Effects of Substrate on Heat Inactivation of Taka-amylase A

Transcription

1 form p. a. als Elutionsmittel gereinigt. Mit dem Eluat der gelben Zone, das das gesuchte Chinon darstellt, wird die chromatographische Reinigung einige Male wiederholt, dann läßt man das Chloroform über Nacht abdunsten und erhält das Methoxychinon als gelbe, amorphe Masse; aus Petroläther umkristallisiert, im Hochvakuum bei sublimiert und erneut aus Petroläther umkristallisiert, liefert sie zartgelbe Nadeln. Die Eluate der übrigen Zone geben nach dem Verdunsten des Chlorforms schmierige, braune Rüdestände, die auf die Farbteste für Chinone negativ reagieren (Chinhydrone; Additionsprodukte von Diazomethan an Oxydiinone) s. Tab. 8). Zu beachten ist, daß man den Überschuß an Diazomethan nicht größer wählt als angegeben, da sonst die dunkelbraunen, öligen Additionsprodukte von Diazomethan an Chinone, die stets auftreten, zum Hauptumsetzungsprodukt werden. Wichtig ist außerdem, daß man eine durch Destillation hergestellte Diazomethanlösung verwendet, da die übliche, durch Dekantieren des Äthers von der alkalischen Entwicklungslösung dargestellte Lösung stets noch so viel Alkali enthält, daß vorhandenes Chinon zersetzt wird. Nach Stdn. zieht man den Äther von der nun Methoxychinon enthaltenden Lösung ab; es verbleibt ein dunkelbrauner, fester, manchmal auch schmieriger Rückstand, das noch stark verunreinigte Methoxychinon. Ausbeuten: Reinigung der Chinone mg mg mg XVIII, XXII, XXXIII. Der D e u t s c h e n F o r s c h u n g s g e m e i n s c h a f t und dem F o n d s d e r c h e m i s c h e n I n d u s t r i e sind wir für die gewährten finanziellen Unterstützungen dieser Arbeit sehr zu Dank verpflichtet. Der obige Rüdestand wird in Chloroform p. a. aufgenommen und an einer Kieselgelsäule (Kieselgel zur Chromatographie 0,05 0,24mm; Merck) mit Chloro- Effects of Substrate on Heat Inactivation of Taka-amylase A GIITI TOMITA a n d SAM SOON K I M Institute of Biophysics, Faculty of Agriculture, Kyushu University, Fukuoka (Japan) ( Z. Naturforschg. 22 b, [1967] ; e i n g e g a n g e n am 4. Juli 1966) Taka-amylase A is highly protected from heat inactivation and denaturation by the presence of its substrate, starch. The effect of PH on the protective action was studied to find the relation between the protective action and the state of ionization of taka-amylase A. The separation of protective effect of substrate from that of hydrolysis products was made by digesting starch with taka-amylase A before incubation to find the difference of protective abilities of substrate and hydrolysis products. The stabilization of the higher order structure of enzyme protein, depending on the state of ionization of enzyme protein, brought about by the formation of enzyme-substrate and -product complexes seems to be responsible for the appearance of the protective effect observed. Some enzymes, for example, D-amino-acid oxidase glutamic dehydrogenase 2, alcoholic dehydrogenase 3, lactic dehydrogenase 3, bacterial a-amylase 4 and taka-amylase A 5-7, are considerably protected from the inactivation and denaturation by the presence of coenzymes or substrates or related compounds. Such a phenomenon is of interest not only from the standpoint of the mechanism of inactivation or denaturation, but also from that of the enzymatic reaction mechanism, since a structural change of Biochem. J. 4 8, [ ]. Sympos. Enzyme Chemistry (in Japanese) 5, 1 K. BURTON, 2 H. 3 K. KUBO, 4 1 [1950]. OKUNUKI, B. SHITA, and T. Japanese) 11, HAGIHARA, YONETANI, 99 I. SEKUZU, M. NOZAKI, J. enzyme protein induced by the presence of coenzymes or substrates or related compounds is thought to be closely related to the appearance of enzymatic activity. The protective effect of starch on the inactivation and denaturation of taka- and bacterial a-amylase by heat, acid and urea has been studied by O K U N U K I and others 4 ' 5. They concluded that the protective effect must be derived from the hydrolysis products of starch, for the reason that starch was completely hydrolysed Sympos. Enzyme Chemistry (in NAKAYAMA, H. MATSUBARA, a n d Biochemistry [Tokyo] 43, 483 T. OKUNUKI, K. OKUNUKI, [1956]. 5 B. HAGIHARA, T. NAKAYAMA, H. MATSUBARA, a n d 6 Biochemistry [Tokyo] 4 3, [ ]. G. T O M I T A and S. S. K I M, Nature [London] 2 0 5, 4 6 [ ]. G. T O M I T A and S. S. K I M, Experientia [Basel] 22, YAMA- [1956]. B. HAGIHARA. T. J. 7 [1966]. Dieses Werk wurde im Jahr 13 vom Verlag Zeitschrift für Naturforschung in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.v. digitalisiert und unter folgender Lizenz veröffentlicht: Creative Commons Namensnennung-Keine Bearbeitung 3.0 Deutschland Lizenz. This work has been digitalized and published in 13 by Verlag Zeitschrift für Naturforschung in cooperation with the Max Planck Society for the Advancement of Science under a Creative Commons Attribution-NoDerivs 3.0 Germany License. Zum ist eine Anpassung der Lizenzbedingungen (Entfall der Creative Commons Lizenzbedingung Keine Bearbeitung ) beabsichtigt, um eine Nachnutzung auch im Rahmen zukünftiger wissenschaftlicher Nutzungsformen zu ermöglichen. On it is planned to change the License Conditions (the removal of the Creative Commons License condition no derivative works ). This is to allow reuse in the area of future scientific usage.

