Isomerases from Three Clostridial Species YUEN WAN SHING, JAMES M

Home , Clostridium pasteurianum, Enolase

JouRNAL oF BACToOLOGY, Apr. 1975, p. 177-184 Vol. 122, No. 1 Copyright 0 1975 American Society for Microbiology Printed in U.SA. Psychrophilic, Mesophilic, and Thermophilic Triosephosphate Isomerases from Three Clostridial Species YUEN WAN SHING, JAMES M. AKAGI,* AND RICHARD H. HIMES Departments of Biochemistry and Microbiology,* University of Kansas, Lawrence, Kansas 66045 Received for publication 27 December 1974

Triosephosphate isomerase was purified to homogeneity as judged by analytical gel electrophoresis from Clostridium sp. strain 69, Clostridium pasteurianum, and C. thermosaccharolyticum, which grow optimally at 18, 37, and 55 C, respectively. Comparative studies on these purified proteins showed that they had the same molecular weight (53,000) and subunit molecular weight (26,500). They were equally susceptible to the active site-directed inhibitor, glycidol phosphate. However, their temperature and pH optima, as well as their stabilities to heat, urea, and sodium dodecyl sulfate, differed. The proteins also had different mobilities in acrylamide gel electrophoresis. This difference in ionic character was also reflected in the elution behavior of the enzymes from hydroxyapatite and in the isoelectric points determined by isoelectric focusing in acrylamide gel. The amino acid composition of these proteins showed that the thermophilic enzyme contains a greater amount of proline than the other enzymes. The ratio of acidic amino acids to basic amino acids was 1.79, 1.38, and 1.66 for the thermophilic mesophilic and psychrophilic enzymes, respectively. This is consistent with the relative iscelectric point values of these three enzymes.

