kinetics of heat inactivation of enzymes in light and heavy water (lysozyme, ribonuclease, and...

JOURNAL OF POLYMER SCIENCE VOL. XXXI, PAGES 281-290 (1958) PRAGUE SYMPOSIUM

Kinetics of Heat Inactivation of Enzymes in Light and Heavy Water*

(Lysozyme, ribonuclease, and desoxyribonuclease)

D. SHUGAR and E. GAJEWSKA, Institute of Biochemistry and Biophysics, Academy of Sciences, Warsaw, Poland.

The use of heavy water as a tool in the study of reaction paths and mecha- nisms has been common practice now for many years. I ts application to the study of the mechanism of enzyme inactivation or protein denaturation has, however, been barely touched upon. Since the mechanism in such processes is believed to involve the rupture of a number of intramolecular hydrogen bonds, it is to be expected that, if the hydrogen in such bonds is replaced by deuterium, the rate-constants for the breakage of these bonds will be lower than those for the corresponding bonds to hydrogen, because of zero-point energy considerations.

This, of course, assumes that it is possible to substitute by deuterium all the hydrogens involved in such bonding by dissolution of the protein in question in heavy water. Morowitz and Chapman2 report that all exchangeable hydrogen in globular proteins is replaced by deuterium within 20 minutes of the time of dissolution in heavy water. Linderstrom- Lang et aL3 and Haggis4 have shown, however, that the situation is not quite so simple, and that the rate and extent of exchange are dependent on the specific protein, the temperature, and pH. Some discrepancies also exist between the quantitative results of different observers due partially, perhaps, to the difference in techniques used. Nonetheless the extent to which exchange does take place is such that one may expect to encounter marked differences in heat inactivation as between light and heavy water.

Two trials along these lines have been hitherto reported. Maybury and K a t ~ , ~ using optical rotation as a criterion of denaturaticn, find that urea denaturation of ovalbumin in heavy water is only one-third as fast as in HzO. While this is about what one would be led to expect, the system used in this case is somewhat complicated by the fact that, aside from the sub- stitution of deuterium for hydrogen in intramolecular hydrogen bonds, three possible types of interaction exist : protein-solvent, protein-urea, and solvent-urea. A simpler arid more quantitative study, which appeared during the course of our work, is that of Guild and v:Ln Tubergen6 on the heat inactivation of catalase at neutral pH in the temperature range 50-

* Presented at the Symposium on Macromolecules, Prague, September 9-15, 1957.

281

282 D. SHUGAR AND E. GAJEWSKA

70°C. The findings of Caldwall et aL7 on the inactivation of catalase in D2O and H2O a t room temperature, referred to by the previous authors, is not really relevant to heat-inactivation studies since the mechanism cannot be the same.8

We have selected for our studies three enzymes, lysozyme, ribonuclease, and desoxyribonuclease, the first two of which have been previously studied in H209-11 and the iast of which we are concurrently investigating. Except a t alkaline pH values above about 8.0, lysozyme is inactivated according to a first-order reaction with thermodynamic constants for the inactivation process similar to those for ordinary chemical reactions and which we have assumed to indicate the lack of importance of hydrogen bonding for enzymatic a ~ t i v i t y . ~ Above pH 8, the dependence of reaction velocity on temperature is not in accord with Arrhenius' law and the activation energies are quite high. In the case of ribonuclease, the reaction order and rate are dependent on enzyme concentration, the order approaching unity as the concentration increases towards 1 mg./ml., indicating that the reaction is rather an intermolecular onelO-ll and showing some resemblance to pepsin in this respect ;I2 nevertheless, the thermodynamic constants are here also of the same order as for ordinary chemical reactions. Desoxyribo- nuclease, on the other hand, with a molecular weight of 60,00013 as com- pared to 15,000 for lysozyme and ribonuclease, exhibits both a high energy of activation and a large entropy change as normally observed for such processes; furthermore, in acid medium a t about pH 3, the heat inactivation of this enzyme is partially reversible on c~ol ing . '~

It therefore appeared to us that a comparison of the behavior of the above three enzymes in water and heavy water might provide some addi- tional useful information as to the mechanism of the inactivation process and also the importance of hydrogen bonding in protein molecules of different molecular weights and structures.