2 within a very short digestion time. Furthermore, they concluded that the protective effect of digestion products was attributed to protection of the secondary structure of enzyme protein from denaturation. In the present investigation, the effect 7 of pn on the protective action of substrate from heat inactivation and denaturation of taka-amylase A was studied to find the relation between the protective effect and the state of ionization of taka-amylase A, and the separation 6 of protective effect of starch from that of decomposition products was performed under the suitable experimental conditions to find the difference between the protective abilities of substrate and hydrolysis products at various states of ionization of taka-amylase A. This is shown in Fig. 1. Curves 1 and 2 show activity-incubation temperature relation in the absence and presence of starch, respectively. The incubation was made for 10 min at pn 5.6 and the activity was measured at pn 5.6, the optimum pn wo to The possible explanations were given to the results obtained. Experimental Taka-amylase A was prepared from 'takadiastase Sankyo' (a preparation obtained from a wheet bran culture of Aspergillus oryzae) and recrystallized three times from aqueous solution containing 0.01 M calcium acetate by the A K A B O R I ' S method 8. Taka-amylase A solutions were prepared in 0.02 M acetate or veronal or glycine buffers containing potassium chloride added to the final ionic strength of The concentration of taka-amylase A was determined spectroscopically assuming the extinction9 of taka-amylase A in acetate buffer (PH = 5.6; // = 0.1) to be =22.1 at nm. The heat treatment for inactivation and denaturation was carried out as follows: aqueous solutions of takaamylase A at various PH values, in the absence and presence of soluble starch, were incubated for 10 min at various temperatures in the thermostat and rapidly cooled at 0 C after incubation. In order to separate roughly the protective effect of starch from that of hydrolysis products, the digestion of starch with taka-amylase A was made at 0 C for various periods before incubation. The activity of taka-amylase A was measured at C by the blue value method 10. Other procedures will be described in the following section. Chemicals employed were of special grade for analytical use. Results and Discussion Taka-amylase A is highly protected from heat inactivation by the presence of its substrate, starch. 8 S. A K A B O R I, T. I K E D A, and B. [Tokyo] 41,577 [1954]. HAGIHARA, J. Biochemistry Incubation temperature [ C] Fig. 1. Change of activity of taka-amylase A with incubation temperature. Incubation time, 10 min; concentration of takaamylase A, 0.05mg/ml; ph 5.6 (acetate buffer). Curve 1, taka-amylase A alone; curve 2, taka-amylase A + starch (6 mg/ml) ; curve 3, difference of activity between curves 1 and 2. In the absence of substrate, activity decreases suddenly over 50 C and is completely lost at about 65 C. On the other hand, the presence of substrate (6 mg/ml of starch) gives rise to a remarkable resistance against heat inactivation, and the activityincubation temperature curve shifts towards higher incubation temperature than curve 1. The amount of this shift is about 3.5 C under the present experimental conditions, and this is thought to be "an effective temperature" lowered as substrate effect. Curve 3 is the difference between curves 1 and 2 and shows a sharp peak at about C. From curve 3, it is understood that the protective action is most remarkable at the "transformation temperature" from native to denaturation. Fig. 2 shows the dependence of activity on incubation time at an incubation temperature of C, which is the temperature with the most effective protective action. Curves 1 and 2 show activity-incubation time relation in the absence and presence of substrate, respectively. It takes a much longer incubation time in the latter case than in the former to obtain an equal degree of inactivation. This is 9 10 T. T A K A G I and T. I S E M U R A, J. Biochemistry [Tokyo] 4 8, 781 [I9]. H. F U W A, J. Biochemistry [Tokyo] 4 1, 538 [1954].