The biochemical basis for the loss in viability Enzyme assay. Triosephosphate isomerase activ- of psychrophiles at moderate temperatures is ity was routinely measured in the direction from now generally accepted to be protein denatura- DL-glyceraldehyde-3-phosphate to dihydroxyacetone tion (4, 8, 12). We found that a phosphate by coupling the product to a-glycerophos- recently phate dehydrogenase and following the oxidation of psychrophilic Clostridium contains a triose- reduced nicotinamide adenine dinucleotide at 340 phosphate isomerase (EC5.3.1.1) that is very nm. The assay mixture (1 ml) contained 0.14 nM heat labile and is probably responsible for the reduced nicotinamide adenine dinucleotide, 1.5 mM low maximum growth temperature of this orga- DL-glyceraldehyde-3-phosphate, 10 Ag of a-glycero- nism (17). The same enzyme from a thermoph- phosphate dehydrogenase (Sigma Chemical Co.), 5.4 ilic Clostridium was previously found to be very mM ethyenediaminetetraacetate, and 20 mM trietha- stable to heat and the one from a mesophilic nolamine hydrochloride (pH 7.9). One unit of activity Clostridium showed a thermostability some- is defined as the amount required to convert 1 Mmol of where between the two extremes (10). To gain D-glyceraldehyde-3-phosphate into dihydroxyacetone phosphate per min at room temperature. The protein more information about the physical and chem- concentration was determined either by a modified ical properties of these proteins and the reasons phenol reagent method (14) or, in the case of purified for their different thermostabilities, we purified proteins, by measuring the absorbance at 280 nm. The this enzyme from the psychrophilic, meso- E17 of purified triosephosphate isomerase was de- philic, and thermophilic clostridial species. termined by performing amino acid analysis on sam- Comparisons of some of the properties of the ples of known absorbancy at 280 nm. Assuming 95% purified enzymes show that the main difference recovery after hydrolysis and chromatography, an is in the ionic character of the proteins. E2%0 value of 13.8 was obtained for all three proteins. Rabbit muscle triosephosphate isomerase is reported to have an El%? value of 13 (13). MATERIALS AND METHODS Chromatography. Diethylaminoethyl (DEAE) - Organisms. Clostridium sp. strain 69 (18), Clos- cellulose (Schleicher and Schuell) was washed with tridium pasteurianum ATCC 6013 (6), and C. ther- 0.5 N KOH and 1 N HCL and after several water mosaccharolyticum strain 3814 (27) were grown at 18, washes was suspended in 0.2 M phosphate buffer, pH 37, and 55 C, respectively, in the media previously 7. Hydroxyapatite was prepared as described by described. All organisms were cultivated anaerobi- Bernardi (2). Sephadex G-100 (Sigma Chemical Co.) cally, harvested by certrifugation at their respective was allowed to swell and to degas overnight in 0.05 M mid-exponential growth phase, and lyophilized. tris(hydroxymethyl)aminomethane (Tris)-hydrochlo- 177 178 SHING, AKAGI, AND HIMES J. BACTERIOL. ride buffer, pH 7.5. Columns of these materials were Amino acid composition. A known amount, ap- packed by gravity and stored at 4 C. proximately 1.5 ml, of the sample solution containing Electrophoresis. Preparative disc electrophoresis 0.2 to 1.0 mg of the protein in 6 N HCl was incubated was used in the final step for the purification of at 110 C in an evacuated sealed tube for 24 h. At the triosephosphate isomerase. A separating gel (6 cm) end of the incubation, the tube was opened and containing 7.5% acrylamide, 0.12% methylene bisac- evacuated overnight in a desiccator containing NaOH rylamide, 0.03% (vol/vol) N,N,N',N'-tetrame- pellets. The dried residue in the tube was dissolved in thylenediamine, 0.07% ammonium persulfate, and 0.4 a known volume of 0.1 M lithium citrate buffer, pH M Tris-hydrochloride buffer, pH 9.0, was first formed 2.2. Samples (1 ml) were used for column chromatog- in a Canalco column PD-2/230. Then a stacking gel (2 raphy according to the method described by Spack- cm) containing 3.5% acrylamide, 0.06% methylene man et al. (21), using a Beckman Spinco model 120 C bisacrylamide, 0.06% (vol/vol) N,N,N',N'-tetrame- automatic amino acid analyzer. The combined con- thylenediamine, 0.5 mg of riboflavin per 100 ml, and tent of cystine and cysteine in triosephosphate isom- 0.06 M Tris-hydrochloride buffer, pH 6.8, was formed erase was determined by estimation of the amount of on the top of it by exposure to fluorescent light. The cysteic acid formed by the performic acid oxidation of electrode buffer contained 0.025 M Tris and 0.19 M these amino acids (9). The content of tryptophan was glycine, pH 8.2. One millilter of the sample solution determined by estimation of the tryptophanyl fluores- containing approximately 20 mg of protein, 5% su- cence obtained from the SDS-treated and mercapto- crose, and 15 gl of tracking dye (0.05% bromophenol ethanol-reduced protein (16). blue in water) was applied to the column. Separation Purification of triosephosphate isomerase. Tri- was performed at a constant current of 20 mA with the osephosphate isomerase was purified to homogeneity cathode on top. The elution was carried out with 0.4 as judged by analytical gel electrophoresis