MATERIALS

Desoxyribonuclease was a Worthington crystalline preparation. Ribo- nuclease from several different commercial sources was used, with similar results. Lysozyme was prepared according to the procedure of Alderton and Fevold15 and recrystallized five times in the isoelectric form. In initial experiments, the heavy water used was 97% DzO which, in some experiments, was reduced to 95% as a result of the addition of H20 solutions of enzyme; in later experiments 99.85% D2O was used. Buffers were usually 0.02 M acetate, phosphate, or borate, as required, and HC1 was used a t appropriate dilutions to obtain pH values in the neighborhood of 3 or lower; pH (or pD, as the case may be) was controlled by means of Radiometer and Soviet pH meters, equipped with glass electrodes.

EXPERIMENTAL

The experimental procedure was largely as previously de~cribed.~-I' Because of the limited quantities of heavy water, ribonuclease, and desoxyribonuclease available, all inactivation experiments were conducted in 1- ml. capacity, 3-mm. diameter tubes, glass-stoppered to prevent evaporation

KINETICS OF HEAT INACTIVATION OF ENZYMES 283

during heating, and with the use of only several tenths of a milliliter of enzyme solution, from which aliquots were removed with calibrated micro- pipettes.

Each experiment included suitable controls from which a calibration curve of enzyme activity versus concentration could be drawn, and most experiments were repeated several times. Experiments in light and heavy water were performed simultaneously, with separate calibration curves for each. Lysozyme activity was measured turbidimetrically, l6 ribonuclease by means of a colorimetric pr~cedure , '~ and desoxyribonuclease by acid precipitation of nonhydrolyzed DNA and spectrophotometric estimation a t 2600 A. of the acid-soluble fraction with the use of a Soviet SF-4 spectro- photometer.

Thermodynamic constants were calculated from the theory of absolute reaction rates.sals

RESULTS

The activity and stability a t room temperature of both lysozyme and ribonuclease were essentially unaltered by dissolution in heavy water. In the case of desoxyribonuclease, however, soIutions in DzO were found to be about 10-1570 more active. Whether this is due to some effect on enzyme, substrate, or both, remains to be determined. The amount of D,O added to the substrate solution with the enzyme was oniy 1% of the substrate volume, and it is difficult to see how this could have any appreciable effect. For catalase, Guild and van Tubergen6 found no difference in activity upon dissolution in heavy water, which is in agreement with previous observations by but not with the observation of Macht and Bryan.20

Lysozyme inactivation was investigated a t a concentration of 1.0 mg./ml. a t two pH values; 7.2, a t which previous observations indicated a rela- tively low energy of activation and concomitant small change in entropy; and pH 8.8 where we had previously found a high activation energy and a correspondingly large entropy increase. The results are illustrated by Figure 1 and Table I.

TABLE I Constants for Inactivation of Lysozyme"

k X E , A H , A F T , AS:, T, kcal./ kcal./ kcal./ cal./"

pH Medium "C. set.-* mole mole mole mole

7 . 2 HzO 72 11 75 14 30 29 25 13 78 22

75 14 37 36 25 33 78 22

8 . 8 HzO 68 10 105 104 25 234 70 26

8 . 8 D,O 68 4 . 0 118 117 25 270 70 11

7 . 2 DzO 72 8 . 9

pH 7.2 in phosphate buffer, pEI 8.8 in borate.


Fig. 1. Heat inactivation of lysozyme in borate buffer a t pH 8.8 in HzO (- -) and D 2 0 (-) a t temperatures indicated.

TABLE I1 Constants for Inactivation of Desoxyribonuclease"

k X E, AH, AFz, AST, T, kcal./ kcal./ kcal./ cal./"

pH Medium "C. set.? mole mole mole mole

2 . 4 HzO 80 5.2 18 85 7.3

2 . 8 Hz0 75 6 .2 DzO 75 7.4

100 8 .9

68 7.7 D20 68 1 .6

68 13.2

68 6 . 6

66 24.0 D20 63 6. I

- - -

- - __ - 3.4 H2O 90 4 . 1 18 17 27 -27

5.6 H2O 65 1.1 151 148 26 366

- - - -

6.2 H2O 66 3.1 168 167 26 416

DzO 66 1 . 3 185 184 26 466

7.3 H2O 63 8 . 7 78 77 25 158

- - - -

" Appropriate dilutions of HCI for pH 2.4-3.4 and phosphate buffer for other pH values.


Fig. 2. Heat inactivation of desoxyribonuclease in phosphate buffer at pH 5.6 and 68" in HzO (- -) and D,O (-).

