) adsorption on inert carriers or synthetic ion-exchange resins, ii) inclusion in gel lattices, iii) covalent cross-linking of the enzyme by an appropriate bifunctional reagent, iv) covalent bonding of non-essential functional groups of the enzyme to a suitable polymeric support, v) encapsulation. In practice, insolubilization sometimes involves a combination of the methods listed above (See Section 25.

The present project is concerned with the effects of insolubilization on the radiation sensitivity of catalase in an oxygenated aqueous environment. The catalase was insolubilized by covalent bonding to poly­ (diazostyrene) because of previous demonstrations of the success of this method in giving a stable, active derivative51• It was also expected that poly(diazo­ styrene) would be effective in scavenging hydroxyl and other radiation-produced free radicals (See Sections 1.3 and 3.4.2). The chemical reactions presumably involved in the preparation of poly(diazostyrene)-bound catalase are set out in Figure 2. The reactivity of covalently insolubilized enzymes with radiation-produced radicals does not appear to have been previously investigated.

1.6.1 The Estimation of Protein in Insolubilized Enzymes

Knowledge of the enzymic content of the conjugates will be critical to a discussion of the results of the irradiation of bound catalase. Any proposed method for estimating the enzymic content must be unaffected by the presence (possible or actual) in the sample of the following groups or ions: Styrene Polystyrene

-(-CH-CHtn

H/N02 >

Poly( ni tro:Jt.>1 1·ene) Poly(aminostyrene)

~atalase Insolubilized > : Catalase

Poly(diazostyrene) 21.

nitro -N0 2 (Fig\U"e 2) arylam.ino Ar-NH2 (Figure 2) tin (IV) Sn4+ (Pig\U"e 2) diazonium -N +•N- (Figure 2) azo -N•N- (Section 3.3.5) triazene derivatives -NH-N=N- (Section 3.3.5) phenol Ar-OH (Sectian 3.3.4)

The presence of these groups in varying amounts precludes the use of two methods, namely total ~itrogen estimation according to Kjeldahl52 and that of Wilikly and Coworkers53 • Wilikly's group estimated the protein content of bound peroxidase suspensions by the method of Lowry and Associates54 • Frotein is estimated in this method from the degree of reduction of Folin's reagent induced by amino acid residues such as the phenolic tyrosine residue. The presence of similar groups in the polymeric support would lead to erroneous results. One of the most widely used methods has been acidic hydrolysis of the bound ~rotein foJUo ■ ed by estimation of either the resulting total amino acids by quantitative ninhydrin methods at 570 nm. (e.g. refs. 55 and 56) or the individual amino acids by autoanalyzer57 or valine by quantitative paper chromatography58, or arginine by 28.

quantitative Sakaguchi reaction59 • All these methods are very time consuming. A number of workers have estimated the protein content of conjugates by difference between the protein content of the original coupling solution and the conjug­ ate•s washings. Thase workers have usually relied upon direct absorption at 280 nm.(e.g. refs. 60 and 61). The major objection to this technique is the inconven­ ience and inaccuracy in collecting and measuring the protein concentration in large volumes of the wash solutions. Yagi and Associates62 used an elegant method in which they coupled 1311 labelled protein to diazotised poly(diazostyrene) and then estimated the protein content from the radioactivity of the resulting conjugate. Similarly, in the case of polymer-bound chromoproteins it should be possible to estimate the protein content by radioactivation analysis of the metal contained in the prosthetic group. Clearly then, a need exists for a method of protein estimation which requires only relatively simple equipment but which is convenient and sufficiently rapid to be used on a routine basis. In the present investigation an attempt was made to meet this need with a pyrolysis-gas chromatographic method, standardised reliably by amino acid analysis of the hydrolysate of the bound protein, 29.

1.7 THE SYSTEM TO BE INVESTIGATED

The major objective of the present study was to investigate the effect of insolubilization by poly­ (diazostyrene) on the radiation sensitivity of catalase in a dilute, oxygenated, aqueous environment. To obtain data on the mode of inactivation of catalase in such systems it was necessary to firstly study the kinetics applying in irradiated solutions. The relative protective ability of the insolubilization, was compared to the protection afforded by conventional, radical scavenging and repair processes, utilizing the peptide, diglycylglycine. Finally, the structural properties of the insolubilized catalase were investigated to facilitate interpretation of the effect of the insolubilization on the radiation sensitivity of catalase. 30.

2 EXPERIMENTAL METHODS

2.1 MATERIALS

Catalase from beef liver w~s a lyophilized powder with a nominal activity of 1200 Kiel units per gram (Koch Light, Colnbrook, England). The powder was stored at -20°c and was sampled with the usual precautions afforded enzyme preparations63 • Hydrogen peroxide (30?', Faulding, N.s.w.) was used without further purification. Phosphate buffer (pH7) was 0.014 Min potassium dihydrogen phosphate and 0.012 Min disodium hydrogen phosphate64 • Both salts were dried at 110°0 for 2 hours before use. Purified water for irradiations: Trace organic impurities were removed by double distillation of ordinary distilled water from a mixture of potassium permanganate and potassium. hydroxide (0.01.MWith respect to both) in all-glass apparatus. The distillate was then irradiated for several weeks before use65 • Potassium permanganate (O.Ol Ntor estimation of catalatic activity was prepared by dissolving the solid (0.079g, Univar grade, Ajax Chemicals, N.S.W.) in dis­ tilled water (150 ml.) and boiling gently for 15 minutes. 31.

After cooling and filtration through sintered glass, the filtrate was diluted to volume (250 ml.) with distilled water. Solutions were prepared twice weekly and stored in a dark, glass-stoppered bottle. Porapak Q polystyrene beads (100-120 mesh, unspecified

~ cross linking, Waters Associates, Texas, ~.S.A.) were used in gas chromatography of the products of pyrolysis of insolubilized catalase. Aerated sulphuric acid (0.40 M, 21.) for radiation dosimetry was prepared by dilution of the concentrated acid (44.5 ml.) with purified distilled water. The sol­ ution was oxygenated for several hours using a clean borosilicate glass delivery tube66 • Diglycylglycine gave a single spot after chromato­ graphy on paper strips in a 1-butanol;acetic acid; water (4:2:4 v/v) system. Styrene was purified by distillation in a nitrogen atmosphere under reduced pressure. The glass apparatus contained copper coils to inhibit polymerisation. The fraction distilling at 40°0/17 mm. was retained. R-Cymene was purified by distillation at atmospheric pressure in glass apparatus. The fraction boiling between 177.5°c and 178.5°c was retained. 32.

Benzene for viscosity measurements was purified by sulphuric acid extraction and redistillation. The fraction boiling at 79-81°c was collected. The purified benzene was stored in glass-stoppered bottles and fil­ tered through a sintered glass crucible immediately before use. nii0 1.5008 (found) 1.5011 (ref. 67) In general, analytical grade reagents were used. 33.

2.2 AP~ARATUS

Ultraviolet and visible spectra were obtained with silica cells in:- (i) A Cary Recording spectrophotometer Model lll4, (ii) A Perkin-Elmer Model 137 recording spectrophotometer. Infrared spectra (solid films or halocarbon mulls on rock salt plates) were recorded on a Perkin-Elmer Infracord spectrophotometer. Gas chromatography on pyrolysis products was per­ formed with a Phillips Series 4000 instrument incorpor­ ating a Curie-point pyrolysis unit. Viscosities were determined with Type A British Standard U-tubes (B.S. 188) in a water bath, the tem­ perature of which was controlled to within± 0.02°c. The pH of buffer solutions was determined with either a Pye Model 11086 pH Meter or a Vane Electronic pH Meter Model 203. Elemental Analyses were carried out by the Austral­ ian Microanalytical Service, c.s.I.R.o., Melbourne. Amino acid analyses were performed by Dr. D.c. Shaw of the John Curtin School of Medical Research, the 34.

Australian National University, Canberra, and Mr. R.W. Sleigh, Division of Food Research, c.s.1.R.o., Ryde, N.s.w. Ampoules for irradiation experiments were made from borosilicate glass, (i.d. 7mm.) thoroughly cleaned and rinsed several times with purified distilled water before drying in an oven(~. 100°0). All irradiations were performed in the 60co y-ray facility operated by the School of Chemical Technology, the University of N.s.w. The cylindrical sot.roe, sheathed in stainless steel and having the dimensions 15 cm. x l cm. (o.d.) was normally shielded by a water-filled tank set into the floor of the "cave•. For use, the source was raised through the floor to the centre of a wooden stand con­ taining the samples. The centres of the source and samples were usually about lli inches above the floor of the cave. The irradiation stand shown in Figure 3, enabled irradiations to be performed at 4.0 cm.from the centre of the source. 35.

Yigure ): Stand used in Irradiation Experiments. (Reproduced at approxiamately one third actual size.) • • 37.

2.3 PREPARATION OF INSOLUBILIZED CATALASE

2.3.1 Polystyrene

Polystyrene was prepared by polymerisation in solution. Dry benzoyl peroxide (0.5g) was added in portions with swirling to a mixture of styrene (50 ml.) and a-cymene (50 ml.) in a round bottom flask (250 ml.) to which a reflux condenser was attached. The flask was then partly immersed in a boiling water bath for 24 hours. After the addition of benzene (200 ml.), the re­ action mixture was poured in a thin stream ibto well­ stirred methanol (approx. 31). The polymer immediately precipitated as while curds and stirring was continued for a further 30 minutes before filtration at the pump. The polystyrene was redissolved in benzene (200 ml.) with very gentle heating and the above purification repeated twice. The polystyrene(£!• 28g, 62~ yield) was finally filtered and dried for several days over potassium hydroxide pellets !,a vacuo before sufficient polymer(.£!· lOg) was removed for further purification and characterization. This small sample was lyophilized from a benzene solution and dried for 50 hours at 100°c over fused calcium chloride in an Abderhalden ~istol. 38.

Calculated for Ca Ha: 92.3$ 7.68~H Found .• The infrared spectrum was identical to that of authentic polystyrene (wave lengths of maximum absorption in cm.-1 : 2970, 2860, 1930, 1850, 1795, 1590, 1480, 1440, 1355, 1175, 1145, 1065, 1025, 905, 840, 755 and 700).

2.3.2 Nitration of Polystyrene68 To ice-cold, fuming nitric acid (20 ml.) in a flask equipped with a magnetic stirrer, was added polystyrene (2g) over a period of 5 minutes. The mixture was stirred vigorously in an ice bath for 3 hours and then at room temperature for 45 minutes. The resulting clear yellow solution was poured in a thin stream into stirred, dis­ tilled water (800 ml.) to give cream granules of poly­ (nitrostyrene). After filtering, the product was sucked dry for a few minutes then crushed to a fine powder, dispersed in distilled water (400 ml.) and allowed to stand with occasional stirring for 5 days in the dark. The product was again filtered at the pump and washed with distilled water until the washings were neutral to universal indicator. Finally the product, a pale yellow, hydrophobic, 39. light-sensitive solid, was dried for 3 weeks in the dark under vacuum over potassium hydroxide and sulphuric acid. Yield:2.7g .(93~). All but one of the polymer-bound enzymes which were irradiated were prepared from this sample of poly­ (nitrostyrene). The remaining conjugate (0.018g protein per gram of polymeric support, C/24) was derived from a sample of poly(nitrostyrene), prepared similarly in all respects to the material described above except the nitration period *was only li hours. Yield: 2.2g (77~). In the infrared spectrum, maximum absorption occurred at the following wavelengths (cm.-1 ) : 2980, 1620, 1530, 1460, 1360 and 707. 2.3.3 Reduction of Poly(nitrostyrene) 69 Powdered poly(nitrostyrene) (0.35g) was blended with hydrochloric acid (lON, 3 ml.) in an erlenmeyer flask (100 ml.) fitted with a magnetic stirrer and reflux condenser. Stannous chloride dihydrate (21g) in hot hydrochloric acid (lON, 17 ml.) was then added, with stirring, through the reflux condenser followed by a hydrochloric acid rinse (lON, 10 ml.) The mixture under gentle reflux was slowly stirred for 22 hours to give the reduced polymer as a yellow translucent aggregate. The filtered polymer, after washing with cold

* In the ice b~th. 40. hydrochloric acid (10N, 3 x 50 ml.) until the washings were negative to cacotheline (detects sn2+)?0 was dissolved in hot, dilute hydrochloric acid (1%, 10ml.) to give a yellow solution for diazotisation. In view of its instability in the dry state, part­ icularly to light, poly(aminostyrene) was immediately converted to the diazonium salt without characterization.

2.3.4 Diazotisation of Poly(aminostyrene)

Hydrochloric acid (5M, 15 ml.) was slowly added to the total volume of the solution of poly(aminostyrene), care being taken to minimise aggregation of the precipitate so formed. After cooling in an ice bath for 10 minutes, cold sodium nitrite solution (10~, 5ml.) was added dropwise with stirring to ensure that the temperature did not rise above 5°c. The reaction mixture was then stirred for a further 30 minutes in an ice bath. In some samples excess nitrous acid was destroyed with urea (10~, 5 ml.) but this was found to be unnecessary since the diazonium salt was subsequently washed until the pH of the washings reached 7.

The suspension of diazonium salt was separated by centrifugation, washed with cold distilled water and then cold phosphate buffer (1lH7) until the washings were neutral; this usually required a total volume of the 41. order of 300 ml. The resulting gelatinous, reddish­ orange diazonium salt was coupled to catalase at pH7 without prior isolation.

2.3.5 Reaction Between Catalase and Poly(diazo!tyrene)69 The diazonium salt was suspended in cold phosphate buffer (pH7) (13 ml.) and a solution of catalase (8.20 x l0-5M) in cold phosphate buffer (pH7) pipetted in while stirring. The volume of catalase solution was adjusted to give the nominal enzyme contents shown in Table l. The reaction mixture was stirred magnetically at 3°-5°0 for 15 hours or until the supernatant li4uid gave negative tests for catalatic activity (by effervescence in hy­ drogen peroxide) and protein (by the Biuret method71)*. The crude polymer-enzyme conjugate was separated by centrifugation, washed with cold phosphate buffer (pH7) (5 x 50 ml.) and finally dispersed in the buffer (10 ml.) as a reddish-brown gelatinous solid and stored at 3°-5°c. TABLE l NOMINAL ENZYME CONTENT OF INSOLUBILIZED CATALASE The weight of catalase added to dispersed ~oly(diazo­ styrene) was expressed as a ratio of the weight of poly­ (nitrostyrene) used in each sample.