from the M Tris-hydrochloride buffer, pH 9.0, containing. psychrophile, Clostridium sp. strain 69, the meso- 0.03% (vol/vol) N,N,N',N'-tetramethylenediamine phile, C. pasteurianum, and the thermophile, C. at a flow rate of 1 ml/min. Each fraction contained 1.5 thermosaccharolyticum. The following procedure was ml of eluate. devised. All operations were performed at 2 to 4 C Analytical gel electrophoresis was used to deter- unless otherwise stated. mine the purity and relative mobility of the triose- (i) Extraction. Dried cells (100 g) were added to phosphate isomerase. The electrode buffer, separating 0.05 M Tris-hydrochloride, pH 8, containing 1.6 mM gel (1.2 ml), and stacking gel (0.3 ml) were as ethylenediaminetetraacet ate to make a 10 to 12% described above except that 10% acrylamide was used (wt/vol) suspension. This suspension was homoge- in the separating gel. Ten to 100 gl of the sample nized twice with a Manton-Gaulin model 15 M-8 TA solution containing approximately 50 gg of protein, homogenizer and centrifuged for 90 min at 30,000 x g. 10% glycerol, and 5 ,ul of tracking dye (0.05% bromo- The residue was discarded. phenol blue in water) was applied. Electrophoresis (ii) DEAE-cellulose column. The cell-free extract was run at a constant current of 2 mA per tube. The was applied to a DEAE-cellulose column (35 by 5 cm). gels were stained with 0.5% napthol blue black in 7% The column was eluted with 0.05 M Tris-hydrochlo- acetic acid solution for at least 1 h and destained ride, pH 7.6. The enzyme did not adhere to the electrophoretically in 7% acetic acid solution. DEAE-cellulose column under these conditions, and Polyacrylamide electrophoresis in sodium dodecyl the effluent containing triosephosphate isomerase sulfate (SDS) was used to determine the subunit activity was collected. molecular weight of triosephosphate isomerase by the (iii) Ammonium sulfate fractionation. The efflu- method of Weber and Osborn (26). ent from the DEAE-cellulose column was adjusted to Electrofocusing. Gel electrofocusing (28) was used pH 9 with 2 N KOH, brought to 60% saturation with to determine the isoelectric point of the triosephos- solid ammonium sulfate, stirred for 2 h, and cen- phate isomerase. The gel (1.4 ml) containing 7.5% trifuged for 90 min at 30,00 x g. The supernatant was acrylamide, 0.2% methylene bisacrylamide, 1% car- adjusted to pH 6.5 with 6 N HCl and then brought to rier ampholytes (LKB; pH 3 to 6), and 0.06% potas- 85% saturation with solid ammonium sulfate. The sium persulfate was prepared in each tube. The upper residue was collected by centrifugation, dissolved in a chamber (cathode) and the lower chamber (anode) minimal volume of 0.001 M potassium phosphate contained 0.4% triethanolamine and 0.2% sulfuric buffer, pH 7.5, and dialyzed against the same buffer acid, respectively. After the gels were run for 30 min to overnight. establish the pH gradient, 100 ,l of a solution (iv) Hydroxyapatite chromatography. Approxi- containing approximately 100 ug of the protein and mately 200 mg of the dialyzed protein in 20 ml was 10% sucrose was layered on top of the gel and under a applied to a hydroxyapatite column (40 by 3 cm) layer of 0.4% carrier ampholytes in 5% sucrose. previously equilibrated with 0.001 M potassium phos- Electrofocusing was continued for 10 h at 200 V and 4 phate buffer, pH 7.5. After washing with 400 ml of C. The pH gradient in the gels was determined by 0.001 M potassium phosphate buffer, pH 7.5, elution cutting a blank gel into 16 equal sections and measur- was carried out with a linear potassium phosphate ing the pH of a 1-ml water extract of each piece. The gradient (0.001 to 0.2 M) at pH 7.5 at a flow rate of 2.5 other gels were stained with 1% napthol blue black in ml/min. The total volume of the gradient was 800 ml. 7% acetic acid for 1 h after the removal of carrier Each fraction contained 7 ml of effluent. Enzymically ampholytes by soaking each gel in 200 ml of 2.5% active fractions were pooled and concentrated by trichloroacetic acid overnight. Destaining was done ultrafultration, using an Amicon PM 30 membrane. with 7% acetic acid electrophoretically. (v) Preparative disc electrophoresis. The concen- VOL 122, 1975 TRIOSEPHOSPHATE ISOMERASES FROM CLOSTRIDIA SP. 179 trated hydroxyapatite chromatography fraction was cally inactivated by glycidol phosphate. The made 5% with respect to sucrose and then applied to a kinetics and stoichiometry of the reaction and polyacrylamide gel column. Electrophoresis was run the structure of the reagent indicate that this is as described earlier. The purity of triosephosphate isomerase in the enzymically active fractions was a specific reaction with the active center of the checked by analytical gel electrophoresis. Fractions enzyme. It has been reported recently that a exhibiting only one band were pooled and concen- glutamyl residue, presumably at the active site, trated as described above. is esterified by this reagent (24). When triose- The purified enzymes were stored in 0.001 M potassium phosphate buffer, pH 7.5, at 4 C. The 8 thermophilic enzyme showed great stability, but the mesophilic and especially the psychorphilic enzymes 6 slowly inactivated under these conditions. 5