I I i

168" I

I i

/ f I

f I

I I

I

Fig. 3. Inactivation of desoxyribonuclease in phosphate buffer a t pH 6.2 in HzO (- -1 and DzO (-) a t temperatures indicated.


It should be noted that, although the velocity constants at alkaline pH are in agreement with those previously obtained, the corresponding rate constants at pH 7.2 are somewhat higher than for our earlier results. The reason for this is not clear, despite the fact that the lysozyme preparation used was a different one. Since, however, the activation energy in HzO in these studies is practically the same as that previously obtained (30 kcal./mole as against 33 kcal. /mole), and in the present experiments measurements in light and heavy water were made simultaneously under identi- cal conditions, we feel that the comparison between HzO and D20 is valid.

Ribonuclease was studied at pH 7.2 at 85" and 90" and at pH 8.8 at 70" and 73". Since no marked differences in rate constants in light and heavy water were observed under these conditions, reference should be made to our previous worklOfll for the thermodynamic constants for inactivation (see also Table 111).

Desoxyribonuclease: The results for this enzyme are tabulated in Table I1 and illustrated partially by Figures 2 and 3.

DISCUSSION From the overall results it will be seen (Table 111) that the isotope effect

kH/kD varies with the activation energy E in the general direction to be expected, i.e., the reaction with the lower E exhibits the lower isotope effect.' In the case of ribonuclease and for lysozyme a t neutral and (by inference from our previous results) acid pH, it may be concluded that the inactivation process is not due primarily to the rupture of hydrogen bonds in the rate-determining step, in accordance with our previous sugges-

This does not, of course, exclude the possibility that some hydrogen bonds are broken during inactivation, but only that, if such is the case, they are not involved in the rate-determining step and are not placed in evidence by the above results because their rupture is not accompanied by any appreciable unfolding of the molecule. This is a possibility which exists in the case of both lysozyme and ribonuclease because of extensive disulfide cross linking21*22; for example, the acid denaturation of lysozyme is not accompanied by any change in viscosity, which indicates a lack of unfolding of the molecule.23

. t i ~ n . ~ - l '

TABLE I11 Isotope Effect for Heat Inactivation of Enzymes versus Activat,ion Energy

Enzyme E',

PH h / k D kcal./mole

Ribonuclease 7 2 8 . 8

Lysozyme 7 . 2 8 .8

5 . 6 6 . 2 7 . 3

Desoxyribonuclease 2.4-3.4

1 25 1 -30 1 33 2 . 4 112

- <0.8 -18 5 . 0 150 2 . 1 175 1 . 5 78


That ribonuclease does, in fact, contain labile hydrogen bonds has been demonstrated spectroph~tometrically~~~~~ and by deuterium exchange studies.26 It would, obviously, be of interest to show whether these actually are ruptured under the conditions prevailing in these studies, and such measurements are now under way on ribonuclease and other proteins in this laboratory.

The theoretically expected isotope effect for temperatures up to about 400" is approximated by:

and should therefore decrease with temperature.' It follows also from this that, for a reaction in which the rupture of hydrogen bonds is the rate- determining step, the activation energy El and consequently AS', will be higher in heavy water. An examination of the data for lysozyme a t pH 8.8 and desoxyribonuclease a t pH 6.2 (Tables I and 11) shows that this is indeed the case.

For catalase, Guild and van Tubergen6 report that both E and AS' are almost doubled in going from light to heavy water. It is, however, difficult to see what physical interpretation may be given to such an enormous increase in AS' (from 191 to 360 cal./mole-degree) if we adopt the usual interpretation of AS' in terms of the "opening up" or unfolding of the protein chain during the course of i n a c t i v a t i ~ n . ~ , ~ ~ It is, of course, possible that solvent effects may be involved, e.g., hydration2* or changes in dissociation constants of various groups (see below), but the results for lysozyme and ribonuclease argue against this.