Sample Number C/24 D/6 C/65 C/74 D/12

Weight Ratio 0.113 0.0555 0.0829 0.0840 0.137 * Control tests on solutions of catalase were positive in each case. 42-

2.4 PROPERTIES OF INSOLUBILIZED CATALASE AND CONSTITUENT POLYMERS

2.4.1 Viscosity Average Molecular Weight of Polystyrene The molecular weight was estimated by viscometry in benzene solution at 30.0°c according to the method given by Sorenson and Campbell7 2• Efflux times were determined for the following concentrations of polystyrene: 0.2064~ w/v, o.3016~ w;v, o.3916~ w;v, 0.8484~ w;v

£.:.i:.2 Tests on Poly(diazostyrene) Aqueous suspensions of poly(diazostyrene) were tested for pH (with universal indicator) and diazonium groups (with 10~ 2-naphthol in dilute sodium hydroxide solution73 ) at the time of preparation and after standing for 2 weeks at room temperature. The thermal stability of diazonium groups was investigated by applying the 2- naphthol test at intervals while boiling a suspension (0.05~) in dilute aqueous hydrochloric acid (O.Oll1). 2.4.3 Amino Acid Composition

Free or bound catalase (£!• 6mg. dry weight) was subjected to amino acid analysis by the method of Moore and Stein74 involving hydrolysis and chromatography on a sulphonated polystyrene ion-exchange resin. Tryptophan was not estimated. 43.

2.4.4 Estimation of Bound Catalase by Ion-Exchange Chromatography The amount of bound catalase in the polymer-enzyme conjugate was estimated from the molar quantity of an "indicator" amino acid found in the hydrolysate from each conjugate. The indicator amino acid was chosen such that its concentration was constant in the hydrolysate from both free and bound catalase. Thus, with a knowledge of the amino acid composition of free catalase (Section 2.4.3), the molar quantity and hence the weight of ~rotein in the sample could be calculated.

2.4.5 Estimation of Bound Catalase by Pyrolysis Gas Chromatography

Dried particles of the insolubilized enzyme were clamped in a wire loop and inserted in the pyrolysis chamber. The products of pyrolysis were separated and estimated by gas chromatography. The ratio of the area of a peak arising from catalase to that of a peak from the polymer was investigated at varying enzyme contents in order to calibrate the method against the results of catalase estimations by ion-exchange chromatography (Section 2.4.4). 44.

The pyrolytic products were successfully chromato­ graphed under the following conditions:

Column 6 feet x 1/8 inch aluminium column packed with Porapak Q crosslinked polystyrene beads (100-120 mesh). Carrier Gas Nitrogen. Flow Rate 60 ± 1 ml. per minute measured at 50°c. Pyrolysis Conditions 770°0 for 10 seconds. Colu.m.n Temperature Programmed from 50°c to 230°0 at 10°c per minute. Chart Speed 20 mm. per minute.

2.4.6 Pyrolysis Experiments on Reference Samples of Insolubilized Catalase Samples of insolubilized catalase were prepared by adding known amounts of catalase to identical weights of poly(diazostyrene) (0.0346g; obtained as the weight of the washed, dried sediment from one of the identical samples). The method of preparation of the resulting conjugates (reference conjugates) was as outlined in Section 2.3. However, after reaction between the enzyme and poly(diazo­ styrene) the crude conjugates were evaporated to dryness in such a way that no catalase was lost. The dry product 45.

was pyrolyzed, the area ratio determined in the usual way (Section 2.4.5) and attempts made to correlate it with the weight ratio of added catalase. The weight ratio, xr is shown in Table 2.

TABLE 2 The Catalase Content of Reference Saaples for Pyrolysis

Weight of Catalasa (g) Weight Ratio, Xz., of C3talase to Poly(diazostyrene) (based on 0.0346g of polymer per sample).

0.0030 0.0867 0.0040 0.116 0.0050 0.145 0.0060 0.173

2.4.7 The Solids Content of SusP,_!nsions of Bound Catalase The weight of insoluble catalase in each sample was determined to facilitate the interpretation of the results of irradiations. An aliquot (0.60 ml.) of the stirred conjugate suspension was transferred to a tared sintered glass crucible, washed with distilled water (5 x 40 ml.) to remove phosphate salts and dried in an oven (100°c) for l¼ hours before cooling in a desiccator and weighing. With one exception, the results were determined in duplicate and a mean taken. 46.

2.5 ESTIMATION OF CATALATIC ACTIVITY

A slightly modified version of the method of Bonnichsen, Chance and Theore1175 was used to determine the activit:i.eJof both free and bound enzyme. The initial concentration of hydrogen peroxide was determined as follows: Hydrogen peroxide solution (0.25N, 2.00 ml.) was pipetted into phosphate buffer (pH7, 50.0 ml.). The resulting buffered hydrogen peroxide (2.00 ml.), the "substrate solution", was pipetted into dilute sulphuric acid (2~, 2 ml.) and titrated with potassium permanganate (O.OlN) from a burette which allowed estimations of 0.01 ml. This was referred to as the "initial titration" and was usually performed in duplicate. Subsequent concentrations of hydrogen peroxide were determined as follows: The catalase solution (or suspen­ sion) (£!• 1/ M) was transferred to a small watchglass with a special wide-mouthed micropipette (capacity::!• 30 f-1• - the exact volume being unimportant as only the relative enzymic activity was required and this was achieved by using the same micropipette for all samples in a given experimant). The watchglass was dropped into this solution and a stopwatch started sirml.taneously. While still swirling gently, a sample was withdramwith 47. a wide-tipped pipette (2.0 ml.) and the aliquot blown into sulphuric acid (2~, 2 ml.). The time (to the nearest second) at which the last of the aliquot left the pipette was noted as the time of deliver3 • This sample withdrawal was rapidly repeated twice more with two clean, dry, wide-tipped pipettes. The aim was to transfer 3 samples within about 45 seconds from the start of the reaction. This was achieved with practice. The acidified samples were held in an ice bath until titrations against potassium permanganate (O.OlN) were completed. (Usually a period of about 20 minutes elapsed from the start of the reaction to the completion of the last titration but longer periods did not cause significant changes). The temperature of the substrate solution was meas­ ured but the slight variations found did not cause significant changes in activity. (Activity increases bi a factor of 1.1 for each 10°c rise76). The activities of irradiated catalase samples relative to a control sample were estimated by comparison of the ratesof destruction of hydrogen peroxide. Since the concentrations of permanganate and peroxide stock 48. solutions were constant for each set of results, the volume of permanganate used was proportional to the peroxide concentration. Hence the relative enzymic activity of each sample was estimated from the initial slope* of a plot of permanganate titration volume (ml.) against time (minutes). When this method was first applied to polymer- bound catalase a blank titration was also performed by adding the conjugate suspension(£!• 10 /"l.) and substrate solution (2.0 ml.) (in that order) to the sulphuric acid. However, results indicated that this was unnecessary and it was deleted from later assays. The possibility of interference by diglyoylglycine in the assay for catalatic activity was tested as follows: A solution of catalase (1.47 mg.) in phosphate buffer (pH7, 0.50 ml.) was mixed with a solution (0.50 ml.) of the peptide (38.6 mg.) in phosphate buffer and assayed by the usual method. A blank was prepared by substit­ uting phosphate buffer for diglycylglycine solution. The results are reported in Appendix 1.2.4.

* In the case of slight curves, the slope was estimated from the tangent drawn by the "mirror method"77 • 49.

2.6 THE EFFECTS OF r:-RA.DIATION

2.6.l Dosimetry

The absorbed dose (in krads) was measured with the Fricke dosimeter by observing the change in absorbance due to the radiolytic oxidation of ferrous to ferric ions and reading the absorbed dose from a calibration curve. The method is applicable to the range, 4-40 krads of absorbed dose66 • "Analar" ferric ammonium sulphate dodecahydrate (0.100 K) in sulphuric acid (0.4 M) was used to prepare the calibration curve65 • Known volumes (measured by mioroburette) of the ferric ion solution (0.10 ml., 0.20 ml., 0.40 ml.) were diluted to 100.0 ml. with sulphuric acid (0.4 M) and the absorbance read at 305 nm. in 0.250 cm. silica cells. The calibration curve was constructed in terms of absorbanoe versus absorbed dose (krads) since66

Dose (rads) • 60.9 [ :Pe 3•] where the ferric concentration is M. This calculation aakes use of the density of the dosimetric solution (1.02 g/ml.) and the yield of ferric ions produced by 60co r-rays (G • 15.5 molecules/100 eV.). The data are shown in Table 3 and Figure 4. 50.

TABLE 3 CALIBRATION OF THE FRICKE DOSIMETER (In every case, the absorbance was read at 19.0°c)

Molar Concentration Equivalent Absorbance of Fe3+ Dose (krads) (at 305 nm.)

0.100 X 10-3 6.09 0.082 0.200 X 10-3 12.2 0.125 0.400 X 10-3 24.4 0.200 - For estimations of the absorbed dose, aliquots (1.00 ml.) of the dosimetric solution (7.56 x 10-4 M in ferrous ammonium sulphate hexah.ydrate and 9.76 x 10-4M in sodium chloride, dissolved in oxygenated 0.1 M sulphuric acid) were pipetted into dry glass ampoules * (1.d. 7 mm.) which were sealed in the flame. Samples were then irradiated in duplicate at 4.0 cm. from the source for accurately known periods. The absorption of the irradiated samples was determined at 305 nm. and the absorbed dose estimated from the calibration curve.

* Theae tubes were used because they were identical to the ampoules in which catalase samples were irradiated. 51 .

0.300

u.200

Absorbance (at 305 nm) 0.100

().l)00..______. ______

,) .oo 10.0 20.0 30.0

Dose (kr.<:td.3)

!!~re _i.:.-~ill!~!2!!.-2f _E_~.-!!!£~~~~~~~£.:.._Y~~~ll2~ --of the------Absorbance of Fe 3+ at 305 nm. with absorbed Dose 52-

2.6.2 Ir!:!~ioo of Catalase and its Conjusates

An aliquot (0.50 ml.) of a solution of free catalase in phos~hate buffer (pH7) or an aliquot (0.60 ml.) of stirred suspension of bound catalase in phosphate buffer (pH7) was transferred with a wide-tipped pipette to a borosilicate ampoule (i.d. 7 mm.). The sample was diluted to 1.00 rnl. with phosphate buffer (pH7) and oxygenated through a fine capillary (ea.- 0.05 mm. bore, 5 bubbles per second) for not less than 5 minutes. The ampoule was quickly sealed in the flame, care being taken to ensure that the sample at the bottom of the tube was not heated. 'rhe samples were irradiated at 15 ± 2°c and 4 .o cm. from the source for carefully measured periods. After irradiation, the samples were placed in a refrigerator (3°-5°c) within 5 minutes of being removed from the source and held for 24 ~ 4 hours.* * This treatment served as a precaution against the

possibility of an irradiation "after-effect 11 4 - it ensured that samples removed from the source at different times received a relatively constant after-effect. The length of the procedure for assay of catalatic activity necessitated this approach. 53.

The ampoules were then opened, shaken, a sample (a constant volume ,2!• 30 fl.) removed and assayed for catalatic activity (Section 2.5). A blank was performed on a sample held at 15 ~ 2°c away from the radiation source but subjected in all other respects to the treatment given the irradiated samples.

2.6.3 Cha,!!!es in Amino Acid Composition of Irradiated Catalase A solution of free catalase (1.50 mg. per ml., 0.60 ml.) in phosphate buffer (pH7) was irradiated as described in Section 2.6.2 and then quantitatively transferred with phoaphate buffer to a borosilicate glass tube for hydrolysis and ion-exchange chromatography according to the method of Moore and Stein74• Tryptophan was not estimated. An unirradiated control sample of identical concen­ tration was treated similarly.

2.6.4 Irradiation of Catalase-Diglycylglycine Solutions Stock solutions of diglycylglycine (0.50 ml.) and catalase (0.50 ml.) each in phosphate buffer (pH7), ware mixed to give a solution containing the desired amount of catalase and 20.0 mg. per ml. (0.106 M) of diglycylglycine. Tne solution was oxygenated and irradiated as described in Section 2.6.2. 54.

3 DISCUSSION OF RESULTS

3.1 THE RADIOLYTIC INACTIVATION OF CATA.LASE IN SOLUTION

In this section the data given in Appendix 1.2 will be reduced to more meaningful proportions in preparation for a consideration of the possible modes of inactivation of catalase (Section 3.2).

3.1.1 Variations in n37 ~ The results of the radiolysis of free catalase in solution (with and without diglycylglycine) have been detailed in Appendix 1.2 and are summarized in Tables 4 and 5 and Figures 5 and 6. It is clear that the curves of residual activity against dose (on a "semi­ log'' plot) are sufficiently linear to allow the calculation of n37doses. Further, when n37 is plotted against the enzyme concentration (Figure 7) the curve is similar to that described in Figure (1) (c) and hence consistent with the empirical equation (Equation 7, Section 1.4.1):

n37 =(A+ F) E0 +BP+ C The values of the terms, A,B,C and F can be estimated from Figure 7. 55.

TABLE 4

The D37 Dose of Irradiated Solutions of Catalas• Based on the data of Appendix 1.2.2 and ligure 5.

Catalas• Concent6ation (K X 10) 6.27 2.92 1.44 0.758 0.184

D37 Dose 445 280 215 245 160 (krads)

TABLE 5 The D37 Dose of Irradiated Oatala!e-Diglycylglycine Solutions Based on the data of Appendix 1.2.4 and Figure 6. The concentration of diglycylglycine was 20.0 mg./ml. ( 0.106 14).

Catalaae Concentration (K X 106) 6.15

D37 Dos• (krads) 1060 620 385

Prom Figure 7, • + p - 143.35 X 106 lcrad 1(-l and A - 48-75 X 106 krad .-l ••• p - 94.6 X 106 krad .-l ilso, as P - 0.106 K

krad krad

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Uatalase Uatalase

M M

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2.92 2.92

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for for

(%) (%)

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Residual Residual Ac Ac

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I I

l2ou l2ou

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~ctivity ~ctivity Residual Residual

. .

VI VI

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-6 -6

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x x

(.;atalase (.;atalase

....---

......

(~i-..da) (~i-..da)

0.184 0.184

ue,~o ue,~o

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(e (e

o·---___, o·---___,

10 10

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5v 5v 60 60

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6

Activity Activity

ResiJu.a.l ResiJu.a.l

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(Kr!:i.ds) (Kr!:i.ds)

0.758 0.758

l>ose l>ose

(d) (d)

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(~ontinued) (~ontinued)

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10 10

80 80

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60 60 40 40

90 90

70 70

100 100

t.>' t.>'

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Catalase Catalase

ti ti

M M

(~) (~)

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400 400

(krads) (krads)

1.44 1.44

Dose Dose

(c) (c)

\ \

t t

10 10

20 20

30 30

60 60

50 50

40 40 70 70

80 80

90 90

100 100

(~) (~)

Activity Activity Residual Residual

• •

CX> CX>

V1 V1

Uatalase Uatalase

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' '

M M

(krgds) (krgds)

10-

x x

Dose Dose

2.78 2.78

(b) (b)

I-

ty ty

30 30

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Acti'1i Acti'1i

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Catalase Catalase

M M

(krads) (krads)

6 6

~~!!2!!~, ~~!!2!!~,

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Dose Dose

x x

~lli~L~!!!i!l-=-~~~~£~!~-f~!-~!~~~=£.!!!l£i±~l£!1!. ~lli~L~!!!i!l-=-~~~~£~!~-f~!-~!~~~=£.!!!l£i±~l£!1!.