RESULTS x

Purification of triosephosphate isomerase. 4 The results of the purification scheme devised for the three proteins are summarized in Table 0 1. The yield was approximately 20 mg of protein from 100 g of dried cells. The recovery of enzyme activity from the crude extract was usually about 15%.

Molecular and subunit molecular weight. 0 1 2 3 0.1 0.2 0.3 0.4 0.5 Estimation of the molecular weight was done by Ve / Vo MOBILITY Sephadex gel filtration essentially as described FIG. 1. Native and subunit molecular weight. (A) by Andrews (1). A value of 53,000 was obtained Gel filtration on Sephadex G-100. Blue dextran (1 mg) for the triosephosphate isomerase purified from and proteins (2 mg each) were dissolved in 1 ml of 0.05 the three organisms (Fig. 1A). The subunit M Tris-hydrochloride buffer, pH 7.5, before being molecular weight was determined by SDS-gel applied to the column (95 by 1.5 cm). Elution was electrophoresis (26). A value of 26,500 was carried out with the same buffer. Fractions of 0.5 ml were collected at a flow rate of 1 ml/min. The marker obtained by this method (Fig. 1B). Thus, each proteins, bovine serum albumin, bovine hemoglobin, of these native enzymes is composed of two and horse heart cytochrome c, were located spectro- subunits. photometrically at 280, 406, and 412 nm, respectively. Effect of glycidol phosphate on the active (B) SDS-gel electrophoresis. The triosephosphate site. Rose and O'Connell (15) found that rabbit isomerase notation refers to the protein from each of muscle triosephosphate isomerase was specifi- the three organisms. TABLE 1. Purification of triosephosphate isomerase from Clostridium sp. strain 69, C. pasteurianum, and C thermosaccharolyticum Clostridium sp. strain 69 C. pasteurianum C. thermosaccharolYticum Fraction Total Total Sp Total Total Sp Total Total Sp protein activity act protein activity act protein activity act (mg) (mU) (U/mg) (mg) (mU) (U/mg) (mg) (mU) (U/mg) Cell extract 11,289 220 19 10,980 840 77 31,800 782 24 DEAE-cellulose 6,440 192 30 6,120 724 118 13,817 725 52 column 60% (NH4)2SO4 2,623 153 58 1,823 584 320 4,784 680 142 supernatant 85% (NH4)2SO4 1,400 108 77 684 396 579 2,443 550 225 precipitate Hydroxyapatite 15.6 9 615 29.6 49 1,655 18.2 33 1,813 chromatographya Preparative 2.4 4 1,750 5.2 19 3,653 2.2 13 5,909 disc electrophoresis

aApproximately 200 mg of the dialyzed protein was applied to the hydroxyapatite column. 180 SHING, AKAGI, AND HIMES J. BACTrERIOL . phosphate isomerase from the psychrophile, was assayed before and after exposure to differ- mesophile, and thermophile was incubated with ent temperatures for various time periods (Fig. this active-site-specific alkylating agent, the 4). The three enzymes showed quite different relative rates of inactivation appeared to be stabilities to temperature. Another way of com- quite similar (Fig. 2). Thus, there appears to be paring the stability is to incubate the enzymes no difference in the accessibility of the active for the same time period at different tempera- site of the three enzymes to this reagent. tures. The results of such experiments showed pH and temperature optima of the reac- that the enzymes from the psychrophile, meso- tion. The optimal pH for the reaction catalyzed phile, and thermophile lost 50% of the initial by the psychrophilic, mesophilic, and ther- activity after 10 min of incubation at 28, 45, and mophilic enzymes were 8.2, 7.9, and 8.6, respec- 64 C, respectively. tively (Fig. 3). The optimum region for the Urea and SDS denaturation. Triosephos- activity of these enzymes was somewhat nar- phate isomerase was assayed before and after rower than that of rabbit muscle triosephos- exposure to different concentrations of urea for phate isomerase, which has a plateau maximal various time periods. The resistance of the three activity between pH 7 to 9 (11). proteins to urea denaturation was found to When triosephosphate isomerase was assayed follow their resistances to heat denaturation at various temperatures under otherwise identi- (Fig. 5). In fact, the thermophilic triosephos- cal conditions, the maximal velocity of the phate isomerase was very stable. This enzyme reaction was found to occur at 25, 42, and 62 C was also unaffected by 8 M urea in the time for the psychrophilic, mesophilic, and thermophilic enzymes, respectively. Although the maximal temperature for the reaction of the

thermophilic enzyme might be limited by the 8- thermostability of the coupling enzyme, these results indicate that these three enzymes have c 60 greatly different temperature optima. Thermostability. Triosephosphate isomerase 40