The behavior of desoxyribonuclease in acid media requires further study. Kunitz14 claims that, following inactivation of a dilute solution of this enzyme a t pH 2.8, full activity is regained a t room temperature over the course of 20 hours, if allowance is made for spontaneous inactivation of a suitable control. We have found that such reversibility indeed obtains over the pH range 2.4-3.2, but only if the initial heat inactivation does not exceed about 50%. To date, however, we have been unable to obtain any evidence for reactivation in heavy water. Since reactivation in ordinary water occurs in the pH range in which carboxyl groups dissociate (as is also the case for the reversible acid denaturation of chymotrypsinogenB), and since it is to be expected that the dissociation constant of the carboxyl groups would be appreciably affected in heavy water,' this may account for the lack of reversibility a t the pH values tried. Although we have been unable to locate any data in the literature on the titration of proteins in heavy water, it is a well-known fact that the dissociation constants of many compound^^^^^^ are reduced in DzO, the ratio k H / k D varying from 2 4 . It would obviously be desirable from this point of view to run a titration curve on desoxyribonuclease in light and heavy water, but the quantity of enzyme available to us a t the moment is inadequate for this purpose.

On the whole, our results, together with those of Maybury and Ka td for ovdbumin and Guild and van Tubergene for catalase, show that for


heat inactivation of enzymes (or denaturation of proteins) reasonable agreement does exist between the isotope effect and the importance of hydrogen bonding for enzymatic activity, as deduced from the thermodynamic constants for inactivation. At the same time, it is clear that no generalizations may be drawn from studies on a single protein and that the interpretation of the behavior of each must take into account its specific physico-chemical properties.

It should be pointed out that, in addition to our present and previous observations indicating the lack of importance of hydrogen bonding in the maintenance of the native configuration of ribonuclease, several other observers have arrived at an essentially similar conclusion on the basis of different experimental findings. Anfinsenlal for example, suggests that, in view of the stability of ribonucleaee in 8 M urea, much of the enzyme structure is nonessential insofar as enzymatic activity is concerned. Rogers and K a l n i t ~ k y ~ ~ conclude from studies on the carboxypeptidase digestion of ribonuclease, that the “active center” of the enzyme is close to the C-termi- nal end of the chain and involves only about 20770 of the molecule. It would obviously be of interest to know whether those phenolic groups of ribonuclease which are strongly h y d r o g e n - b ~ n d e d ~ ~ , ~ ~ are contained in this active centre.

For lysozyme the situation is somewhat more complicated, inasmuch as the appreciable isotope effect observed in alkaline medium, although in agreement with the observed high values of E and AS’, suggests a more drastic unfolding of the molecule. Since lysozyme and ribonuclease have similar molecular weights, and since both consist of a single polypeptide chain cross linked by -S-S- groups,21s22 further comparative data might be of value in drawing some conclusions about any differences in structure between the two. In particular, a comparison of deuterium exchange for both these enzymes might be of some help in this direction. Some pre- liminary measurements on deuterium exchange in lyeozyme, made by Mr. Wierzchowski of our institute in the laboratory of Prof. Linderstrom-Lang, indicate some similarity to that of ribonuclease. However, more precise measurements are necessary, particularly in view of the claim of Haggis33 that lysozyme shows greater exchange than ribonuclease.

This brings us once more up against the problem of the relationship of the above results to deuterium exchange in the proteins studied. Our assumption has been that such exchange is sufficient to embrace a t least some of the bonds involved in heat inactivation, in view of the high temperatures reached in heat-inactivation studies. Haggis, 3 3 however, reports that, for ribonuclease, exchange is only 90% complete even at 60°C., although these results are not in agreement with those of Hvidt.26 The effect of pH is also of importance; Lenormant and Blout found that a por- tion of the peptide hydrogens of bovine serum albumin do not exchange with deuterium at the isoelectric point or in acid medium.34 The effect of incomplete deuterium exchange will be reflected by a lower value for the isotope effect than that to be expected. It is clear, therefore, that suitable data on deuterium exchange are necessary for an adequate interpretation of the results of inactivation.


References 1. K. B. Wiberg, Chem. Reve., 55,713 (1955). 2. H. J. Morowitz and M. W. Chapman, Arch. Biochem. Biophys., 56, 110 (1955). 3. K. Linderstrom-Lang, Symposium on Peptide Chemistry, The Chemical Society,

4. G. H. Haggis, Biochim. et Biophys. Acta, 19,545 (1956). 5. R. H. Maybury and J. J. Kate, Nature, 177, 629 (1956). 6. W. R. Guild and R. P. van Tubergen, bcience, 125,939 (1957). 7. M. L. Caldwall, S. E. Doebbling, and S. Manion, J . Am. Chem. Soc., 78,84 (1956). 8. H. Neurath, J. P. Greenstein, F. W. Putman, and J. C. Erickson, Chem. Rev., 34,