6.15 6.15

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Activity Activity Residual Residual 59.

90 ju

7tJ 60 5J

40 He:1idual Activit.v ~) ( 30

20

V 400 800

i)o.3e ( kraJ!-3)

(c) 1.4d x 10-6 M Catal~sa 60.

1000

Boo D37 Dose (krads)

600

400 , I I / G) 200

0 0 2.00 4.00 6.00 6 Catalase Concentration (M x 10 )

a Gatalase Solution A Catalase - (Gly)J (0.1J6M) Soluti,:rn

Q~~1: Y~E~.'.;!~~E!.2f~ 37 .Do~~-~!~ th~9~~_£~ntr!:~l2.!! of Catalase in Solution. and from Figure 7,

BP + 0 • 180 krads and C • 145 krads then B • 331 kradslt\1 To pnmmar~ze, the exponential nature of the residual activity - dose curves and the linear variation of the D37 dose with catalase concentration are compatible with catalase being inactivated by radiation-produced solvent radicals. For the most part, these radicals are just as likely to•act with active catalase molecules as with inactive catalase molecules. The results, being consistent with equation (7), also indicate that, al­ though diglycylglyoine acts to some extent as a simple radical scavenger during its protection of catalase, an appreciable proportion of this effect can be explained by aasUD1ing the repair of inactive catalase molecules by the peptide. J.1.2 The Protection Quotient Before calculating the value of the protection quotient, Q (Section 1.4.2) allowance should be made for the dose absorbed by the oxygenated buffer, 145 krads (Section 3.1.1) This is best accomplished by subtracting this figure from the values of DP and De in the expression for Q (equation 8). The values so obtained will be referred to as corrected Q values,

QC (Table 6).

TABLE 6 Calculation of the Protection Quotient (Diglycylglycine concentration 0.106 M)

Eo De Corrected DP Corrected QC (mg./m.l.) (krads) De (krads) (krads) DP (krads) a a a,b a,c a,b a

1.53 445 300 1080 935 0.158 0.71 280 135 595 450 0.0775 0.35 215 70 380 235 0.0412 0.185 245 100 285 140 0.00370 0.045 160 15 205 60 0.00675 a Symbols: E0 is the catalase concentration, De is the D37 dose in the absence of diglycylglycine, DP is the D37 dose in the presence of diglycylglycine, QC is the corrected protection quotient. b Corrected by subtracting 145 krads from the figures in the preceding coluans (see Section 3.1.2).

C These figures have been read from the curve of D37 versus catalase concentration (Pigure 7) at concentrat­ ions identical to those used for the determination of 63.

Table 6 Continued

De• This procedure was adopted as catalase solutions were prepared daily and hence solutions with slightly different catalase concentrations were used for radiolysis in the presence and absence of diglycylglycine

Figure 8 shows that Qc is related to E0 by the empirical expression,

(11) where K is a constant~ But in view of equation (l0)(Section 1.4.2),

1 = Q (10) it is interesting to consider the relationship between the reciprocals of Qc and E0 • Thus by dividing both sides of equation (11) by Q~ and rearranging, one finds that,

= (12)

Since the results of Figure 8 are therefore consistent with equation (12) and not with equation (10) it appears (in agreement with the conclusions of Section 3.1.1) that the protection afforded catalase by diglycylglycine is not due to free-radical competition alone. 64.

0.160

0.120 Corrected Protection Quotient

·~C 0.080

0.040

0 0.400 o.eoo 1.20 1.60

C:at.'ilase Concentri:ition (mg./ml.)

f~~E~-~: Variation of_the_Corrected_Protection Quotient -----with Catalase------Concentration, 65.

3.1.3 Coaparison with Published Work

ilthough the influence ot diglycylglycine on the aenait1vity of catalase to {-radiation baa not been studied previously, its effect has been studied under x-radiation78 • A solution of lyophilized catalase powder (8 x 10-8 M) in 0.05 K phosphate buffer (pH 7.0), was x-irradiated at room temperature with a dose rate ot 9 x 104r/minute (ea. 5.3 x 103 krad/hour). The catalase solution contained- diglycylglyoine at concentrations ranging from Oto 5 mg./m1. Unfortunately it was not specified whether the system was aerated or not; however, in view of the fact that it was not stated and that Luoite vessels were used it is probable that the samples were neither de-aerated nor sealed. In general, the dose-inactivation curves were not exponential so the kinetic analysis used in Section 3.1.1 cannot be applied. !he authors stated that diglycylglycine acts as a radical scavenger but produced only meagre evidence to support this; they claimed that a radical scavenging mechanism operates since the D50 dose (1.e. the dose required to produce 50~ residual activity ot the enzyme) increases with diglycylglycine concentration to a limiting value. Thus in contrast to the previous study, the conclusions ot Sections 3.1.1 and 3.1.2 66.

derived from carefully controlled experimental conditions and kinetic examinations give a firm. basis !or attribut­ ing the protection of catalase by diglycylglycine to radical scavenging but also demonstrate the importance of repair processes.

3.1.4 The Inactivation Yield It is clearly established that the G value, partic­ ularly for the inactivation of catalase, is dependent on such factors as the concentration of catalase and any iapurities, the type of irradiation used and the dose rate 79 , 80• Consequently, coapariaon of published G Yalues for catalase is difficult. In addition, the problem is further complicated by the fact that different methods of calculation {with different degrees of pre­ cision) have been used in the calculation of the G values; however, the published figures do represent the order of magnitude of the G value. Thus, 0.044 x 106 M catalase solutions in aerated phosphate buffer {0.125 M, pH 7.9) when irradiated with 6000 trays at a dose rate ot 63.4 krads/hour gave an inactivation yield of 1.7 x 10-3 moleoulea/100 eV. (ref. 81). This value was calculated from the slope of an inactivation-dose curve which obeyed first order kinetics. 67.

It has been shown previously (Section 1.4.1, equation 4) that the G value can be calculated from the following formula when the residual activity dose curves are exponential: -1 -1 G moles l rad *

= ( ) This equation is very similar to that used by Hopkins and Spikea81 and will serve to illustrate the difficulties in estimating G which were discussed in the preceding paragraph. Table 7 and Figure 9 summarize the G values found at different catalase concentrations.

TABLE 7 The Variation of Inactivation Yield with Catalase Conceniration

Catalase Concentration,E0 (M x 106)(from Table 4) 6.27 0.18

D37 (krads)(from Table 4) 445 280 215 245 160

Dose Rate (krads/hour) (from Appendix 1.2) 137 137 158 138 138

G (aolecules/100 eV.) 10.1 6-45 3.00 1.08 X 103

* D37 is expressed here in rads - not krads as elsewhere in this thesis. 68.

G (Molecules/100 eV.) X 103

8.00

4.00

0 0 2.00 4.00 6.00 Catalase Concentration ( M x 10)6 -

flg~Ll= y~~at~.£E!-~!~~!;_~B~ctiv~ti£~~IieldL_QL_~~!h ------Cat~lase Concentration, 69-

The agreement with the G value obtained by Hopkins and Spikes, at lower concentrations is reasonable in view of the much lower dose rate used in their investigat­ ion (see Section 3.2.2). However, a more precise result minimising any interference from buffer constituents (Section 1.4.1) is obtained by inverting the slope (A) of the graph of D37 versus catalase concentration (ligure 7). In this way G has the value 19.8 x 103 molecules/ 100 eV. The variation from the values in Table 7 is due to the dose absorbed by the oxygenated phosphate buffer (145 krads from Figure 7). In other words the results illustrate the dilution effect (Section 1.4.3) and provide further evidence for the conclusion that free-radical competition is at least partly responsible for the inactivation and protection of catalase in aqueous buffer in the presence of diglycylglycine.

3.1.5 Yield for Degradati~ of Diglycylglycine In triply distilled water, the G value for chemical changes in peptides is usually low(~. 5 molecules/ 100 eV.) (rats. 82,83) the major portion of which is accounted for by reactions of the terminal amino group and the pe~tide bond(s). In fact, the G value for peptide bond rupture of a 0.05 M diglycylglycine solution 10.

in triply-distilled water after 60co y-irradiation, was reported as 1.75 molecu1es/100 eV. (ref. 84). It has been shown (Section 1.4.1) that the G value for degradation of diglycylglycine can be deter­

mined from the ordinate intercept when the n37 dose is plotted against catalase concentration. Accordingly, since B • 331 krad M-l (Section 3.1.1) G for (Gly)3 • 2.92 x 103 molecules/100 eV. This yield is rather large and indicates that diglycylglycine is involved in a chain reaction probably with phosphate buffer85 • It has been shown (Section 3.1.1) that the oxygenated phosphate buffer in the present experiments absorbed 145 krads and it is not unreasonable to assume that the radicals produced in this way could degrade diglycylglycine. Previous workers have noted the enhancement of radiation effects in phosphate buffer and the 0P031t radical ion has been identified by E.S.R. studies during the radiolytic conversion of dihydrouracil to uracil in phosphate buffer86; pertinently, the rate constant for the re­ action of e;4 with the H2Po4 ion is quite high (6.0 x 109 x-1 sec.-1 : Appendix 2.2). 71.

The possibility of the G value of diglycylglycine being affected by interference by this peptide with the assay method for catalase, has been eliminated by experiment (Appendix 1.2.4). (This is indirectly supp­ orted by the fact that the related compound glycine has no effect on catalatic activity87 ).

3.1.6 Swmarz

It can be concluded that the indirect effect is the method by which catalase is inactivated after radiolysis in oxygenated aqueous solution. ilthough previous workers78 have claimed that catalase can be solely protected by the radical scavenging action of diglycylglycine under these conditions, the present work indicates that this is only partly true, repair of inactivated catalase molecules by diglycylglycine also being involved in the process. These conclusions will prove useful in the consideration ot the radiolytio inactivation of insolubilized oatalase (Section 3.4). 12.

3. 2 SPECIES RES?O:fSIBLE FOR 1rHE R.ADIOLTric IHAC'fIVATION OF cITALASE IN SOLUTION

The processes of radical scavenging and repair can be explained in terms of the reactivity of several s~ecies present in the irradiated solutions.

3.2.1 Primary Radical Products other than Q!!. The primary radical products formed by radiolysis of uilute aqueous solutions have been described (Section 1.3). Pulse radiolysis of deaerated aqueous solutions of catalase (.£!:· 1 /'Min catalase, 2 x 10-3M in Na2HP04*) enabled the reactivity of e-aq with catalase to be estimated88 • The rate constant determined from the decay of the characteristic absorption of e aq at 720 nm. was 3.7 x 109M- 1 sec.-1 (ref. 88). In oxygenated solutions (as in the present study) it is ~robable that reaction between catalase and e-aq will be negligible in view of the fast reaction between e-aq and oxygen:

e aq +

k = X 1010 M-1 sec.-1 (See Appendix 2.2)

Furthermore, reactions between e aq and catalase will only become significant when oxygen concentration has

* there w~s no detectable reaction between either e 8(! or 0:1• and Hl04 ions. 73.

fallen to extraordinarily low levels since the solubility of oxygen in water at 20°c is 1306 x 10-6 M. (ref. 89) and the cat~lase concentration used here ranged from 2!·

(0.2 - 6) X 10-6 M. The conjugate base of the perbydrox.yl radical o2, formed for example by reaction between oxygen and e-aq , decomposes as follows90 :

+

The product H02 is the conjugate base of hydrogen peroxide (pKa 11.62; ref. 67) which would be the species formed in neutral solutions:

+ The reactions of o2 with organic molecules have not been thoroughly investigated but appear to be generally alow10•12 and hence the disproportionation should prevai190 (particularly in neutral solutions). This general conclusion is supported by the results of an investigation into the kinetics of the x-ray induced inactivation of catalase in aerated, neutral aqueous solutions which showed that o2 is only of minor importance (if any) in the inactivation of catalase. Consequently o2 will not be given further consideration but the role of hydrogen peroxide will be discussed in detail in the next section. 74.

It has been shown92 that H• atoms do not inactivate oatalase in dilute aqueous solution. Indeed, in oxygenated solutions H• is quickly scavenged:

0 HO• + 2 ~ 2 k • 2 x 1010 •-l sec.-1 (See Appendix 2.2)

The resulting perhydroxyl radical readily dissociates to form. its conjugate base in neutral solutions90 • +

Thus, it is clear that of the primary radical pro­ ducts produced in oxygenated irradiated aqueous solutions, both e;q and H atoas are converted by oxygen to the relatively unreactive perhydroxyl radical and its conjugate base. It is significant that this latter species is expected to form. hydrogen peroxide, the sub­ strate tor the catalatic reaction, and,as indicated below (Section 3.2.2), an.effective scavenger of e;q. 3.2.2 Hydrogen Peroxide

Only two workers have systematically investigated the effect of hydrogen peroxide on the sensitivity of catalaae to radiation. Sutton91 found that catalase was protected from x-ray induced inactivation in neutral, aerated, aqueous solution by an intermediate with a 75. lifet1ae similar to that of eydrogen peroxide. He pursued this problem by adding hydrogen peroxide to the catalase solution during the irradiation and found that the rate of radiolytic inactivation of catalase varied inversely with the equilibriwa concentration of hydrogen peroxide. ~urther analysis93 showed that the protective efteot of hydrogen peroxide was explained partly by complex formation between it and the iron ato11S in the haem groups and partly by some other aechanisa possibly radical competition between catalase and hydrogen peroxide. This work was extended by Magdon80 who x-irradiated aqueous solutions of catalase in phosphate buffer (pH 7.6). He found that appreciable amounts of hydrogen peroxide were only formed in irradiated oxygenated solutions and that the yield of hydrogen peroxide increased linearly with dose rate. Magdon also found, in agreement with Sutton9l,9J, that hydrogen peroxide protects catalase from the effects of irradiation and confirmed the inter­ dependence of dose rate, oxygen and hydrogen peroxide concentrations on the radiationsensitivity of catalase. Thus he showed that the ionic yield for inactivation of catalase was constant with increasing dose rate in deoxygenated solution but decreased over a fourfold range in oxygenated solution. In particular, it was ahown 76. that oxygen became increasingly protective as the dose rate was increased above 3.27 krads/hour to 131 krads/ hour, the maxiJLWR rate investigated. ilthough 0.rays were used here, the latter value is similar to the dose rates used in the present study and hence it is expected that the oxygen converted to hydrogen peroxide on irradiation would have a protective influence on catalase. Kagdon attributed the protective effect to the ability of complexes formed by hydrogen peroxide with the iron centres of catalase to scavenge and deactivate oxidizing radicals. Hydrogen peroxide itself also reacts rapidly with e -aq:

+

and other things being equal should prove al ■ost as effect­ ive as oxygen in scavenging e;q (Section 3.2.1). Thus, in the present experiments it might be expect­ ed that an appreciable fraction of the oxygen originally present would be converted to hydrogen peroxide which in turn would protect catalase from radiolytic inactivation. 77.