3 Incubation time [min] Fig. 2. Change of activity of taka-amylase A with incubation time. Incubation temperature, C ; concentration of takaamylase A, 0.05mg/ml; ph 5.6 (acetate buffer) ; concentration of starch, 6 mg/ml. Curve 1, taka-amylase A alone; curve 2, taka-amylase A + starch; curve 3, difference of activity between curves 1 and 2 ; curve 4, difference of incubation temperature between curves 1 and 2. shown in curve 4 obtained from the difference between the incubation time in curves 1 and 2 at the same inactivation. From the extrapolation of curve 4 to zero activity, it is known that the longer incubation time by about 9 min is necessary in the coexistence system of substrate than in the substratefree system for complete inactivation. Curve 3 is the difference between the activity in both systems at the same incubation time. The optimum activity is observed after about 9 min of incubation. PH Fig. 3. Effect of PH on activity of taka-amylase A. The activity of non-heat-treated taka-amylase A : curve 0 and 0', measured at the PH values given and at pn 5.6 after overnight treatment at the PH values given at C, respectively. The activity of taka-amylase A heat-treated in the absence of starch at the pn values given: curves 1, 2 and 3, measured at the PH values given; curves 1', 2' and 3', measured at PH 5.6. The activity of taka-amylase A heat-treated in the presence of starch at the PH values given: curves a, b and c, measured at the PH values given; curves a', b' and c', measured at PH 5.6. The heat-treatment was made at 50, 55 and C, respectively, for 10 min. Concentration of taka-amylase A, mg/ml; concentration of starch, 6 mg/ml. The presence of substrate yields the protective action from heat inactivation not only at the optimum pn but also in wide pn regions. This is shown in Fig. 3. Curves 1, 2 and 3, and a, b and c show the dependence of activity of heat-treated takaamylase A, measured at the pn values given, on PH in the absence and presence of substrate, respectively. The activity of non-heat-treated native takaamylase A, measured at the pn values given, also depends strongly on pn, showing a slight decrease below PH 5.6 and a profound decrease in the pn region , as seen in Fig. 3, curve 0. Above PH 8.0, the enzymatic activity completely vanishes. Therefore, the curves mentioned above (1, 2, 3 and a, b, c) are thought to show the combined effects of heat and pn inactivations. Curve 0' is the activity of non-heat-treated taka-amylase A measured at the optimum pn (5.6) after exposure overnight to the PH values given at C. The inactivation is completely reversible up to about pn Above pn 10.5, however, the pn inactivation is irreversible due to an irreversible structural change of takaamylase A molecule. From measurements of ultraviolet absorption, optical rotation and enzymatic activity as a function of pn, T A K A G I and I S E M U R A 9 have found that the most of the phenolic hydroxyl groups in taka-amylase A are not freely ionizable but ionize irreversibly above pn 10.5 and the native structure of taka-amylase A does not change below pn 10.5 but above this PH it is degradated by the irreversible ionization of the phenolic hydroxyl groups which may be hydrogen-bonded * to carboxylate groups and may play a part in maintaining the stable structure of enzyme protein. On the other hand, the heat inactivation is irreversible in the whole pn regions as in the cases of ordinary enzymes. In order to eliminate the irreversible part of pn inactivation, the activity of taka-amylase A heattreated at various ph values was measured at pn 5.6. Curves l ', 2' and 3', and a', b' and c show the activities, measured at pn 5.6, of taka-amylase A heat-inactivated at the pn values given in the absence and presence of starch, respectively. * A molecule of taka-amylase A has 28 phenolic hydroxyl groups, of which groups are considered to be hydrogenbonded to the carboxylate groups and masked, cf. Reference 9 and T. T A K A G I and T. I S E M U R A, J. Biochemistry [Tokyo] 4 9, 4 3 [ ].