20 Clostridium sp. strain 69 C. pasteurianur C. thermoacarlSu 6 )77 8 9 10 6 7 l8 9 10 6 7 8 9 lo 111l

FIG. 3. pH optimum for the triosephosphate isomerase activity. Triosephosphate isomerase was assayed at 25 C as described in the text. Triethanol- was cr amine buffer used from pH 6.5 to 8.1. Diethanol- amine buffer was used from pH 8.2 to 11.0. -z

o5 cf

_ 0 10 20 30 40 50 60 LW I~I 1 M1 NUTES 0 10 20 30 40 50 60 0 10 20 30 40 50 60 0 10 20 30 40 50 60 MI NUTES FIG. 2. Effect of glycidol phosphate P on triosephosphate isomerase. The enzyme solutions were FIG. 4. Heat inactivation of triosephosphate iso- adjusted with 0.02 M triethanolamine-hydrochloride, merase. The enzyme was assayed before and after the pH 7.5, to have an initial activity of 10 U/ml. Two incubation of 0.1 gg of the protein in 1 ml of 0.02 milliliters of the solution was incubated with 10 mM M triethanolamine-hydrochloride buffer, pH 7.5, con- glycidol phosphate P at 0 C. Enzyme activity was taining 1 mg of bovine serum albumin at the tempera- assayed after various periods of time. tures shown for various time periods. VOL. 122, 1975 TRIOSEPHOSPHATE ISOMERASES FROM CLOSTRIDIA SP. 181

0- -~~~~~--0 three enzymes were determined by acrylamide C. thermosaccharolyticum gel electrofocusing according to the method of 90 O. Wrigley (28). Values of 5.6, 6.2, and 6.4 were obtained for the thermophilic, the psychrophilic, and mesophilic triosephosphate isomerases, respectively. -0 C.Cpasteurianum Amino acid composition. The amino acid 60 composition of the enzymes was determined (Table 2). In most instances, there were no significant differences between the proteins. However, the content of some amino acids Clostridium did sp. strain 69 vary significantly. Aspartic acid varied; the highest amount was found in the thermophilic 30 enzyme, whereas the lowest was found in the .\ mesophilic enzyme. A greater amount of proline was also found in the thermophilic enzyme. The 10 glycine and alanine contents were lower in the thermophilic enzyme. The psychrophilic enzyme contained significantly less tyrosine and MINUTES more half-cystine than the other two enzymes. FIG. 5. Urea denaturation of the triosephosphate Table 2 also contains the published amino acid isomerase. Approximately 2 jg of the enzyme in 1 ml content of triosephosphate isomerase from rab- of triethanolamine-hydrochloride buffer, pH 7.5, was bit muscle (7). Many similarities are noted incubated at 0 C with 3 M urea. Samples (5, l) were when compared with the clostridial triosephos- withdrawn periodically for assay. phate isomerases. The molecule weight calculated from the studied and lost only 25% of its activity after 30 amino acid composition was 53,600, 53,000, and min in 10 M urea at 25 C. 54,000 for the thermophilic, mesophilic, and The stability of triosephosphate isomerase in psychrophilic enzymes, respectively. These val- SDS was also examined. The psychrophilic ues are in good agreement with those deter- enzyme was much more susceptible to denatu- mined by gel filtration. ration by SDS. In 1.4 mM SDS, after 10 min this enzyme was 90% inactivated whereas the mesophilic and thermophilic enzymes were completely stable. In the presence of 5 mM DISCUSSION SDS, all three of the proteins were unstable, but The purified thermophilic, mesophilic, and the mesophilic enzyme was inactivated at a rate psychrophilic triosephosphate isomerases show about twice that of the thermophile enzyme. great differences in their susceptibility to dena- Electrophoretic mobility. The purified tri- turants. This was especially evident when the osephosphate isomerase from the psychrophile, effects of temperature and urea was examined. mesophile, and thermophile each showed a Presumably, this is due to the presence of single band in analytical gel electrophoresis. structural differences in the proteins. Although Three separated bands were obtained from the some differences in chemical composition and mixture of these three isomerases, indicating physical properties were noted, it is difficult to that they were different in molecular charge assign to these differences a causative effect of (Fig. 6). protein stability. For example, the three en- Hydroxyapatite chromatography. Triose- zymes were found to have different electropho- phosphate isomerase from the psychrophile, retic mobilities and isoelectric points, which mesophile, and thermophile showed different were 6.2, 6.4, and 5.6 for the psychrophilic, elution behavior in hydroxyapatite chromatog- mesophilic, and thermophilic enzymes, respec- raphy during the purification. When the mix- tively. These values are consistent with the ture of the three pure enzymes was applied to a ratios of acidic to basic amino acids, which were hydroxyapatite column and eluted with a linear 1.66, 1.38, and 1.79, respectively. The amide potassium phosphate gradient, they came off content, however, is not known. The variation consecutively in the order thermophile, psy- in ionic character was also reflected in the chrophile, and mesophile (Fig. 7), which sug- elution behavior of these enzymes from the gests that they have different ionic characters. hydroxyapatite column. The differences in ionic Isoelectric point. Isoelectric points of these character suggest that electrostatic interactions 182 SHING, AKAGI, AND HIMES J. BACTERIOL.