3.2.3 1:1,Ydroyl Radicals

Although little direct evidence is available for the effect of the hydroxyl radical on catalase in aqueous solution, a large number of results can only be consist­ ently and ~lausibly explained in teras of the reactivity of the hydroxyl radical. Henglein and Coworkers88 found that the rate con­ stant for the reaction of oa• with catalase in deoxygen­ ated aqueous solution of pH ,!!!•9 was 8.3 x 1010 M-1 sec.-1 i.e. twenty times that of e;q with catalaae. Sutton92 found qualitatively that saturation of aqueous solutions of catalase with hydrogen gas to remove oH• (ref. 11) had a marked protective effect when the solutions were irradiated. However, saturation with oxygen which removes H• and •;q had a much smaller protective effect. Thus it is reasonable to assume that oa• is the radical aainly responsible for inactivation of catalase.

3.2.4 The Sites of Radical Attack on Catalase

The results of the present study make possible a correlation between changes in the amino acid residues and the activity of irradiated catalase. Thus, catalase (6.2 x 10-6 M) wast-irradiated in oxygenated aqueous solution with a total dose (557 krads) slightly in excess of the n37 dose C!:,!• 445 krads). The results are 78.

TABLE 8 Radiation-Induced Changes in the Amino Acid Composition ot Catalase (Residues in irradiated samples expressed as a percent­ age of those in unirradiated control samples).

Residue Shimazu and Tappe194 This study 105 rads 106 rads (Appendix 1.2.3) 5.57 X 105 rads Methionine 23 15 142 - Cyatine 38 o.o 60a Phenylalanine 47 32 107 Histidine 66 53 118 Isoleucine 66 50 101 Leucine 77 69 l.03 Lysine 77 72 91 Valine 79 76 98 Glutamic acid 86 82 100 Serine 90 47 97 A.spartic acid 90 70 103 Tyrosine 93 64 131 Threonine 99 46 70 Glycine 101 80 103 A.rginine 104 93 94 Alanine 121 91 99 Proline - 111 a A3 i (Cystine) 79.

shown in Table 8 and when considered in the context of the l°" accuracy of the results it is apparent that a dose which reduces the activity of catalase to roughly one third of its initial value caused an appreciable change to only the half-cystine content of the enzyme. (The results for methionine, tyrosine and threonine in Table 8 are outside the expected order of accuracy and a9pear to be spurious because of erroneous values for each of these residues in the analysis of the control sample. The results for free catalase in Appendix 1.1.3, to which the control sample from Appendix 1.2.3 has been compared, appear to be more reliable mainly because of the greater nwaber of replications performed, but also, in the case of methionine, because of the supplementary estimation of methionine sulphone. The difficulty of accurately estimating, by ion-exchange chromatography, methionine, tyrosine and tbreonine in protein hydrolysates is widely reoognised74 ). Thus it appears that the cystine and cysteine residues, have a marked influence on the oatalatic activity. Recent work by two groups of investigators has indicated that these residues are indeed implicated in the catalatic activity but the details of the mechanism are still subject to disagreeaent95 , 96• 80.

It is pertinent to compare the results of the present study with published material. The literature on this topic can be classified into two groups: (a) Radiation-induced changes to the protein portion of catalase as evidenced by either, absorption spectroscopy of the enzyme, or amino acid analysis of its hydrolysate. (b) Radiation-induced changes to the haem pros­ thetic groups of catalase as demonstrated by ultra-violet/visible absorption spectroscopy. Shimazu and Tappe194 found marked changes to the protein portions of catalase (4 x 10-6 M) had occurred after (-irradiation initially at pH7 in aqueous solution under an atmosphere of nitrogen. The solutions were not buffered and neither the residual activity of the irradiated catalase nor the dose rate was .determined. After irradiation, the enzyme was hydrolyzed with hydrochloric acid and the products quantitatively estimated by paper chromatography. The proportion of each residue left after irradiation is shown in Table 8. The listing is in decreasing order of !ability at 105 rads. Although it can be seen that this order changes at 106 rads, the most labile residues appear to be 81. methionine, cystine,* histidine, phenylalanine and isoleucine. It is apparent that Snimazu and Tappel have rP.co!'ded gross changes in the amino acid residues of catalase. In contrast to Shimazu and Tappel's work, catalase was irradiated here in oxygenated phosphate buffer and the difference in effect of similar radiation doses (e.g. the results for,£!:• 105 rads) is assumed to be due to the protective influence of oxygen discussed earlier (Section 3.2.1). The failure of Shimazu and Tappel to reyort a dose rate will not invalidate this comparison since Magdon80 has shown that the dose rate only influences the inactivation of catalase in the presence of oxygen. Consec1uently it appears most likely that the changes in the amino acid residues of catalase when irradiated under deaerated conditions are, in contrast to the dominant role of OH" in aerated solution (Section 3.2.3), caused by a number of the primary radical products of irradiated water. As discussed in Section 1.3, information on the reactivity of amino acids is useful in the prediction of the reactivity of the corresponding residues in the protein. Further study of Section 1.3 will reveal * Although the chromatographic method97 used was capable of resolving cystine and cysteine, the latter compound was not mentioned in connection with the irradiation of catalase9~. 82.

that the most reactive amino acids with OH• are for the most part included in the list of the most labile residues from Shimazu and Tappel's work. Similar correlations apply for the other electrophilic radical, a·, tryptophan, cystine, tyrosine, methionine and phenylalanine being most reactive98 while for the nucleophilic e; 4, the most reactive residues would be assumed to be cystine and cysteine, followed by phenylalanine, tyrosine, tryptophan, arginine and histidine99 • Cysteine and tryptophan were evidently not estimated in Shi:mazu and Tappel's work but of the remaining residues, the notable exceptions from the list of labile residues are tyrosine and arginine; the fact that most of the tyrosine residues are sterically hindered, being in the interior of the catalase molecule 100 , is consistent with these results. However, at doses in excess of 106 rads, tyrosine was degraded readily94 presumably after gross conformational changes in the catalase exposed more of the tyrosine residues to the radiation-produced radicals. It will be shown in Section 3.3.5, that arginine is also unexpectedly inert in reactions with diazonium salts, the two results, possibly indicating that most of the arginine residues, too, are in the interior of the catalase molecule. 83.

The literature also contains two cases in which radiation dam.age to the catalase protein was demonstrated by ultra-violet/visible absorption spectroscopy93,lOl. Although in both cases, the activity of the enzyme was significantly decreased, it was not possible to draw an unequivocal conclusion regarding the influence of changes in protein structure on catalatic activity since there was simultaneous dam.age to the haem groups. However when hydrogen cyanide was used as a protector during x-irradiation in aerated aqueous solution93, it was possible to demonstrate that the catalatic activity was more sensitive to radiation-induced changes in the haem groups than in the protein. This can be ration­ alised in terms of attack by the electrophilic oH• radical as porphyrins in general react readily with electrophiles102• Other studies103•104 have demonstrated the susceptibility of porphyrins in aqueous solution to radiation-induced oxidation (as would be caused by oH• radicals). Thus it is apparent that the most important sites for oH• radical attack on catalase in aerated solutions are the haem prosthetic groups. The results of this investigation also now show that either or both of

cys11i.ne and cystine are the most likely points of initial attack in the protein portion of the molecule and in view of the foregoing discussion, this can now be reconciled with attacK by the OH• radical. 85.

3.3 THE PROPBRTIES OF FOLYMER BO~ CATALASE

The prime objective of this discussion is to lay the foundation for a subsequent consideration of the effects of insolubilization on the radiation sensitivity of catalase (Section 3.4). It is therefore opportune to look at methods of determing the protein content of insolubilized enzymes as well as methods for predict­ ing the composition of a conjugate prepared by a known procedure from a given ratio of protein to polymeric carrier. Other aspects germane to the objective stated above include investigation of the type of bonding between the enzyme and the ~olymer as well as the overall structure of the insolubilized enzyme.

3.3.1 Estimation of Protein Content !!l Gas Chro!!togr!£hic Pyrolysis

In view of the inadequacies of methods used by previous investigators for the determination of the protein content of insolubilized enzymes (Section 1.6.1), a new method, based on gas chromatographic pyrolysis was developed. The results of the ~yrolysis experiments were calibrated in terms of an accepted method for the estimation of protein, amino acid analysis of the protein hydrolysate. 86.

The general variables in pyrolysis gas chromato­ graphy have been reviewed by Levy105 • It is relevant at this point to summarise the development of the method given in Section 2.4.5. The estimation of the catalase content of the conjugate is, in principle, similar to quantitative analysis of the composition of copolymers. Barlow and Associates106 have devised a general procedure for attacking a problem of this type so that a copolymer's composition can be estimated from the sizes of peaks characteristic of each component. Firstly, the optimum pyrolysis temperature is determined from a series of trial pyrolyses. Secondly, pyrolyses of weighed samples of each homopolym.er enable a calibration curve to be made of polymer weight against peak area. Finally, weighed samples of the copolymer are pyrolysed and the composition estimated from the calibration curve prepared with the homopolymers. At a later point, attention will be directed to the assumptions underlying this ~rocedure. The design of the pyrolysis unit used here as well as the physical nature of the conjugates did not allow the ~yrolysis of known weights of sample. Under the circumstances and further, as it seemed inherently more accurate, it was preferable to use an internal 87. standard for the estimation of the catalase content. Consequently the results of the pyrolysis experiments were expressed as the ratio of the area of a peak repres­ entative of catalase to that of a peak representative of the polymeric carrier, the latter serving as an internal standard. Clearly, the accuracy and precision of the analysis would at least be facilitated, if the chromatographic peaks representative of the carrier and enzyme were resolved, symmetrical, large and not too different in size. Samples of polystyrene (in benzene solution) were coated onto wires by evaporation of the solvent at room temperature and pyrolysed at 358°0, 510°0, 610°0 and 770°0 respectively. The most satisfactory temperature in relation to peak size and resolution was 770°0 (Figure 10). Repetition of the·experiments with an aqueous sol­ ution of catalase showed that the only satisfactory temp­ erature for the enzyme was 770°0 (Figure 11). However, the only significant peaks from catalase had retention volumes which were low and identical with peaks from the polymer,thus necessitating a search for circumstances which would allow better resolution of the early peaks. ligure 10: Influence of Plrollsis Temperature on l,!! Gas Chromal9gr9.!...0f Pollstlr!!!!.•

(a) Pyrolysis at 510°0 for 2 second•~ ChromatograP!!ic Condi~!ocs:

12 ft. column of 2~ Carbowax 4000 on Chromosoro - W operated isothermally at ao0 c. Carrier gas:- nitrogen at 60 ml./ - I t r- i ....-➔---- _..., __ . ·------·- ----+-­ .. r •

.,.____,--- -·-•-- .

: -­.·+¥'. I

..

' -

1 X5~0

- •·.

! - -

•----

-, • i •I

. h------'- ' ! -- J ···------­.' .. . 3.00 2.00 1.00 0 Retention Time (min.) 90.

Figure 10 {Continued)

{b) Pyrolysis at 770°c for 2 seconds.

Chromatographic Conditions as for part {a). ------_...... ,_,..__....,._ ------· - .

. . --~ ---- . ' --~ ------1I... 1 -----

T I . 11' ------. --t ::1 .• - I ___t ::i~,.. ... >------~----

..I• •• r·

j. I L

I . - - tt r, ,- I- . - __I____ -- ..-- •- ,__ --

1 ....

J~: t

4.00 3.00 2.00 1.00 0 Retention Time (min.} 92.

1!'igure 11: Gas Chromatogram from Trial Pyrolysis of lree Oatalase.

Q.2.ndi tiona : Pyrolysis of a catalase film for 2 sec. at 770°0. Ohromatographed on a 12 ft. colwan of 2~ Carbowax 4000 on Chromosorb-W operated isothermally at ao0 c with 60 m1./min. of nitrogen. X500

------•------!}------

.. -. --+-.

6.00 5.00 4.00 3.00 2.00 1.00 0 Retention Time (min.) 94.

Preliminary tests also showed that the size of the peaks increased with the duration of pyrolysis up to 10 seconds. A period of 10 seconds was therefore used in the final method. Of the stationary phases tested, the silicone oil SE 30, gave poor resolution and induced "tailing" in many peaks while the more polar phases, Carbowax 4000 ( a polye¥4ne glycol) and ethylene glycol succinate gave little improvement in the resolution of the early peaks. The cross-linked polystyrene resin, Porapak Q, allows rapid elution of water and other polar compounds with little or no "tailing"; it is recommended for the separation of mixtures of hydrocarbons and polar com­ pounda107 and has been used, after coating with poly­ ethyleneimine for the separation of amines (the main products from pyrolysis of proteins108). Although poly­ ethyleneimine was unavailable, Porapak Q seemed to offer the best chances of successful resolution in the present study. The resolution of the early peaks was best at a temperature of 50°c. Yet isothermal chromatography at this temperature was inefficient for resolution of 95.

the later peaks so the temperature was programmed from 50°c to 230°c to overcome this deficiency (Figures 12 and 13). The results of the pyrolyses and catalase estimations (from amino acid analyses) are summarized in Table 9 and Figure 14. (This data is originally from Appendices 1.1.5 and 1.1.4, respectively).

TABLE 9

Variation of the Area Ratio with Enzl!!eto Polimer Ratio for Insolubilized Catalase Sample Mean Weight Ratioa Mean Area Ratio,b Enzyme to Polymer, x Y,

C/24 0.018 0.0197 D/6 0.018 0.0300 C/65 0.058 0.0532 C/74 0.060 0.0533 D/12 0.106 0.0820 a From ion-exchange chromatography. b From pyrolysis gas chromatography.

As can be seen from Figure 14, with the exception of C/24, the mean weight ratio of enzyme to polymer, x, 96.

Figure 12: Influence of the Temperature Programme on the Pyrolysis Chromatogram of Insolubilized Catalase.

(a) Initially Chromatographed at 200°0 for 3 minutes.

Conditions:

Sample C/74 (0.060 g protein/g polymeric support) pyrolyzed at 770°0 for 10 sec. then chromatographed on 6ft. Porapak Q with 60 ml./min. nitrogen. Temperature programme: 200°0 held for 3 min. then increased at 40°C/min. before holding at 230°0 until completion of the chromatogram.

E is the peak representative of the enzyme. P is the peak representative of the polymer. 97.

0

Q 0 • rl ' C> . ··r-·- 0 1 • . -♦--- C\J 0 J 0 ·- L. •- \ ·J U", ,' . ~ I rr\ 0 0

I .•. ♦• 0 0 • . . . . ' . . U", .. -. r ... 0 0 ..._, \.0 (I)

•• ♦ ••. 0 8 0 -r-4 8 c­ C .• I o 0 0 ...... µ - co C: (I) 0 .µ J Q) (r. O'I 0

A4 . I. 0 r-f 0

I. •-·-- ·--- -♦ rl I i- - I rl l I . • I 0 . 1l i I . • I - . .. t·' . C\J rl t I l 0 l I ...... '""' .L. 0 . ~ rl 98.

Fi~E!-1,g (Continued)

(b) Initially Chromato d at 50 for 7 nutes.