4 The heat inactivation is minimum at about pn 7.0, and steeply increases with decreasing pn in acidic regions and gradually with increasing pn in alkaline regions. Above about pn 10.5, the heat inactivation shows a remarkable increase as the result of the combined effects of heat inactivation and irreversible pn inactivation. From the curves (l', 2', 3' and a', b', c, and 0') in Fig. 3, the degree of protection by substrate from heat inactivation defined by (difference between the activity decrease by heat-treatment in the absence and presence of starch)/(activity decrease by heattreatment in the absence of starch) is obtained at various pn values, for various incubation temperatures and plotted in Fig. 4. The values of degree of protection for the incubation temperatures given fall roughly on a curve which shows a slight decrease carboxylate or phenolic hydroxylate groups of carboxylate-phenolic hydroxylate hydrogen bonds playing an important part in stabilizing the native structure of taka-amylase A and the protective ability is completely lost by the irreversible ionization of these groups. The protective action observed in the presence of starch is thought to be derived from the superimposed effects of starch and its hydrolysis products. The separation of protective effect of starch from that of hydrolysis products was made by digesting starch for various periods at 0 C before incubation. The dependence of activity on digestion time is shown in Fig. 5, curve 1. The incubation temperature and time are, respectively 59 C and 10 min, at which the protective action is most remarkable, as shown in Figs. 1 and 2. Fig. 4. Effect of PH on degree of protective action of substrate from heat inactivation of taka-amylase A. The degree of protective action was defined by (difference between the activity decrease by heat-treatment in the absence and presence of starch) / (activity decrease by heat-treatment in the absence of starch). The heat-treatment was made for 10 min at the PH values given, and the activity was measured at PH 5.6 after heat-treatment, x, A and o, heat-treated at 50, 55 and C, respectively; concentration of taka-amylase A, mg/ml; concentration of starch, 6 mg/ml. with increasing pn in the region PH 6 10, and the sudden decrease in both sides below pn 6 and above PH 10. A slight decrease of protective action in the range pn 6 10 may be due to the changes in calcium binding properties in this region, as reported by KATZ and KLOTZ 11 in bovine serum albumin. The decrease of protective action in the acidic and extreme alkaline regions suggests that the protective ability is weakened by the reversible ionization of Fig. 5. Changes of activity of taka-amylase A with digestion time before heat-treatment and of starch concentration with digestion time, and contributions to protective action from starch and hydrolysis products. Incubation temperature, 59 C; incubation time, 10 min; concentration of taka-amylase A, 0.05 mg/ml; PH 5.6 (acetate buffer); concentration of starch, 6 mg/ml. Curve 1, taka-amylase A + starch; curve 2, starch concentration at various digestion times; curve 3, protection by starch; curve 4, protection by hydrolysis products; a, b, c and d are, respectively, the activity after heat-treatment in the presence of dextrin, maltose, glucose and limit dextrin. In the absence of starch, activity decreases to 32% after heat treatment. The amount of substrate (degree of polymerization n> ~6) 12 in the course of digestion, which was measured by iodostarch reaction, is shown in Fig. 5, curve 2. As shown in Fig. 6, the decrease in activity by heat treatment is proportional to the concentration of substrate in the region of low substrate concen- 11 S. KATZ and I. KLOTZ, Arch. Biochem. Biophysics 44, 351 [1953]. 12 The minimum degree of polymerization of amylose to colour on treatment with iodine, cf. M. A. SWANSON, J. biol. Chemistry 172,825 [1948].