9. D. Shugar and E. Syruczek, Bull. acad. polon. sci., Classe ZZ, 2, 73 (1954). 10. E. Gajewska and D. Shugar, Bull. acad. polon. sci., Classe ZZ, 3, 117 (1955). 11. E. Gajewska and D. Shugar, Bull. acad. polon. sci., Classe ZZ, 4, 157 (1956). 12. E. J. Casey and K. J. Laidler, J . Am. Chem. SOC., 73, 1455 (1951). 13. G. Gehrmann and S. Okada, Biochim. et Biophys. Acta, 23, 621 (1957). 14. M. Kunitz, J . Gen. Physiol., 33, 349 (1950). 15. G. Alderton and H. L. Fevold, J . Biol. Chem., 164, 1 (1946). 16. D. Shugar, Biochim. et Biophys. Acta, 8, 302 (1952). 17. D. Shugar, Bull. acad. polon. xi., Clmse ZZ, 1, 39 (1953). 18. S. Glasstone, K. J. Laidler, and H. Eyring, The Theory of Rate Processes, McGraw-

19. D. L. Fox, J . Cellular Comp. Physiol., 6, 405 (1935). 20. D. I. Macht and H. F. Bryan, Proc. SOC. Exptl. Biol. Med., 34, 190 (1936). 21. C. Anfinsen, R. R. Redfield, W. L. Choate, d. Page, and W. R. Carroll, J . Biol.

22. H. Fraenkel-Conrat, J . Am. Chem. Soc., 73, 625 (1951). 23. B. Jirgensons, Arch. Biochem. Biophys., 41,333 (1953). 24. D. Shugar, Biochem. J., 52, 142(1952). 25. C. Tanford, J. 1). Hauenstein, and I ) . G. Rands, J . Am. Chem. Soc., 77, 6409

26. A. Hvidt, Biochim. et Biophys. Actu, 18, 36 (1955). 27. H. Eyring and A. E. Stearn, Chem. Rev., 34, 190 (1939). 28. M. A. Eisenberg and G. W. Schwert, J . Gen. Physiol., 34,583 (1951). 29. C. K. Rule and V. K. LaMer, J . Am. Chem. Soc., 60, 1974 (1938). 30. G. Schwarzenbach, Z . Elektrochem., 44, 46 (1938). 31. c. Anfinsen, Compt. rend. trav. lab. CUrkbeTg, Sbr. chim., 30, 13 (1956). 32. W. I. Rogers and G. Kalnitsky, Biochim. et Biophys. Acta, 23, 525 (1957). 33. G. H. Haggis, Biochim. el Biophys. Acta, 23, 494 (1957). 34. H. Lenormant and E. R. Blout, Bull. SOC. chim. France, 1954,859.

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( 1955).

Synopsis A study has been made of the kinetics of heat inactivation in light and heavy water, of

lysozyme, ribonuclease, and desoxyribonuclease. In all instances, the isotope effect for inactivation ( k ~ / k ~ ) is found to be related to the activation energy and to decrease to approximately unity for low activation energies. For lysozyme at acid and neutral pH and for ribonuclease a t acid, neutral, and slightly alkaline pH, the results indicate that hydrogen bonding is not of great importance for enzymatic activity, in agreement with the results of previous studies in ordinary water. For desoxyribonuclease a t neutral and alkaline pH, the isotope effect varies from 1.5-5.0 which, in agreement with the high values of the thermodynamic constants for heat inactivation of this enzyme, suggests that the rupture of a considerable number of hydrogen bonds is involved in this process. Although the heat inactivation of this enzyme in the pH range 2.4-3.2 in H,O is partially reversible on cooling (if 50% inactivation is not exceeded), no such reversibility was found in D20; i t is believed that this may be due to a decrease in the pK of the protein carboxyl groups in heavy water. It was also found that the isotope effect decreases


with temperature, as is to be expected, the result being a small increase in the activation energy and entropy change for the inactivation process in heavy water. The results are discussed, and it is shown that no general conclusion may be drawn from studies on a single enzyme (or protein), but that the findings must be considered individually in relation to structure, deuterium exchange in solution, titration curve in heavy water, etc. Suggestions for further studies are advanced.