Conditions:

Temperature programme: 50°c held for 7 min. then increased at 40°C/min. before holding at 230°c until completion of the chromatogram. Other conditions as for part (a). •

-

......

-~­

!

II

h.

1.00

· I

I

I 1

I 1

l Li'

I

;1

i[ :

I

J

.

I

r.

..

'

• •

.

i:-

..

2.00

c

I ' '

. .

: :

-~ll.

-,--·

l-

;

: : :

3.00

x200·

-.

I

-4------

• -

--.

..

4.00

- '

---

-·

.

-

•

I I I

•

- · I

0

5.00

~--~E

-

·-

_

I

I ~

I

6.00

..

__

-

-

1--

-

I

. .

I

i t t==~~~..l,...... ;~~**=t==!=::-:.

t1

7.00

I

•

I .

+--·: 7

f t

~~

8.00

- .

(min.)

-

I I ,.

-

--·

--

' '

'

~ I

'

1-----

111

9.00

~

Time

J~-

I

---'-'J-~

~ ------19.0

\

20.0

·:

Retention

------ll

___

L

.

21.0

_

22.0

____

I .,,__

__

...

23.0

--

~

---·

24.0

25.0

P=t+,

'

---

~---~

26.0 100.

1igure 13: Typical Pyrolysis Chromatogram from Insolubilized Catalase.

Conditions:

Sample D/12 (0.106 g protein/g polymeric support) pyrolyzed at 770°0 for 10 sec. then chromatographed on 6ft. Porapak Q with 60 ml./min. nitrogen. Temperature programme: from 50°0 to 230°c at 10°0/ min. then held at 230°c until completion of the chromatogram.. 101.

- . - I I l . : :.I .... I ...... I... I :• ' I .... 0 I l, : 1'..: l ' ' 0 i 0 ' • r-t ...... ~ ·r· ...... 0 • 0 - • (\J 0 0 • "' 0 0 • . . . q- I 0 . . . . . 0 I • I I!'\

I -1 -- . El I \0 .... I t 0 ,..... ---- ··-- .. 0 • r-! d 0 ·rt 0 ...... ,e CX)• 0 ~ 0 ·rl • .µ

°'0 C • :, 0 ·M (\J .µ C Q) 0 .µ : t r: ... • r-t Q) (\J ex= 0 I • N (\J 0 • ..... / ""(\J

I! 0 v C\J

0 • Lt\ N ' ... '.I

0 •· • • \0 N 102.

:, o.oao

0.0600 '

0.0400

0.0200

,. ' 0 0 0.0200 0.0400 0.0600 0.0800 0.100 X ------··--- -· ------Fieure 14: Calibra,!!on C..:~~...!2!~!.2.!lsis­ Q:=!,_Qli~2mat~fE~~hl- (y is the-mean area ratio of a peak represent- ative of the enzyme to one representative of the polymer; x is the weieht ratio of enzyme to polymer from· ion-exchange chromatoer4vhY of the hydrolysate of the bound catalase.) 103.

and the mean area ratio, y, are linearly related, the equation of the line being

y - 0.018 + 0.60x The failure of sample C/24 to obey this empirical relationship is not entirely unexpected as the duration of the nitration of the ~olystyrene used in the prepar­ ation of conjugate C/24 was only half that of the nitration used in the preparation of the remaining conjugates. (Zenft:man68 has shown that the degree of nitration increases with the duration of the nitration reaction). This could result in an ano:malou3 area ratio for sample 0/24. Further, results which are in­ dependent of the pyrolysis method support the pro~osition that sample C/24 differs from the other 1nsolubili3ed enzymes (Section 3.3.3). Although the mean values of the area ratios used in Figure 14 fit the line quite well, the raw data from which the means are derived show appreciable scatter about the line. This is not surprising since the retention volume of the peak characteristic of catalase is unavoidably low and it is uaually accepted that the relative que.ntitives of such volatile ~yrolysis products are comparatively sensitive to fluctuations in the 104.

experimental conditions109 • Notwithst~nding these deficiencies in precision, the method is clearly applica­ ble to the routine estimation of the catalase content of conJugates prepared from similar poly(nitrostyrenes) so long as at least five replicate pyrolyses are performed on each sample. This is not inconvenient as a pyrolysis can be completed in about half an hour and contrasts with the established ion-exchange method which takes, in all, about 2 days (on specialised equipment).

les of

Apart from the correlation discussed in the pre­ ceding section, it was of interest to investigate the possibility of a correlation existing between the area ratio from pyrolysis and the known quantity of catalase added to form a series of specially prepared conjugates (reference samples); such a correlation would avoid the necessity of calibration by ion-exchange methods and would further establish if specific experimental conditions would give conJugates of the anticipated compositions. Although an extensive set of data was compiled in these tests (Appendix 1.1.6), "curve fitting" by visual 105-

inspection was not sufficient to clearly indicate a relationship between the known enzyme content and the area ratio. Least squares regression analysis was then performed on the replicated data and the following relationship established110: log y where xr is the weight ratio of added catalase to polymer in the reference samples, and y is the area ratio of a _peak representative of catalase to that of a peak representat- ive of the polymer.

Analysis of the variance of the regression showed that the line was valid111• It is pertinent to compare the calculated catalase content (from Section 3.3.1) of these reference samples, with the figures obtained from their preparation (Section 2.4.6). It follows from Table 10 that either the protein estimation by ion-exchange chromatography is incorrect or that the assumed catalase content of the reference conjugates is erroneous. The ion-exchange method is unlikely to be greatly in error, because it is a recognised method and has been previously used for the 106.

accurate estimation of the protein content of conjugates (see Section 1.6.1).

TABLE 10

The Catalase Content of the Reference Co,!!iug&t!!, Weight Ratio of Mean Area Ratio Ratio of Enzyme Enzyme to Folymer (data from to Folymer (Section 2.4.6) Appendix l.1.6) (from Figure 14) Xz---- y X 0.0867 0.0314 0.0215 0.116 0.0414 0.0380 0.145 0.0559 0.0620 0.173 ------0.0930 0.106

On the other hand, the pyrolyses on the reference conjugates are based on two assumptions, namely, a) that the samples are sufficiently homogeneous to allow effective sam~ling, and b) that any unbound catalase present pyrolyzes in exactly the same way as the conjugated catalase.

In fact, the reference conjugates were far from homogen­ eous as the particles of ~olymer were coated with varying a~ounts of a stickly residue which also covered the walls of the flask after evai.,oration. The validity 107.

of the sampling of the reference conjugates then, is open to doubt and this is reinforced by the low order of precision in the .Pyrolysis experiments with the reference conjugates - a degree of .l?recision much lower than that found in the pyrolysis of the samples which were ,Jre.i:Jared in the usual manner (and later subJected to a~ino acid analysis). Thus, the invest­ igation of the reference conjugates ~rovides indirect su~..r?ort for the necessity of firstlJ calibrating the .Pyrolysis method against amino acid analysis of the conjugate's hydrolysate. Obvio~sly the validity of the second assumption should be tested by comparing the area ratio from pyrolysis of a conjugate with that from pyrolysis of a physical mixture of catalase and poly(diazostyrene) of sinilar com~osition. For reasons to be advanced later (see Section 3.3.4) this was impossible. d.:.3.3 Correlati~~!E_!he "Nomina~_Q~alase Con!!B! and the R~~~_2f Amino Acid Analys_!! The systematic inveRtigation of the radiation chemistry of bound catslase was ereatly hindered by the difficulty of ~redicting the catalase content of a conJub~te ~re~~red under given conditions. To over­ come this froble~ and to facilitate further work in this area, the weight of catalase added during cou_pling, expressed ~s a ratio of the initial weight of ;oly(nitro­ styrene)* h~s been correlated with the catalase content of the conjugate estimated from the results of ion­ exchanee chromatogra~hy or the hydrolysate from the bound catale..se. The data on which the correlation is based are shown in Table 11 and Figure 15.

TABLE 11 The Variation of Actual Catalase Content with Nominal Catalase Content

Sample Number x Nominal x8 Actual n a Content Oontentb C/24 0.113 -0.947 0.018 D/6 0.0555 -1.256 0.018 c/65 0.0829 -lo081 0.058 C/74 0.0840 -1.076 0.060 D/12 _2.137 --=£!.863 Ool06 a The wei;ht ratio of catalase to Joly(nitrostyrene) Section 2.3.5. b The weijht ratio of catalase to ~oly(diazostyrene) A,i)pendix 1.1.4. * The "Nominal" catalase content (Section 2.3.5). O.lOJ

0.0800

,.) • 0600

u.O4OO

-1.200 -1.100 -1.000

f!.~~15: Y~iation~£_tn!_~£!~!~!:.EalFis~~!l!~!.1.-!a'

~!. th t_!2~_rfo~i n~~-C:.i 1~!.!!~2n te !l!.i.2n, £Lili (; o !!J. uga!!_.,

------110.

If samJle C/24 i3 again excluded (for reasons simils.r to those advanced in Section 3.3.1) it can be calculated that the results are consistent with the following empirical eq_u<'1 tion:

= 0.30 + 0.22 log x n It would appear from this expression that either appreci~ble quantities of catalase have been lost during cou~ling or that the ~olymeric carrier has considerably increased in weight during its conversion from ~oly(nitro­ styrene) to ~oly(diazostyrene). Catalase is known to adsorb slowly onto glass surfaces from aiueous solution112 so tl::..e .i?Ossibili ty was invest­ igated that catalase adsorbed onto the walls of the flask during the long coupling reaction. Svendsen112 has shown that sfter equilibrium is established Pyrex glass can adsorb 1.5 x 1012 molecules of catalase per cm. 2 from solutions containing electrolytes such as phosphate buffer. The coupling reaction was carried out here in phos~hate buffer contained in a small Pyrex conical flask with an internal surface area of approxi~ately 33 cm. 2• These conJitions should allow an adsorbed catalase concentration of£!· 3 x 10-9 M which is no more than 1~ of the concentration of catalase present in the reaction 1 1 1 •

mixtures. The time of contact of catalase solutions with other glass surfaces was not sufficient to allow apprecia.ble a.ddi tional losses.

A second ?Ossibility is that some catalase could have been lost in the supernatant from the coupling reaction, after failing to combine with the polymer. The negative Biuret tests obtained on the supernatant (3ection 2.3.5) allow the elimination of this possibility. If no significant quantity of catalase has been lost before coupling to the polymer, the alternative proposition is that the polymer has greatly increased in weight. Normally one would not ex~ect this to occur since the weight of a repeat unit would be only slightly affected if it were substituted by a diazonium group rather than a nitro group. However, it is interesting to speculate on the effect of the occlusion of tin salts on the weight of the polymer. It is accepted11 3 that low molecular weight amines can combine with the hexachlorostannate (IV) ion formed after the use of tin (II) chloride and hydrochloric acid as reductants:

+ 201- ~snc16]= + [snc1J= ~ (RNH3) 2 [sn c1J 112.

The occlusion of hexachlorostannate (IV) ions in the amino or diazo polymer could significantly increase the average repeat unit weight of these polymers. This would obviously result in the actual catalase content being only a fraction of the nominal catalase content and could thus serve as a partial explanation of the discrepancies described above. In fact, subsequently, recent work in these laboratories has shown that occluded tin is not removed by even the most exhaustive washing114 • The situation is further complicated by decreases in the weight ratio arising from the removal of diaper- sable poly(diazostyrene) in the washings. The results of Appendix 1.1.2 indicate that portion of the poly(diazo­ styrene) formed a stable dispersion in aqueous solution and hence would have been lost during washing. To summarize, the actual enzyme content of the conjugate can be related to the logarithm of the nominal enzyme content by an empirical equation. It appears that the relationship is influenced by the occlusion of tin in the polymeric carrier and possibly by the loss of poly(diazostyrene) through dissolution during washing. 113.

3.3.4 The Stability of Diazonium Groups

It is evident from the results of Appendix 1.1.2 and consistent with the results of previous workers69 , 115 that the diazonium groups in the polymer are stable on standing at room temperature for two weeks and congequently were present in the samples of insolubilized catalase submitted to irradiation in the present experiments. The gelatinous nature of the co~jugate (as for poly(diazostyrene)) also reflects the presence of unreacted diazonium groups. The existence of diazonium groups in the conjugate was tolerated because of this stability and also because the conjugates were usually irradiated within one or two days after yreparation and storage at 0-5°0 i.e. before much change had occurred in the number of diazonium groups. This was considered to be better than the alternative of blocking the diazonium groups with a totally foreign but effective molecule such as 2-naphtho169 or the more closely related but ineffective glycine 115 • An important problem to arise from the presence of unreacted diazonium groups in the polymeric carrier was the imyossibility of preparing physical mixtures of 114.

catalase and polymeric carrier for use in comparative experiments in radiation chemistry or pyrolysis. Obviously one should have a physical mixture with the same chemical structure as the conjugate but with the possibility of chemical bonding between the enzyme and the polymer excluded. The presence of diazonium groups does not allow this.

From theoretical considerations, the diazonium groups in poly(diazostyrene) should couple with activated aromatic side chains in proteins. However, direct proof of this expectation is very difficult to obtain with polymer-bound proteins. The usual methods for detection of the resulting azo and related groups demand either a soluble compound (ultra-violet/visible spectroscopy116), or the absence of interfering diazonium groups (titration with reducing agents such as titanous chloride117 and gasometric methods dependent on nitrogen evolution117). Moreover identification of the azo group and character­ ization of the catalase-polymer bond cannot be effected by removal of the interfering diazonium groups with compounds such as 2-naphthol since these reagents will 115.

introduce further azo groups. Infra red spectroscopy has also proved unsuccessful118 • Consequently, those workers who have investigated the bonding of proteins to poly(diazostyrene) have relied on indirect evidence. Yagi and Associates62 found that bovine serum albumin was more firmly held by poly(diazostyrene) than by either poly(aminostyrene) or by a poly(diazostyrene) sample which had been subjected to prior exhaustive coupling with 2-naphthol. The bonding in the latter two cases was attributed to adsorption whereas with poly(diazo­ atyrene) the authors felt that bonding was both chemical and physical. In the only other reported study of this type, Brandenberger118 concluded that catalase could be adsorbed by both poly(aminostyrene) and poly(diazostyrene) but he could not obtain sufficient evidence to decide if chemical bonding occurred between catalase and poly(diazo­ styrene); this is a clear demonstration of the difficulties involved in proving chemical combination between proteins and diazotised polymers. This situation can be contrasted with that involving low molecular weight diazonium. salts and proteins where evidence for chemical combination is readily forthcoming. 116.