5 tration, where the incubation temperature and time are, respectively, C and 10 min and incubation was started immediately after mixing the substrate with amylase solution. This proportionality makes it possible roughly to separate the total protected amount (expressed in activity difference between curve 1 and the activity after heat treatment in the absence of substrate) into two parts: protected amount by substrate ( n > 6) and that by decomposed compounds ( n < 6 ). These are shown in Fig. 5, curves 3 and 4, respectively. D <o I -5 t- o o E T=> o -8? Digestion time [min] * 100 "tj Starch concentration [mg/ml]-*- Fig. 6. Dependence of activity on starch concentration after heat-treatment. Incubation temperature, C ; incubation time, 10 min; concentration of taka-amylase A, 0.05 mg/ml; PH 5.6 (acetate buffer). In the short period of digestion, the protection is mainly due to the effect of substrate (a > 6 ). The decomposition products from starch, a-limit dextrin and maltose are also effective for protection of amylase from heat inactivation, but glucose has no protective effect. In the presence of these substances (6 mg/ml in each), the activity decreases by heat treatment (at 59 C and for 10 min) to 38, 37 and 32%, respectively, as shown in Fig. 5. Limit dextrin and maltose seem to stabilize the secondary structure by combination with amylase, and to protect it from heat inactivation and denaturation, though the effect is not so great. The dependence of protected amount difference between activities (measured at pn 5.6) of amylase heat-treated in the absence and presence of starch on digestion time before incubation is shown in Fig. 7. The protected amount at zero digestion time is normalized to unity. Curves 1 and 2, and a and b are the protected amounts when incubated for 10 min at pn 5.6 and 9.0, respectively. The amount of substrate in the course of digestion, which was Fig. 7. Changes of protected amounts (difference between activities of taka-amylase A heat-treated in the absence and presence of starch) with digestion time before heat-treatment. Curves 1, 2, and a, b, heat-treated at pu 5.6 and 9.0, respectively; curve 3, starch concentration at various digestion times before incubation, and also means the amount protected by starch; curves 1', 2', and a', b', amounts protected by decomposition products from starch, corresponding to the cases of curves 1, 2, and a, b, respectively. The digestion was made at PH 7.0 at C in all cases. The heat-treatment was made for 10 min at the temperature shown and at ph 5.6 or 9.0, immediately after digestion. The protected amount at zero digestion time is normalized to unity. The activity was measured at ph 5.6. Concentration of taka-amylase A, mg/ml; concentration of starch, 6 mg/ml. measured by iodostarch reaction, is shown in Fig. 7, curve 3. Curves 3 and l ', 2', a', b' are the amount protected by starch and those by decomposition products, respectively. The proportion of protection by digestion products to that by starch at pn 5.6 is larger than that at pn 9.0. This means that digestion products are less effective for the protection from heat inactivation of taka-amylase A in pn-inactivated or -denatured states. According to L E V I T Z K I and others 13, pancreatic a-amylase can form an insoluble complex with macro-dextrin. The yield of insoluble complex is dependent on the size of dextrin and the optimal precipitation occurs only within a limited range of the enzyme/dextrin ratio. Excess amylase or excess dextrin inhibits completely precipitation of complex. Taka-amylase A can also form at 0 C an insoluble and stable complex with starch within a limited range of enzyme/substrate ratio, as shown in Fig. 8. The yield of enzyme precipitated by the formation of insoluble complex was calculated from the amount of enzyme protein left in the supernatant after the mixture of taka-amylase A and starch was centrifuged in the cold. 13 A. L E V I T Z K I, J. H E L L E R, and M. S C H R A M, Biochim. biophysica Acta [Amsterdam] 81,101 [1964].

6 Concentration of starch [mg/ml] Fig. 8. Effect of starch concentration on formation of insoluble complex. Concentration of taka-amylase A, 0.29 mg/ml. Solutions of taka-amylase A and starch at ppi 8.0 were mixed at 0 C. Under the present experimental conditions of heat inactivation, the taka-amylase A/starch ratio is too small to yield precipitation of complex. In such an excess-substrate zone, the formation of insoluble complex is prevented and only soluble complex is formed. Therefore, the insoluble complex does not take part in the protective action observed in the present investigation. molecules to combine to aggregates. For this reason, the taka-amylase A solution becomes turbid by the occurrence of heat denaturation. The turbidity is a measure of aggregation of denatured enzyme molecules. The turbidity 14 due to heat denaturation was measured by the optical density at 500 nm and is shown in Fig. 9. The turbidity runs parallel to the inactivation and is inhibited by the presence of substrate. From these results, it is considered that the formation of enzyme-substrate and -product complexes not only highly stabilizes the higher order structure of taka-amylase A molecule, but prevents the formation of aggregation of enzyme molecules induced by heat denaturation. The dependence of turbidity of heat-treated