Rdsumd Une 6tude a 6% faite sur la cin6tique d’inactivation par la chaleur du Iysosyme, du

ribonucl6ase et du d6soxyribonucl6ase dans l’eau 16ghre et lourde. Partout l’effet isotope de l’inactivation ( k ~ / k ~ ) a 6tB trouv6 en relation avec 1’6nergie d’activation et d6croit approximativement jusqu’A l’unit6 pour de basses Bnergies d’activation. Pour le lyso- syme aux pH acide et neutre, e t pour le ribonucl6ase aux pH acide, neutre et 16gerement alcalin, les r6sultats montrent que la liaison hydrogene n’a pas grande importance dans l’activit6 enzymatique, en accord avec les r6sultats ant6rieui-s dansl’eau ordinaire. Pour le d6soxyribonucl6ase aux pH neutre et alcalin, l’effet isotopique varie de 1.5 A 5.0 et, en accord avec des valeurs 6lev6es des constantes thermodynamiques pour l’inactivation thermique de cet enzyme, suggere que la rupture d’un nombre consid6rable de liaisons- hydrogene a lieu au cows de ce processus. Tandis que l’inactivation thermique de cet enzyme au pH 2,4-3,2 dam l’eau est partiellement r6versible en refroidissant (si on ne d6passe pas 50% d’inactivation), aucune r6versibilit6 n’a 6t6 trouv6e dans D20; on croit que cela peut Atre dQ A une diminution du pK des groupes carboxyles de la proteine dans l’eau lourde. On a trouv6 6galement que l’effet isotopique diminue avec la tempbratwe, comme pr6vu, le r6sultat &ant une legere augmentation de 1’6nergie d’activation et un changement d’entropie pour le processus d’inactivation dans l’eau lourde. Les r6sultats sont discutBs et on montre qu’aucune conclusion g6n6rale ne peut &re faite aux d6pens des 6tudes sur un seul enzyme (ou prot6ine) mais que les r6sultats doivent &re consid6res individuellement en relation avec la structure, 1’6change de deuterium en solution, la courbe de titration dans l’eau lourde, etc. Des suggestions sont faites pour des 6tudes ult6rieures.

Zusammenfassung Es wurde eine Untersuchung der Kinetik der Hitzeinaktivierung von Lysozym, Ribo-

nuclease und Desoxyribonuclease in leichten und schweren Wasser durchgefuhrt. Der Isotopeneffekt der Inaktivierung (kH/kn) steht, wie festgestellt wird, in allen Fallen in Beziehung zur Aktivierungsenergie und fiillt bei kleinen Aktivierungsenergien etwa zum Wert eins ab. Die Ergebnisse an Lysozym bei saurem und neutralem pH und an Ribo- nuclease bei saurem, neutralem und schwach alkalischem pH sprechen dafur, dass Wasserstoff brucken keine grosse Rolle fur die enzymatische Aktivitat spielen; das steht in mereinstimmung mit den Ergebnissen fruherer Untersuchungen in gewohnlichem Wasser. Bei Desoxyribonuclease andert sich der Isotopeneffekt im neutralen und alkalischen pH-Bereich von 1,5 auf 5,0, was, in tfbereinstimmung mit dem hohen Wert der thermodynamischen Konstanten fur die Hitzeinaktivierung dieses Enzyms, darauf hinweist, dass bei diesem Prozess eine betrachtliche Anzahl von Wasserstoffbruckeu gelsot wird. Obwohl die Hitzeinaktivierung dieses Enzyms in pH-Bereich 2,4-3,2 in HzO beim Abkuhlen teilweise reversibel ist (wenn eine Inaktivierung von 50% nicht uberschritten wird), wurde in D,O keine solche Reversibilitat gefunden; es wird angen- ommen, dass das auf eine Abnahme des pK-Wertes der Carboxylgruppen des Proteins in schwerem Wasser zuruckgefuhrt werden kann. Es wurde auch gefunden, dass der Iso- topeneffekt mit der Temperatur abnimmt, was, wie zu erwarten, eine kleine Zunahme der Aktivierungsenergie und -entropie beim Inaktivierungsprozess in schwerem Wasser ergibt. Die Ergebnisse werden diskutiert und es wird gezeigt, dass aus Untersuchungen an einem einzelnen Enzym (oder Protein) keine allgemeingiiltigen Schlusse gezogen werden konnen, sonden dass diese Befunde individuell in Bezug auf Struktur, Deuterium- austausch in Losung, Titrationskurve in schwerem Wasser usw. betrachtet werden miis- sen. Es werden Anregungen fur weitere Untersuchungen gegeben.

kinetics of heat inactivation of enzymes in light and heavy water (lysozyme, ribonuclease, and...

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