The results of extensive investigations performed by a number of workers116•119•120 show that the reactive sites in proteins coupling to low molecular weight diazonium salts are residues of histidine, t¥rosine, lysine, cysteine, arginine, tryptophan as well as terminal glyciae, proline and hydroxyproline residues. Further, chromatography of the hydrolysates of such cou~1ed proteins can reveal the extent of the reaction with low molecular weight diazonium. salts121• It can be seen from Appendix 1.1.3 that the hydroly­ sate from bound catalase is deficient in lysine, histidine, cysteine (cysteic acid) and tyrosine. (It would appear that the low methionine content of the hydrolysate from bound catalase is due to oxidative losses since the content of methionine sulphone is not decreased by a similar amount; methionine sulphone is not susceptible to oxidation under the conditions of the analysis74 • Similarly the use of the cysteic acid rather than the half (cystine) content should have &Toided errors due to oxidative losses in cystine and/or cysteine74 ). It has been assumed that the magnitude of these deficiencea represents the extent of the reaction between the lysine, histidine, cysteine and tyrosine 117.

residues of catalase and poly{diazostyrene). The total number of these residues lost on binding to poly(diazo­ styrene) is ,2!• 140 per mole of catalase {computed as the sum of the results in column 4, Table 15, Appendix 1.1.3) * • It is interesting to compare the coupling conditions here with those used by previous workers for low molecular weight diazonium compounds116• The very long reaction time of 15 hours (compared to up to one hour) and the high weight ratio of diazonium salt to protein used here should, within the limitations of steric hindrance, lead to virtually maximal coupling of the protein. Further, it is interesting to make a comparison of the number of bound residues per ~ole of catalase with the weight ratio of polymer to enzyme in the conjugate, since this enables a calculation of the molar ratio of polymer to catalase to be made. In fact, assuming a molecular weight of the order of 100,000 for

* The arg\lments advanced here were developed prior to publication of the results of a similar investigation on other enzymes by Goldstein and Coworkers122• 118. poly(diazostyrene) * and knowing the weight ratio of catalase to polymer (A~pendix 1.1.4) as well as the number(£!• 140) of residues per mole of catalase bound to poly(diazostyrene), it can be readily calculated that the molar ratio of polymer to catalase in these experiments varies from£_!• 20 to 100 and that from l to 10 residues are bound per mole of poly(diazostyrene) (the greater the mole ratio, the less the number of residues bound per mole of polymer). The results of Higgins' group116 indicate that the most reactive residues are usually histidine and tyrosine.

* The value of 100,000 seemed reasonable as a first approxiamation since the molecular weight of polystyrene was 76,600 (degree of polymerization, 737; Appendix 1.1.1) and, in view of the conclusions of Section 3.3.3, it is expected that the repeat unit weight of poly(diazo­ styrene) will be greater than that of poly(nitrostyrene), and, in turn, greater than that of polystyrene. Little would be gained by accurately estimating the molecular weight of poly(diazostyrene) since the sample contained polymer molecules with a wide range of molecular weight as a result of its origin in the radical polymerization of styrene (Section 2.3.1). 119.

However in this study, lysine seemed more reactive than both histidine and tyrosine possibly indicating that many lysine residues in catalase are on the surface of the molecule. The unexpectedly low result for tyrosine in this regard is consistent with the findings of YagilOO that most of the tyrosine residues in beef liver catalase are folded into the interior of the molecule. Similarly one might conclude that virtually all of the arginine residues are confined to the interior of the molecule since there was no significant combination between arginine and poly(diazostyrene) although previous investigations on other proteins indicated that it should be reactive. In summary, it seems reasonable to assume that the low recoveries of lysine, histidine, cysteic acid and tyrosine in the hydrolysate from bound catalase are due to the formation of chemical bonds between, on the one hand, the residues corresponding to these acids in the protein and on the other, the diazonium groups in the polymer. Moreover, it appears that, in the insolubilized form, the surface of the catalase molecule is ttgrafted" to a large number of poly(diazostyrene) molecules each of which is linked in~ever~lplaces only to the catalase molecule. A structure such as this must surely provide an barrier against the diffusion of reactants towards the catalase molecule. 121 •

3.4 THE RADIOLYTIC INACTIVATION OF INSOLUBILIZED_,2ATALASE

3.4.1 The Inact~ion Yield and Protection Quotient

Although heterogeneous enzymic systems more nearly simulate cellular conditions, the experimental difficulty involved in their study is reflected by the paucity of results123, 124 • Heterogeneous systems of the type described in the present study, i.e. insolubilized enzymes, do not appear to have been irradiated before. The results for the irradiation of insolubilized catalase are summarized in Table 13(a) and Figure 16 (from Appendix 1.2.5). The corresponding results for free catalase are given in Table 13(b).

TABLE 13(a) Radiation Sensit1vi!Z-2L~~~!,!d Catalase (From Appendix 1.2.5). Sam,12le ___2L~- D/_6 C/_24 D/_12

Catalase content (M X 106) 0.90 0.82 0.82 3.9 n37 Dose (krads) 780 900 910 1150 Corrected Qa,c 0.73 0.29 0.29 0.45 Corrected G Valuea,c 3 (Molecules/100-- eV.) X 10 1.4 1.0 1.0 --3.8 122.

_ca_l_c_u_l_a_t_e_d_R_a_diation Sensitivitl of Catalase~olutions With and «ithout Added Dig~£~llcine (Gll) 3 i_gO mg./ mi.) ~atalase Concentration (M x 106) 0.90 0.82 0.82 3.9

D37 Dose (krads) without (Gly) 3 192 187 187 337 D37 Dose (krads) with (Gly) 3 305 295 295 750 Corrected Qc 0.028 0.026 0.026 0.11 Corrected G Valuec (molecules/ 3 --100 eV x 10 ) -- 19.8d a In order to calculate these parameters, it was assumed it'3 that catalase did not lose/integrity on insolubilization. For example, the molecular weight used in the calculation of the G value was that of catalase. Although not strictly correct, it was felt that this approach was justified since it gave a reasonable basis for compar­ ison of the radiation sensitivity of catalase in both free and ~olymer-bound forms. b Based on Figure 7 at catalase concentrations comparable to those of catalase in the insolubilized form. C These values were corrected for the dose absorbed by the bllffer (145 krads - Section 3.i.1). d From Section 3.1.4. :- 100 90 80 70

60 100 50 ... \ <;.• 90 80 40 70 6v 30 50 -N Residual .-. Activity ,- """ (") . 40 20 '\.._._'..,. Residual \" -, Activity(") 30 ' O Dose (f~ids) - ' ...... (b) Sam¥le D/6 (Ap~endix 1.2.;) ' ...... -... 0 '---., 800. 1600-- 2400 3200 4000 Dose (k.rads) (a) Sam.~le c/65 (A~pendix 1.2.5)

· F1e!ir• 16 : !!!.!22!.!-~~!!~!t_:_1!_~~'!.-~!.:..~!.~.!~~:~~::~~~~~~~~!!,. . 124.

100 90 80 70 60 50

40

30 Residual Activit3 (~) 20 \

,. h

10 9 8 7 6 0 800 l Dose (krads) (c) Sa~9le C/24 (Ap~endix 1.2.5)

Fiffi:!re...1§ (Uontinued) 125.

100 90 80 70 60 50

40 0 Residual Activity ( t;) 30 v 8uu 1600 Dose (krads) (d) 3am~le D/12 (Appendix 1. 2.5)

Fiaure-~-- 16 (Continued) 126.

It would be ex~ected that dilute aqueous dUspensions of insolubilized enzyme would be inactivated via the indirect effect; however, the heterogeneity of the system could render the kinetic criteria for diagnosis of the indirect effect (Section 1.4) ina~~licable. Results from comparable Jystems such as catalase adsorbed on glass125 and deoxyribonuclease adsorbed on aromatic ion-exchange resins126 in aqueous environments give support for the incidence of the indirect effect in this study. The residual activity-dose curves (on a semi-log plot) (Figure 16) are linear and allow the n37 dose to be estimated. The values obtained are greatly in excess of those obtained for similar concentrations of free catalase (even in the presence of diglycylglycine) indicating that the insolubilization has effectively protected the enzyme. Comy~rison of the corrected protection quotient for catalase protected by insolubilization with that for catalase ~rotected by the soluble radical scavenier, diglycylglycine, reveals that on a weight basis the ~olymer diverts a greater proportion of the dose from 127.

the enzyme than does diglycylglycine.* Further evidence for the protection afforded the insolubilized enzyme 1s. shown by the corrected G values * in Table 13;from 5 to 20 times as many catalase molecules are inactivated by unit dose in the free than in the insolubilized form. It is interesting at this point to consider the ~ossible reasons for the ~rotection of the enzyme in its insolubilized form.

As stated above, it is reasonable to inter~ret the results in terms of the indirect effect and hence to partially (at least) ex~lain the protective effect of the ~olymer in terms of radical com~etition between the en~yme anJ the ~olymeric suy~ort. Conversely,energy (and/or cnarge) transfer (by either intramolecular or intermolecul~r mechanisms) is not ex~ect~d to be dominant here as it has been shown to be insigDificant in dilute a·1ueous solutions of polymers127• Revair (Section 1.1) of catalase radicals either intermol~cularly by intact molecules of insolubilized en~~1 me or intramolP,C:.11,.,rly by i!'.lteraction of the poly-

* -~he values obtained for botn fr2e anJ insolubili~ed cat~l·13e were corrected for the dose 3bsurbed by tne (di~zostyrene) moeity with the catalase could be im~licated in the ~rotection of the insolubilized enzyme. It has been concluJed (Section 3.2) that catalase

by oH• radical3. Unfortun~tely, the reactivity of the OH 0 radical (or any other radical s~ecies for that mcj,tter) with .f'Oly(diazostyrene) has not been inve3tigated previously; however the reactivity can be inferred from studies on com~~rable lo½ molecular weight com~ounds.

The electrophilic attack of OH 0 radicals on v~ricus monosubstituted benzene derivatives has been correlated with Hammett's cr--function128, 129 (also see Section 1.3). It would tllus be ex;,ect,~d that electron-withdrawing substi tuents such as N.tij, N0 2 and N; would imiJede the attack uf ~s· radicals ~hile electron-releasing substituents such as NH 2 and Oh would enhance it. Althou~h this is found in ~ractice, the effect is not marked as the ~H• radical is so highly reactive with . t· b enzene d t t ·t 1· 7 er1va 1ves ha i snows itt~e se 1 ec t·1vity . 1?3,129 - • It is noteworthy that the nucleo~hilic attack o: tne hydrat~d electron on benzene deriv~tives is ~lso i~ c1.ccc.,r:..iance with ;..i:ammett's a-function but the e aq_ is muon more se.1i?cti ve in its reacti vi t,y, ranging from 6 -J. -1 a rhte constant of 4.J x 10 M sec. for ~henol to 129.

3.0 x 1010 r::-1 sec.-l for nitromnzene130• It would be expected, by an~logy with conventio~al nucleophilic substi tutiona of arom~,tic comJounds, that the :iiazonium group would be even ,rJore strongly activating in the reactions of e- than the nitro group131 and hence aCi that compounds of this ty~e would be more efficient scavengers of e - than oxygen ( k = 1.9 x 1010 M-1 sec. -1 aq_ - Section 3.2.1). It has been shown (Section 3.2.1) that e is effectively scavenged by oxygen; consequently, &(! the role of e- in the radiation inactivation of poly- aq (di~zostyrene) bound ca.talase must be minimal. It is 1-•robable that OH. radicals cause inactivation of insol­ ubilized catalase. It is evi,ient tt.at the predictions of the radical scavenging ca~abilities of ~oly(diazostyrene) advanced above would be modified by such factors as the physical form of the bound enzJme, its structure and the possibility of ''complex" formation betvveen .i;ioly(diazostyrene) and the active site of the catalase molecule. Although it would be ex~ected that radiation­ ~rojuced radicals would react more slowly with the insolubilized tllan the free enzyme, the highly gelatinous, swollen nature of the conJugate used here would offset tl1is ten·.iency. 130.

It is a~parent that tne insolubilized enzyme may be ~ictured as a globular catalase molecule to which is att~ched a large number of poly(diazostyrene) molecules (20 to 100, depending on the sample) (Section 3.3.5). In the light of the comments above on the radical scavenging ability of the FOly(diazostyrene) it would seem that the .i:;olymer would provide an almost impenetrable barrier to the ap)roach of reactants to the surface of the catalase molecule. Nevertheless, the facts that the insolubilized enzyme still retains some activity and is not com~letely resistant to radiation inactivation indicate that the active site at least, is not completely sterically hindered by the polymer. One would not exiiect any "comiJlex" formed between ~oly(diazostyrene) and the active site to offer an explanation of t~e observed protection of active catalase as such an interaction would surely cause perm~nent inactivation of the active site and would not be detect­ ed by the relative esti~&tes of activity before and after irradi~tion (as used in tuis study). InterestingiJ, insolubilization of the type described here should im~ede conformational changes in the enzyme. It is conceivable that insolubi1ization with poly(diazostyrene) 131.

could "lock" catalase into a conformation which is less reactive than free catalase towards OH• radicals. This aspect has been discussed by Melrose50 with regard to the catalytic reactions of insolubilized enzymes with their substrates and it is reasonable to suppose that similar considerations apply to the reactivittesof these enzymes with radiation-produced radicals. (See also ref. 13a) In addition, it is possible that insolubilizat­ ion induces protection by hindering the gross conformat­ ional changes and denaturation which often follow the attack of radiation-produced radicals on proteins (Section

The critical feature of most of the possible modes of protection outlined above is that the protector is actually linked to the target compound. This idea has been suggested previously133 as a means of improving the effectiveness of radical scavenging protectors since it is obvious for steric reasons that such a system is likely to give enhanced efficiency of the scavenger. Neverthe- less, the use in this way of a polymeric radical scavenging ~rotector .i!L!!!!:2 is novel and has the important attribute of introducing conformational and other steric processes as possible modes of ~rotection additional to those advanced in Section 1.1. 132.

4 CONCLUSION

In this vroject, aspects of the radiation chemistry and ~rotection of the ~oly~eptide chain of catalase nave been investigated. The ~-irradiation of dilute, o~ygenated, a4ueous solutions of catalase resulted :in degradation of the c1steine and/or cystine residues as well as enzymic inactivation. These effects, ascribed to radiation- vroduced hydroxyl radicals, were diminished by the radical scavenger, diglycylglycine. It appears that this ~eptide also protects catalase by rei:,airing damaged molecules of enzyme. Insolubilization by p~ly(diazostyrene} was shown to be a novel and effective method of protecting catalase from the effects of 0-radiation in dilute, oxygenated c:1.1,ueous ,ys tP.ms. The effectiveness of this protective technique is a11,t1arently due to a combination of convent­ ional radical scaven~ing and repair processes with ~roximity, conformational and other steric factors.