takaamylase A solutions, in the absence and presence of starch, on pu is shown in Fig. 10. In alkaline region, The destruction of enzyme structure caused by heat treatment leads to inactivation and denaturation, and makes it possible for denatured enzyme t~ \a / Xw\,30?t * r / Fig. 10. Effect of pn on turbidity. Curves 1 and a, the turbidity measured by the optical density at 500 nm in the absence and presence of starch, respectively; curve 2, difference between curves 1 and a ; concentration of taka-amylase A, mg/ml; concentration of starch, 7.5 mg/ml. The heat-treatment was made for 10 min al C. 3i 1/b V k ' '5 80 Incubation temperature[ C] Fig. 9. Effect of incubation temperature on turbidity and activity. Curves 1 and a, the activity of heat-treated taka-amylase A in the absence and presence of starch, respectively; concentration of taka-amylase A, mg/ml; concentration of starch, 6 mg/ml. Curves 2 and b, the turbidity measured by the optical density at 500 nm in the absence and presence of starch, respectively; concentration of taka-amylase A, mg/ml; concentration of starch, 7.5 mg/ml. The heat-treatment was made for 10 min at ph 5.6 and the activity was measured at ph 5.6 (acetate buffer). 14 G. TOMITA and S. S. KIM, Experientia [Basel] 22, 392 [1966] P H 1/ / L. M I C H A E L I S and M. L. M E N T O N, Biochem. Z. 49, 333 [1913]. D. K E I L I N and T. M A N N, Proc. Roy. Soc. [London] Ser. B 122, 119 [1937]. turbidity is nearly zero, but increases with decreasing pn in acidic region. It is of interest to note that curve 2, the difference between curves 1 and a has a peak at the pn of the optimum activity (pn 5.6). The formation of an intermediate enzyme-substrate complex in enzyme-catalysed reaction was first postulated by M I C H A E L I S and M E N T O N 15. This concept has been experimentally proved for kinetics of enzyme reactions by various workers, in various systems. K E I L I N and M A N N 1 6, and others , have 17 K. G. STERN, J. 18 B. CHANCE, J. [Washington] 19 D. G. DOHERTY [1952]. biol. Chemistry biol. Chemistry 109,4 and 114, , 553 [1939]. [1943] ; Science [1949]. F. VASLOW, J. Amer. chem. Soc. 74, 931

7 shown spectroscopically the existence of complexes of peroxides with catalase and peroxidase and calculated the thermodynamic quantities of enzymesubstrate complex such as free energy, entropy and enthalpy. Recently, the change of higher order structure of enzyme on the formation of enzyme-substrate complex has been investigated by means of optical rotation and difference spectrum. These experimental results show that the structure of enzyme protein changes from disorder to order on the formation of enzyme-substrate complex. Such a structural change is thought not only to conjugate intimately with the appearance of enzymatic activity in enzyme-catalyzed reaction, but also to play an important role in the protective action of substrate from heat inactivation and denaturation. A difference spectrum of taka-amylase A has not been observed, but, in the cases of lysozyme and a-chymotrypsin ~ 25, difference spectra on the formation of enzyme-substrate complex are well established. On the formation of enzyme-substrate complex, ultraviolet absorption spectra of enzymes usually show "red shift" and this means the conformation change of enzyme protein from disorder to order. This conformation change may conjugate with the appearance of enzymatic activity. The ordered and rigid structure of enzyme protein may be essentially responsible for enzymatic activity. Furthermore, this structure may be highly stabilized and be protected from external disturbance such as thermal agitation. This seems to explain why takaamylase A can be protected by the presence of its substrate. The presence of hydrolysis products also stabilizes the higher order structure of enzyme protein by the formation of enzyme-product complex and protects enzyme from heat inactivation. As suggested by ISEMURA and other 26, the site with which decomposition compounds are combined, unlike substrate, is not necessarily the active point of enzyme. Therefore, the induced stabilization responsible for the protective action is considered to be probably similar to that by calcium ions. The protective action appears in the limited pa region where the carboxylate and phenolic hydroxylate groups ionize reversibly and reversible conformation change can occurs and pn inactivation is reversible. However, it can not be observed in the extreme acidic and alkaline regions where the irreversible conformation change leading to the irreversible pn denaturation occurs. In this connection, it is considered that there is an intimate relation between the induction of the protective effect and the state of ionization of enzyme protein. The authors wish to express their sincere thanks to the Ministry of Education for a grant covering a part of this research. J. F. WOOTON and G. P. HESS, J. Amer. chem. Soc. 83, 4234 [1961]. 21 J. F. WOOTON and G. F. HESS, J. Amer. chem. Soc. 84, 4 [1962], 22 J. F. WOOTON and G. P. HESS, Nature [London] 188, 726 [I9]. 23 H. A. SCHERAGA, Protein Structure, Academic Press, New York 1961, p G. H. BEAVEN, E. R. HOLIDAY, and E. M. JOPE, Discuss. Faraday Soc. 9, 6 [1950]. 25 E. R. HOLIDAY and G. H. BEAVEN, Advances Protein Chemistry?^^ [1952]. 26 S. FUJITA-IKEDA and T. ISEMURA, J. Biochemistry [Tokyo] 47, 537 [19].