FIG. 6. Analytical gel electrophoresis of the purified triosephosphate isomerase from C. thermosaccharolyticum (1), C. pasteurianum (2), Clostridium sp. strain 69 (3), and the mixture of these three (4). Electrophoresis was performed as described in the test.

7 might play a role in maintaining the structure of 1 C. thermosaccholytlcwm 2 Clostridium sp strain 69 native proteins. In fact, several proteins such as 6 3 C. pasteurianum a-amylase (5), glyceraldehyde-3-phosphate de- I hydrogenase (20), and 5 II ferredoxin (22) from 20 II thermophilic organisms have been found to 4 ~ ,20 contain a higher level of acidic amino acids than those of the proteins isolated from the other ;-9 3- -K sources. The salt bridge has been assumed not to contribute significantly to protein stability. I~ ~~~~~910~~~~~~~~~~~~~~~1 However, if ionic bonds are strategically placed, r1- the contribution of electrostatic forces to the 9 stability of the proteins might be significant. 60 70 80 90 100 110 120 100 140 150 160 F RAC TI ON NUMB[ R In an attempt to measure the capacity for FIG. 7. Hydroxyapatite chromatography of the triosephosphate isomerases. One milliliter of a solution volume of the gradient was 600 ml. Fractions of 1.8 ml containing approximately 0.1 mg of each triosephos- were collected and each was monitored for enzyme phate isomerase in 0.001 M potassium phosphate activity and conductivity. The identity of the peaks buffer, pH 7.5, was applied to a hydroxyapatite was determined from the thermostability of the en- column (45 by 1.5 cm) equilibrated with 0.001 M zymes. The enzyme activity of peaks 1, 2, and 3 potassium phosphate buffer, pH 7.5. After washing retained 100, 12, and 85%, respectively, of their initial with 300 ml of this buffer, elution was carried out with activity after 10 min of incubation at 40 C. The values a linear potassium phosphate gradient (0.001 to 0.3 for the incubation at 45 C were 100, 5, and 70%o, M) at pH 7.5 at a flow rate of 1 ml/min. The total respectively. VOL. 122, 1975 TRIOSEPHOSPHATE ISOMERASES FROM CLOSTRIDIA SP. 183