A rapid and novel ~ethod was develo~ed for enti~~ting the catalase content of a poly(diazostyrene)-catalase con.,ugate. Pyrolysis and eas chromatography of the insolutiliznd enz ✓me allowed a calculation to be made of the ·J.re:i of the ratio/of a Jeak ari~ine from the enzyme to that 133.

of :J. .i.)eo.k t.rising from the 1--'olymeric 9U_;_.1fort. This ~re~ r~tio v~ried linearly with the protein ccntent which was estimated independently from ion-exchange chromatozrsphy of the hydrolysate of the bound catalase. In further work, the protein content was linearly correVited with the rel2.tive ::i.mounts of cata.lase and ~oly(nitrostyrene) used in the ~reparation of the insolubilized cat~l~se. The technique was a~~licable to conjugates ~refared from similar poly(nitrostyrene) sam~les. For insol~bilized catalase, irrespective of the 1,rotein content, an average total of ~-140 residues (histidine, lysine, cysteine and tyrosine) per ca.talase molecule were bound to foly(diazost~rene). Although generalizations are complicated by the range of molecular sizes 2resent in the ~oly(diazostyrene) (by virtue of its oriein in the 2olymerization of styrene), on the average, eacu c8.tRlase molecule was bound to a large number of ~oly(di~zostyrene) molecules (of the order of 20-100). lJ4•

5 ACKNOWLEDGEMENTS

The writer wishes to acknowledge the advice and constructive criticism rendered by his supervisor, Dr. G.J.H. Melrose of the Department of Applied Organic Chemistry, the University of New South Wales as well as the aid given by the head of this department, Associate Professor E.R. Cole in the form. of encourage­ ment and the use of facilities. Thanks are due to Professor F.W. Ayscough of the School of Chemical Technology, the University of New South Wales for permitting the writer to use the 60co (-ray facility and to Mr. C. Samways, Professional Officer in the same school, for his co-operation during the irradiations. The writer is indebted to the New South Wales Public Service Board for a part-time scholarship granted to him in 1969. The skills and patience of Mrs. L. Landon, who typed the thesis, are deeply appreciated. It is a pleasure to acknowledge the assistance of Mr. R. Wakeford of the Chemistry Department, Hawkesbury Agricultural College, Richmond, New South Wales in the production of Figures 3 and 10-13. 135.

Finally, the writer is especially appreciative of the forbearance and encouragement of his family throughout the duration of this project. 136.

6 REFERE:H;ES

1. A.O. Allen, "Radiation Chemistry of ·,,ater and Aqueous Solutions", Van Nostrand, Princeton, 1961. 2. L.G. Augeostine, ~• Enzymol~ ~i, (1962) 359.

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APPENDIX 148.

l RESULTS

1.1 PROPERTIES OF INSOLUBILIZED CATALASE

1.1.1 Viscosity Average Molecula~ight~f Polystyrene

The efflux times of pure benzene and solutions of polystyrene in benzene were determined (Section 2.4. 1) in order to calculate values for the following parameters:

relative viscosity, = t; "7 rel to inherent viscosity, ln / inh = if'/rel/c specific viscosity, -1 i sp - 1Jrel reduced s_pecific viscosity, 7sp/c

(t is the efflux time in seconds of the polystyrene solution; t 0 is the efflux time in seconds of pure benzene and C is the concentration of the polymer solution in g/100 ml.). The results are shown in Table 14. The plots of Js/C and"7inh versus C shown in Figure 17 were extrapolated to infinite dilution to obtain the intrinsic viscosity [ '7] (0.399 dl. g-1 ). The dependence of the intrinsic viscosity on the molecular weight can be expressed by the Mark-Houwink equation72 :

[-,] = ~ • • • log M = ¼ (log [ '] - log K) 149-

The constants Kand a have been determined for _pol,/styrene having molecul

log K = 4.013 a = 0.74 Substitution of these values into the Mark-Houwink equation gives a viscosity-average molecular weight of 76,600 for the sample of polystyrene.

TABLE 14 Viscometric Data for Solutions of Polystyrene in Benzene Polystyrene 0 0.2064 0.3016 0.3916 0.8484 Concentration (~ w/v) --- -- Mean Efflux 196-2 211.2 217.2 222.4 241.8 Time a (sec.)

Relative Viscosity 1.076 1.108 1.133 1.232 'inherent --- Viscosity 0.356 0.339 0.319 0.246 (dl. g-1)

Specific 0.076 0.108 0.133 0.232 Viscosity --- Reduced 0.368 0.358 0.340 0.274 S!J8Cific Viscosity ( dl. g-1) a Each value is the mean of at least 3 results agreeing within 0.2 second. 150.

0.400

u.3110 Viscosity Est i -nc:1. te Reu.uced s .. ,ecit'ic Vi_:.: CU.Ji ty

0.320

Inherent Viacosity

u u.4ou u.6vv

1.1.2 Tests on Poly(diazostyre!!!,)

An aqueous dis~ersion* of freshly prepared, orange poly(diazostyrene) (0.05~) at pH2 settled to an orange precifitate (with a colourless supernatant) a.i'ter standing for two weeks at room temperature. The pH remained constant. The dispersion, and the precipitate obtained after standing for two weeks, turned red on the addition of 2-naphthol, indicating the presence of diazonium groups in both materials. Although the fresh dispersion (pH2) was precipitated by boiling, 30 minutes vigorous boiling was required before a negative 2-naphthol test was obtained.

1.1.3~!!!!2. Acid .Q,£mpos~~ Table 15 shows the effect of reaction with poly­ (diazostyrene) on the amino acid composition of catalase.

* At higher concentrations a gel was readily formed. 152°

TABLE 15

Amino Acid Com~osition of Free and Bound Catalase (Exl-'ressed as residues x 10-2 in 250,000 g of enzymea)

Amino Free Catalase Bound Catalase Residues Lost Acid (l\iean of 6 (Mean of 8b Number % results) results)

Lysine 1. 34 ( 1.2c) 0.48C 0.12 60 Histidine 0 ■ 572(0.49C) 0.28 0.21 43 Areinine 1 .02 (0.88c) 0.92 0 0 As_partic acid 2.24 2.4 0 0 T.areonine 1.13 1 • 1 0 0

Serine 1.15 1.2 0 0 Glutamic acid 2.41 2.3 0 0 1-'roline 1.17 1.1 0 0 Glycine 2.00 2.0 0 0

Alanine 2.02 2.0 0 0 Cysteic aci.dd 0.457 0-348 O.12 8 26 8 Valine 1. 60 1.6 0 0 Methionine 0.457 0.38 0.011 17 Methioni~e 0.349 0-348 oe oe Sulphone

Isoleucine 1.03 1.0 0 0

Leucine 1.86 1.8 0 0

1l'yrosine 0°639 0.30 0.34 53 Tyrosine o.2sf 0.36 56 1 53.

Table 15 Uontinued Amino Acid Free Catalase Bound Catalase Residues Lost (Mean of 6 (Mean of 8b Number ~ results) results)

Phenylalanine 0.937 0.92 0 0 Unknown 0 0.32 0 0 Unknown 0 o.33f v 0 a Based on the assumption that the enzyme is composed entirely of protein. Any error introduced by neglecting the 4 haem prosthetic groups in this way is well within the experimental error(! 3~ ref. 74) since these groups constitute only 1~ of the weight of the catalase molecule. b The 8 analyses consisted of a single analysis on each of conjugates C/24, D/6 and C/65 with duplicate estimations on C/74 and triplicate estimations on D/12. The results of the different samples were sufficiently similar to warrant pooling. C Since only small quantities of bound catalase were available for analysis all results were obtained on a more sensitive but less precise(~.± l~) expanded scale. The figures shown in parentheses for free catalase were then obtained in the same way, s~ecifically for comparison with the results from the bound catalase. 154.

Table 15 Continued d Estimated in the hydrolyzate after oxidation by performic acid135 • e Single determination only. f Mean result from sample D/12 only.

1.1.4 Estimation of Bound Catalase by Ion-Exchange Chromatograpbz The results of the estimation of the protein content of insolubilized catalase by ion-exchange chromatography are shown in Table 16. TABLE 16 Protein Content of Polymer Bound Catalase Sample Weight of Weight of Weight of Ratio of Protein Number Conjusate Protein Carrier in to Carrier Weiihts (mg.) (mg.) ConJugate Actual Means ------·------{mg.) Results C/24 1.203 0.0208 1.182 0.0176 0.018

D/6 5.013 0.0874 4.926 0.0177 0.018 c/65 5.095 0.2768 4.818 0.0575

~/74 5.070 0.2962 4.774 0.0622 0.060 c/74 4.790 0.2634 4.527 0.0582

D/12 5.118 0.4969 4-621 0.107 0.106 D/12 5.118 0.5071 4.611 0.110 D/12 5.075 0.4645 4-611 0.101

* Insufficient ~uantities of the first three samples were available to permit more than single determinations. 155-

1.1.5 Bstimation of Bound Gatalase by Pyrolysis~ Chromat~raphy The retention volumes (ml.) of the peaks, selected as representative of the enzyme and polymer contents of the sample respectively,werE 300 and 1350 (both based on the flow rate at 50°c). The results are shown in Table 17 where the peak representative of the enzyme is denoted by E and that of the polymer by P.

TABLE 17

Estimation of Bound Catala!! by Pyrolysis-Gas_Q.hromatoE~ (Analytical conditions are described in Section 2.4.5) ------·------·------Sample Area (cm. 2 ) Area (cm. 2 ) Ratio E/P Number of Catalase of Polymer ------Peak* E Peak* P C/24 0.89 37.0 0.0240 1.58 97.7 0.0162 1.88 108 0.0174 1.09 46-5 0.0234 3.50 207 0.0174 c/65 J.60 12.1 0.0495 2.28 46.6 0.0490 2.57 47.2 0.0544 4.38 76.0 0.0580 4.68 84-7 0.0552 * Attenuation x 100 156.

Table 17 Continued Sample Area (cm. 2) Area (cm. 2) Ratio E/P Number of Catalase of Polymer Peak* E Peak* P ------Ll/74 1.45 21.3 0.0531 0.85 14.1 0.0605 2.89 63.1 0.0458 12.5 232 0.0537 3.93 73.8 0.0533 D/6 2.64 79.4 0.0333 1.45 57.2 0.0254 1.53 61.6 0.0248 0.10 21.4 0.0326 2.69 79.8 0.0337 D/12 1.05 10.8 0.0974 0.32 3.57 0.0910 3.48 54.1 0.0644 11.7 151 0.0779 8.57 108 0.0792 -- --6.60 76-5 0.0862 1.1.6 Pyrolysis of Reference Samples of Insolubilized Catalase The conoi tion3 fo1· .t'yrolysis-gas chromatography c~ reference sam~les were identical to those of Appendix 1.1.5. The results are shown in Table 18.

* Attenuation x 100. 157.

TABLE 18

Pyrolysis-Gas Chromatography of Reference Samples of Insolubilized Catalase (Analytical conditions are described in Section 2.4.6)

Weight Ratio Area (cm. 2) Area (cm. 2) Area Ratio of Enz,YU to of Catalase of Polymer E/P Polymer- Pea~ E Peak* P (Section 2.4.6)

0.0867 3.11 83.1 0.0374 3.19 94.4 0.0338 4.50 165 0.0274 3.17 92.4 0.0344 2.52 79.8 0.0316 1.68 93.8 0.0179 2.86 86.9 0.0329 7.48 211 0.0355 0.116 0.35 7.88 0.0447 2.61 54.4 0.0480 11.7 249 0.0470 2.79 572 0.0488 3.12 85.2 0.0366 2.08 57.7 0.0359 5.94 153 0.0389 3.98 104 0.0382

* Attenuation x 100 158.

Table 18 Continued - Weight Ratio Area (cm. 2) Area (cm. 2) Area Ratio of Enzyme to of Catalase of Polymer E/P Pol,-er. Peak* E Peak* P (Section 2.4.6)

5.98 128 0.0467 2.15 72-5 0.0296 0.145 9.85 184 0.0536 16.6 217 0.0765 2.85 42-9 0.0664 7.98 121 0.0662 6.12 125 0.0488 3.38 87-4 0.0386 3.78 92-4 0.0409 0.173 0.56 7.2 0.0789 4.55 10.0 0.0650 18.4 127 0.144 4.02 64-4 0.0624 8.40 78-6 0.107 4.75 47.4 0.100

* Attenuation x 100 159.

1.1.7 Solids Content of Suspensions of Bound Catalase

The results of estimations of the solids content of insolubilized catalase are recorded in Appendix 1.2.5. 160.

1 • 2 'l.1 HE EFFECTS OF tRADIAT ION ,l-2-1 Dosimetry

After irradiation at 4.0 cm. for 8.0 minutes, duplicate samples of the dosimetric solution had absor- bances of 0.190 (20.4°c) and 0.178 (20.0°c). These values corresponded on the calibration line (Figure 4) to total doses of 22.1 and 20.6 krads respectively and to dose rates of 166 and 155 krads per hour respectively. The mean, 160 krads per hour, after correction for natural decay of the source material 60cc (half-life 5.27 years67 ), was used in calculations of the dose absorbed by samples of free and bound catalase. 1.2.2 Irradiation of Solutions of Catalase

The changes in enzymic activity on irradiation of solutions of catalase have been expressed as the variations with time of the volume of potassium perman­ ganate solution re~uired to titrate the residual hydrogen peroxide. The slope (ml./min.) of the curve is a relative measure of the catalatic activity (Section 2.5). The detailed results, of which a typical set

(2.92 x 10-6 ill catalase) is shown in Figure 18(a) and Table 19, have been summarized in Table 20. 161.

2.10 .. ,,,,- 2.08 Volume of O.OlN 2 •.06 i0Ano4 (ml.) 2.04 0 L-----J..--,,--__JL-----,--....1.------+-- 10 20 30 40 Reaction 'rime (sec.)

(a) Catalase Solution (2.92 x 10-6 M) after Absorption of 185 krads of Radiation.

1.65

Volume tJf O.OlN KMn04 (ml.) 1.60 Ci)

1.55 L------L--.....---..,______. 0 10 20 JO 40 Reaction time (sec.) (b) Bound Uatalase Suspension (sa~ple D/12;Appendix 1.2.5) afta-Msorption of 1092 krads of Radiation.

F!_gure ~: '.£~,12i_,£al Ti trat!_2B_Da ta f2!~_!!2L.2! ~!!.ll,Llctiviti. 162.

TABLE 19

Radiation-Produced Changes in the Activity of Catalase in Solution (2.92 x 10-6 M Catalase; dose rate from Appendix 1.2.1: 137 krads/hour.)

Absorbed Titration Results Slope (ml./min.) Dose (krads) 'Catalatic--VO!ume of (activity) Reaction o.OlN KMno4 Time (sec.) (ml.)

0 Initial 2.07 0.162 9 2.05 25 2.00 38 1.97

Initial 2.08 0.138 11 2.07 41 -----1.97 97.3 Initial 2.09 0.120 11 2.06 26 ?.05 ---3t. 2.01 185 Initial 2.10 0.078 12 2.08 27 2.07 42 2.05 163. Table 19 Uontinued

Absorbed Titration Results Slo 1)e (ml./min.) j)ose (rl:rads) ~atalatic Volume of (activity) ~-=teact ion 0.01N KMn0 4 rrime (sec.) (ml.)

366 Initial 2.11 0.048 9 2.10 26 2.10 39 2.08 -- 530 Initial 2.11 0.026 9 2.10 21 2 .10 41 2.09 --- TABLE 20 The Hesidual Act!vitl of Irradiated Catalase Solutions

Gatalase .Absorbed .Activity Residual Concent:gation Dose (slope) Activity ;,o' (M X 10 ) (krads) (ml .7min.)

6.27 0 0.568 100 31.5 0.534 93.8 97.3 0.484 85.2 189 0.480 84.4 415 0.250 44.0 599 0.126 22.2 799 0.090 15.8 164.

Table 20 Continued

Catalase Absorbed Activity Residual Concentration Dose (slope) activity:' (M X 106 ) (krads) (ml./min.)

·------0 0.162 100 35.6 0.138 85.2 97.3 0.120 74.1 185 0.078 48.2 366 0.048 29.6 530 0.026 16.0 0 0.534 100 31.6 0.366 68.6 126 0.288 54.0 253 0.162 30.4 379 0.114 21.4 505 0.090 16.9 Duplicate Irradiation

0 100 375 0.028____ ,_.;....;:....,_17.3 0 0-462 100 32.2 0.408 88.4 164 0.180 39.0 284 0.150 32.9 370 0.120 26.3 453 ___2.·J __7_2 ____15.3 __ 165.

Table 20 Continued

<.;atalase Absorbed Activit) Residual Uoncentr&.tion Dose (slope Activity ,, (M x 106 ) (krads) (ml./min.)

0.184 0 0.378 100 31.7 0.306 81.0 92.5 0.228 60.3 150 0.162 42.8 291 0.060 15.9 548 0.048 __12.7

1.2.3 Radiation-Produced C~anses in the Amino Acid Composition of Free Catalase

The amino acid composition of catalase after irradiation in solution is shown in Table 21. TABLE 21 The Effect 04-Radiation on the Amino Acid Com~osition of Free Gatalase Dose rate; 132 krads/hour; totdl dose absorbed: 557 krads; 150 mg. catalase/ml. (6.15 x 10-6 M). Expressed as residues/100 total amino acid residues. Residue Control Sa~~le Irradiated Sample Lysine 3.20 2.90 Histidine 1.57 1.85 Arginine 2.30 2.16 166.

Table 21 Continued

Residue ~rol Samile Irradiated SamJ2le Aspartic acid 11.3 11.7 Threonine 8.14 5-69 Serine 5-61 5.41 Glutamic acid 11.9 11.9 Proline 4.81 5.36 Glycine 9.92 10.2 Alanine 11.3 11.2 Half Cystine 0.55 0.33 Valine 8.40 8.23 Methionine 1.49 2.12 Isoleucine 4.98 5.06 Leucine 8.68 8.95 Tyrosine 2.02 2.65 Phenylalanine 4-.08 4.39

1.2.4 Irradiation of Catalase-Diglycyl~!.Ee Solutions

Diglycylglycine did not interfere in the assay for catalatic activity. The results are shown in Table 22. 167.

TABLE 22

The Effect of Diglycylglycine on the Assay for Catalatic Activity

Catalase + (Gly) 3 Blank (1.47 mg. catalase/ml.; (1.47 mg. catalase/ml.) 38.~mg. (Gly)) /ml.)

Titration Results Slope Titration Results Slope

Catalatic Volume (ml/min.) Catala tic Volume ~ml/min.) Reaction O.OlN (activi- Reaction O.OlN activity) Time (sec.) KMn01 ty) Time KMnOt ----(ml. ---(sec.) (ml. Initial 2.08 0.550 Initial 2.08 0.560 8 1.98 10 1.98 22 1.88 22 1.86 38 1.75 39 1.71 ------

The changes in enzymic activity on irradiation of a tyyical set of solutions of catalase (6.15 x 10-6 M) and diglycylglycine are shown in detail in Table 23 and summari~ed for this and the other solutions in Table 24. 168.

TABLE 2l Changes in the Activity of Free Catalase Irradiated in the Presence of Diglycylglycine (Diglycylglycine concentration: 20.0 mg./ml.) (6.15 x 10-6 M Catalase; dose rate from Appendix 1.2.1: 135 krads/hour)

Absorbed Titration Results Slope Dose (krads) catalatic---Voiume (ml./min) Reaction O.OlN KMn04 (activity) Time ( sec • ) ( ml • )

0 Initial 2.01 10 1..93 26 1.78 42 1.63 Initial 2.00 0.444 8 1.94 22 1.84 -- 36 1.73 173 Initial 2.02 0.444 10 1.95 26 1.83 ------41 ------1.70 620 Initial 2.01 0.300 8 1.98 22 1.89 37 1.82 Table 23 Continued

Absorbed Titration Results Slope Dose {krads) Catala tic Volume {ml./min) Reaction O.OlN KMn04 Time {sec.) {ml.) -- 948 Initial 2.03 0.174 9 2.02 21 1.97 39 1.92 1039 Initial 2.01 0.198 8 1.99 24 1.93 39 1.87 1122 Initial 2.01 0.180 10 1.99 44 1.89 170.

TABLE 24

The Residual Activity of Irradiated Catalase-Diglycyl­ glycine Solutions (The concentration of diglycylglycine was 20.0 mg./ml.: 0.106M)

Catalase Absorbed Activity Residual Concentration Dose (slope) Activity 1, (krads) (ml.7min.) ll!! X 106 ) 6.15 0 0.528 100 83.7 0.444 84.0 173 0.444 84.0 620 0.300 56-9 948 0.174 32.9 1039 0.198 37.5 1122 0.180 34.1 0 0.162 100 81.0 0.138 85.2 166 0.126 77.8 ______10_2_2 ______0 .o 3_6 _____2_,2 __ ._2 __ _ 0 0.090 100 198 0.042 46-7 338 0.036 40.0 488 _____o_._0_23 ______2,5. 6__ 171.

1.2.5 Irradiation of Insolubilized Catalase

The effect of y--1rradiation on the enzymic activity of insolubilized catalase is shown in detail for a typical experiment (conjugate D/12; 10 mg. conjugate containing 0.96 mg. catalase) in Table 25 and Figure l8(b). ill the results are summarized in Table 26.

TABLE 25 Radiation-Produced Chan!!!_in the Activity of Bound Catalase (Conjugate D/12; 10 mg.oo.njugatea containing 0.96 mg. catalaseb; dose rate from Appendix 1.2.1: 138 krads/hour)

Absorbed Titration Results Slope Dose (krads) Catalatic Volumeof (ml./min) Reaction o.OlN KMn04 (activity) ----Time (sec.) (ml.) 0 Initial 1.62 0.156 11 1.60 26 1.59 -- 40 ------·--·---1.59 85.5 Initial l.62 0.132 9 1.60 22 1.59 36 1.58 a Results of total solids estimations according to Sect­ ion 2.4.7. b The 4uantity of catalase found in the amount of conjugate shown (from Appendix 1.1.4). 172-

Table 25 Continued -- Absorbed Titration Results Slope Dose (krads) Oatalatic Volume of ~ml./min.~ Reaction O.OlN Dno4 activity Time (sec.)_i!J..) 279 Initial 1.62 0.114 8 1.61 21 1.61 L 1.60 Duplicate Titration Initial 1.62 --- Initial 1.61 680 Initial 1.63 0.090 8 1.61 19 1.60 33 1.60 Duplicate Irradiation 0 Initial 1.63 0.354 7 l.60 16 1.58 Duplicate Titration Initial 1.63 173.

Table 25 Continued

Absorbed Titration Results Slope Dose (krads) Catala tic vo!uieof ~ml./min. ~ Reaction 0.011' Dn04 activity Time (sec.) (ml.) 1092 Initial 1.62 0.132 7 l.61 21 1.59 36 1.59 Duplicate Titration Initial l.63 9 1.61 174-

TABLE 26 The Residual Activiti of Irradiated Bound Catalase Susiensi2!!!

Sample Quantities& Dose A.ctivityb '/. Residual Irradiated (krads) (mi./min.) Activity

C/65 4 mg. 0 0.212 100 containing 0.22 mg. 30.2 0.216 100 154 0.114 53.8 1067 0.072 33.9 Duplicate Irradiation 0 0.234 100 305 0.222 94.9 . 614 0.126 53.8 3670------ea. 0.020 ca.10 D/6 12 mg. 0 0.108 100 containing 0.20 mg. 86-9 0.090 83.3 139 0.084 77.8 772 0.04,8 44.5 1082 -ea. 0.04 £!•35 Duplicate Irradiation 0 0.168 100 21.8 o.15Q_ 89.2 C/24 12 mg. 0 0.342° 100 containing 0.20 mg. 0 0.354° 641 0.144 41.4 175.

Table 26 Continued

Sample Quantitiesa Dose Aotivityb % Residual Irradiated (k.rads) (ml./min.) Activity

894 0.148 42-5 3721 £!.0.02 oa. 6 Duplicate Irradiation 0 0.186 100 318 0.102 54.8 477 0.126 67 .8 636 0.108 58.1 D/12 10 mg. 0 0.156 100 containing 0.96 mg. 85.5 0.132 84.6 279 0.114 73.1 680 0.090 57.7 Duplicate Irradiation 0 0.354 100 __1_0~9_2 ____0_._1~3_2 ___n~-- a The quantities irradiated as a suspension in 1 ml. of aqueous phosphate buffer (pH7); the first weight is that of the total conjugate and the second is

that of tne catalase contained in it. (See Sections

b These values are the slopes of the respective curves of residual hydrogen peroxide content versus time 176.

Table 26 Continued

and are proportional to the activity of the enzyme (Section 2.5). C The mean (0.348) of these values was used. 177.

2 PUBLISHED REACTION RATE CONSTANTS 2.1 REAC~I0HS OF ---·-----THE HYJROXYL RADICAL Rate constants for some pertinent reactions of the hydroxyl radical are shown in Tables 27 and 28.

TABLE 27

Rate of Reaction of OH• with Various Organic Substrates ---::.A;eous Solution16; 129 •136•137 Valu~s of pH and pK are given where they have a sig- a nificant effect on the rate of the reaction or the structure of the substrate.

Substrate .e!! ~&24,67 Reaction Rate (M ~r- sec. -I)

Acetonitrile 9 2.12 X 106

Acetamide 9 7.8 X 106 Acetat,-{on 9 4.75 4.25 X 107 Acetone 9 4.33 X 107 Methy}kcetate 9 6.54 X 107 Methane 9 1.43 X 108

Nitromethane 9 1.85 X 108

Methanol 9 6.10 X 108

Methylamine 12 10.66 1.43 X 109

5 1.1) X 107 Nitrobenzene 1 3.2 X 109 178.

Tab~7 (,ontinue-- d ~24,67 Substrate £E! Reaction Rate (M-1 sec. -1) 3enzene 9 sul;ihona.te ion 7 4.7 X 10 9 Benzonitrile 7 4.9 X 10 ,', X l);;' Benzcic aci:i 3 4.19 4.3

~r ('_,.. "t~ Fi.0 .-. 109 :3~ nzo d. t ,2 icn 6 - 9 Benzene 7 7.8 X 10 X lOlO Anif'0le 7 1.2 X 10lO ?henol 7.4 - 7.7 9.89 1.4 9 Thioethanol 6 - 7 5.1 X 10 7 Alanine 5.5 - 6.0 2. 35, 9. 69 4.6 X 10 8 Arginine 2.0 - 2.2 2.17,9.04,12.48 4.0 X 10 6.5 - 7.5 2.1 X 109 7 As,i)artic aciu 6.8 - 7.0 2.09,3.86,9.82 4.5 X 10 9 Cysteine 1 1.71,8.33,10.78 7.9 X 10 9 Cystine 2.0 - 2.2 1.65,2.26,7.85,9.85 3-2 X 10 7 Glutamic acid 2.0 - 2.2 2.19,4.25,9-67 7.9 X 10 6 Glycine 2.8 - J.O 2.34,9.6 6 X 10 5.8 - 6.0 1.0 X 107 9.5 - 9.7 1.1 X 109 Histidine 2.0 - 2.2 1.82,6.0,9.17 1.15 X 109 6.0 - 7.0 3.0 X 109 2 ') X 109 Isoleucine 2.0 - • l.. 2 • .36,9.68 1.05 179.

--Table 27 ~ontinued--- ~a24,67 Suostrate ~ Eeactio:1 Rate (l~-1sec. -I~ L~ucine 2.0 - 2.2 2.36,9.60 9.4 X 10 8 5.5 - 6.0 9.8 X 10 9.7 - 9.9 2. 25 X 109

Lysine (HC~ 2.0 - 2.2 2.18,8.95,10.53 3.7 X 108

Methionine 2.0 - 2.2 2.28,9.21 3.7 X 109 5.5 - 5.7 4.9 X 109

Phenylalanine 2.0 - 2.2 1.83,9.13 3.4 X 109

5.5 - 6.0 3.5 X 109

:Proline 2.0 - 2.2 1.99,10.60 1.75 X 108 Serine 2.0 - 2.2 2.21,9.15 1.5 X 108

5.5 - 6.0 1.9 X 108

Threonine 2.0 - 2.2 2063,10.43 2. 25 X 108 Try}:Jtophan 2.0 - 2.2 2.38,9.39 6.5 X 109

6.1 - 6.3 8.5 X 109

Tyrosine 2.0 - 2.2 2.20,9.11,10.07 5.8 X 109 Valine 2.0 - 2.2 2.32,9.62 4.15 X 108

Glycylglycine 2.2 - 2.4 3.14,8.24 9.5 X 107

5.5 - 6.0 1.3 X 108

C:-lycylglycyl- 2.8 - 3.0 3.22,8.09 1.45 X 108 glycine 5.5 - 6.0 2.0 X 1C8

8.5 - 8.7 1.05 X 109 180.

TABLE 28

Rate of Reaction of OH.:_!ill So~_fnorg~ic Substrates in

Aqueo~~tion12 Substrate Rate (;onstant (M-1 sec. -1)

e 3.0 X 1010 aq_ H" 7 X 109 ott· 5 X 109 7 H2 6 X 10 7 H202 2.3 X 10 181 •

2.2 MISCELLANEOUS R::SACTIONS

Table 29 depicts the rate constants for several pertinent reactions which occur in r--irradiated aqueous solutions.

TABLE 29

Rate of some Radical Reac!!,2~.!.!L,!!:,!adiated Ague~ Solutions12 Reactants Rate Constant ( M-I sec. -1)

e Ho+ 2.4 X 1010 aq + 3 H• 1010 e aq + 2.5 X 1010 eaq- + H202 1.2 X 1010 eaq- + 02 1.9 X

e H Po 6.0 X 109 aq + 2 4 9 eaq + eaq 4.5 X 10

H. + H. 2 X lOlO + H• + 02 2 X lQlO H o Ho+ (half-life ca.10-14 2 + 2 sec.)