TABLE 2. Amino acid composition of clostridial TABLE 3. Values of hydrophobicity parameters of triosephosphate isomerase1 triosephosphate isomerase No. of residues/53,000 g H4,avea (nearest integer) Source (Kcal/ NPSb residue) Amino Acid C. ther- C Clos- mosac- C. pas- tridium Rabbit C. thermosaccharolyticum ...... 1.170 0.297 charoly- teur- sp. strain muscleb C. pasteurianum ...... 1.168 0.284 ticum anum 69 Clostridium sp. strain 69 ...... 1.121 0.260 Lysine ...... 40 .49 .47 .42 a Total hydrophobicity divided by the number of Histidine ...... 8 9 7 8 total residues (3). The hydrophobicity of each amino Arginine ...... 15 10 11 16 acid has been determined (26) and the values are Aspartic acid ... 62 40 54 42 known. Total hydrophobicity is the sum of these Threonine ...... 21 25 24 30 values of each amino acid in a protein. Serine ...... 20 16 20 24 bCalculated according to Waugh's definition by Glutamic acid ... 51 54 54 54 counting the Try, Ile, Tyr, Phe, Pro, Leu, and Val Proline ...... 22 14 16 20 residues and expressing the sum as a fraction of the Glycine ...... 32 47 44 48 total number of residues (28). Alanine ...... 32 47 48 54 Valine ...... 47 45 42 50 erases (Table 3). However, whether this is Methionine ..... 12 9 7 4 not known. Isoleucine ...... 33 34 40 30 significant is Leucine ...... 36 35 38 30 Evidence has been presented in this study to Tyrosine ...... 14 15 6 10 show that certain structural differences must Phenylalanine 13 19 16 16 exist in triosephosphate isomerase obtained Half-cystinec .... 5 5 11 10 from thermophilic, mesophilic, and psychro- Tryptophand .... 11 11 10 8 philic sources. However, it is clear that in order to be able to explain the molecular mechanism Total residues ... 474 484 495 496 underlying protein thermostability, comparisons of the primary structures are necessary. More a Amino acids were determined after acid hydrolysis as described in the text. importantly, since differences in stability of ° Calculated from the amino acid composition de- proteins can be caused by few or many changes termined by Corran and Waley (7). in the chemical composition as well as in the c Half-cystine was determined as cysteic acid after physical interaction of amino acid residues, it oxidation of the protein with performic acid (11). will not be possible to explain differences in d Tryptophan was determined by estimation of the stability until the three-dimensional structure tryptophanyl fluorescence obtained from the SDS- of the proteins is known. treated and mercaptoethanol-reduced protein (18). ACKNOWLEDGMENTS This investigation was supported by grants from the hydrophobic interaction in a protein from its University of Kansas Research Fund and Biomedical amino acid composition, several parameters, Sciences Support Fund and by a research grant from the such as HOave (3) and NPS (25), have been National Aeronautics and Space Administration. J. M. A. is the average hydrophobicity, received Public Health Service career development award proposed. HOkave GM 30,262, and R. H. H. received Public Health Service which represents the average free energy change career development award GM 13,320, both from the Na- occurring in transferring the amino acid resi- tional Institute of General Medical Sciences. dues of a protein from a water environment to an organic environment. NPS is the ratio of the LITERATURE CITED Re- 1. Andrews, P. 1965. The gel-filtration behaviour of pro- nonpolar residues to the total residues. teins related to their molecular weight over a wide cently, Singleton and Amelunxen (19) have range. Biochem. J. 96:595-606. made a calculation of the values of these param- 2. Bernardi, G. 1971. Chromatography of proteins on hy- eters from a large number of thermophilic and droxyapatite, p. 326-327. In S. P. Colowick and N. 0. that al- Kaplan (ed.), Methods in enzymology, vol. 22. Aca- mesophilic proteins. They recognized demic Press Inc., New York. though minor differences occur, there is no 3. Bigelow, C. C. 1967. On the average hydrophobicity of definite correlation between these parameters proteins and the relation between it and protein and the thermal stability of the proteins exam- structure. J. Theor. Biol. 16:187-211. there are some correlations 4. Burton, S. D., and R. Y. Morita. 1963. Denaturation and ined. It seems that renaturation of malic dehydrogenase in a cell-free between these parameters and the thermal extract from a marine psychrophile. J. Bacteriol. stability of the clostridial thermophilic, meso- 86:1019-1024. philic, and psychrophilic triosephosphate isom- 5. Campbell, L. L., and G. B. Manning. 1961. Thermostable 184 SHING, AKAG3I, AND HIMES J. BACTERIOL.

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