STUDIES ON THE EFFECT OF TEMPERATURE ON THE CATALASE REACTION. III. TEMPERATURE EFFECT AT DIFFERENT HYDROGEN ION CONCENTRATIONS. IV. A THEORY OF THE CATALASE REACTION. BY SERGIUS MORGULIS, M. BEBER, AND I. RABKIN. (From the Department of Biochemistry, College of Medicine, University of Nebraska, Omaha.) (Received for publication, March 23, 1926.) Effect of Temperature at DiJ’bent pH on Catalase Activity. It has been shown by a number of investigators (Michaelis and Pechstein (l), Sorensen (2), Bodansky (3), Morgulis (4), Biechy (5)) that the catalase reaction is greatly depressed in an acid medium. The study of the influence of changes in hydrogen ion concentration on the catalase reaction over a wide range of pH is beset with practical difficulties owing to the fact that a variety of buffers is needed for this purpose, and this brings into play the influence of different salt content which affects the final quantitative results. It frequently becomes impossible to tell whether ‘the observed quantitative effect is due primarily to the change in pH or to the change in salt composition of the mixture. The direct adjustment of the reaction of the mixture by means of acids and alkalies is, in our opinion, a still less desirable pro- cedure, and is not to be preferred to the use of appropriate buffers since it does not remedy the situation arising from a dif- ference in salt content and composition, and is generally less reliable. We employed the Kolthoff seriesof buffers (6) covering a range from pH 3 to pH 9, and for the experiments where greater alkalinity was tested we used Sorensen’s glycine-NaOH buffer which covers a range from pH 9 to practically pH 13. However, we also experimented with mixtures directly adjusted to various pH from 8 to about 14 by means of NaOH. In general, we can 547 by guest on May 21, 2018 http://www.jbc.org/ Downloaded from
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STUDIES ON THE EFFECT OF TEMPERATURE ON THE CATALASE REACTION.
III. TEMPERATURE EFFECT AT DIFFERENT HYDROGEN ION CONCENTRATIONS.
IV. A THEORY OF THE CATALASE REACTION.
BY SERGIUS MORGULIS, M. BEBER, AND I. RABKIN.
(From the Department of Biochemistry, College of Medicine, University of Nebraska, Omaha.)
(Received for publication, March 23, 1926.)
Effect of Temperature at DiJ’bent pH on Catalase Activity.
It has been shown by a number of investigators (Michaelis and Pechstein (l), Sorensen (2), Bodansky (3), Morgulis (4), Biechy (5)) that the catalase reaction is greatly depressed in an acid medium. The study of the influence of changes in hydrogen ion concentration on the catalase reaction over a wide range of pH is beset with practical difficulties owing to the fact that a variety of buffers is needed for this purpose, and this brings into play the influence of different salt content which affects the final quantitative results. It frequently becomes impossible to tell whether ‘the observed quantitative effect is due primarily to the change in pH or to the change in salt composition of the mixture. The direct adjustment of the reaction of the mixture by means of acids and alkalies is, in our opinion, a still less desirable pro- cedure, and is not to be preferred to the use of appropriate buffers since it does not remedy the situation arising from a dif- ference in salt content and composition, and is generally less reliable. We employed the Kolthoff series of buffers (6) covering a range from pH 3 to pH 9, and for the experiments where greater alkalinity was tested we used Sorensen’s glycine-NaOH buffer which covers a range from pH 9 to practically pH 13. However, we also experimented with mixtures directly adjusted to various pH from 8 to about 14 by means of NaOH. In general, we can
corroborate the earlier findings, namely that a strong catalase reaction occurs over a range of pH 6 to pH 8; and, considering that the catalase activity is very little inhibited even at pH 5 or pH 9, it may be said that there is really no optimum pH for catalase activity in the strict sense of the word. Catalase acts well over an extent of hydrogen ion concentrations ranging from pH 5 to pH 9, but its activity diminishes decidedly on either side of these limits, the diminution being much more abrupt on the acid than on the alkaline side. Thus, at pH 4 one-half of the activity is still present, at pH 3 only one-fifth to one-seventh, and at pH 2 the activity is completely lost. On the alkaline side the loss of activity does not proceed quite so rapidly and the diminution up to pH 13 is gradual and continuous, but even at pH 13 considerable activity still remains (at least one-half). At pH 14 the catalase activity is completely suppressed. Bo- dansky (3) found that the catalase activity was entirely inhibited at pH 13, but we were unable to corroborate this with our kidney catalase preparation even when we followed his method of adjust- ing the reaction by appropriate additions of NaOH to the reac- tion mixtures. Considering what has been said with regard to the inherent impossibility of securing perfectly uniform series of quantitative results over the entire pH range, we may, never- theless, sum up our experience along this line by presenting a slightly schematized curve of catalase activity at different pH values and temperatures.
Rising very rapidly from about pH 2.5, the curve forms almost a plateau between pH 5 and pH 9 (more correctly this plateau has a small curvature) and slopes off gradually to pH 13, this being much more gradual at high than at low temperatures. Between pH 13 and 14 there is a very steep and abrupt drop in the curve since all activity ceases at pH 14. On the acid side, more particularly up to about pH 3.5, there is comparatively very little difference in activity under various temperatures.
By far the greatest temperature effect is manifested over a range of pH 5 to pH 9, i.e. over hydrogen ion concentrations associated with the greatest cat,alase activity, thus demonstrat- ing that the largest catalase destruction occurs under conditions most conducive to catalase activity. This fact tallies also with the observation recorded in Paper I of this series (7) that the
destruction of the catalase increases parallel to the reaction velocity. We must conclude, therefore, that either the part of the catalase molecule which causes decomposition of the hydrogen peroxide is also readily subject to destruction, or that the de- struction of catalase by oxidation is greatly enhanced by the presence of OH ions in the system.
Furthermore, a glance at the curves in Fig. 1 leaves no doubt that although the catalase activity diminishes on either side of
FIG. 1. This represents the catalase activity at 10, 20, and 30°C. over a range of pH values from 2 to 14. The curves are based upon many experi- ments and represent therefore composite curves slightly schematized in that’ they have been drawn to coincide as nearly as possible with the average for the experimentally determined points.
the pH range of 5 to 9, the effect at pH 2 to 5 is obviously radically different from that taking place at pH 9 to 14. Thecatalase at a pH less than 5 undergoes an alteration which manifests itself, for one thing, in a loss of sensitivity towards temperature effects, whereas on the alkaline side, even at pH 13, this effect is still very pronounced. Apart from this, however, the influence of an excess of either H or OH ions manifests itself in another, even more essential and fundamental manner, not revealed in Fig. 1,
but shown by the curves in Fig. 2; namely, a difference in the latent period of the reaction. This point, however, will be dis- cussed fully in the next section.
Michaelis and Pechstein (1) suggested that catalase is an ampholyte which ionizes with the formation of both anions and cations. The molecule is assumed to be active, and the activity attains almost an optimum at the isoelectric point. On the acid side of this isoelectric point catalase fails to decompose hydrogen peroxide because it is present in the form of the cata- lytically inactive cations. The anion is assumed to be also active as the molecule, this assumption being necessitated by the fact that the catalase activity continues to increase even beyond the isoelectric point. This argument will hardly bear serious criticism. In the first place, the location of the isoelectric point of catalase is a purely gratuitous assumption. But even grant- ing the main premises of the argument, the cations being inactive, it is not clear how the formation of anions would account for the increased activity as the sum of active particles would still remain the same as at the isoelectric point. Nevertheless, the Michaelis and Pechstein interpretation, though undoubtedly erroneous in detail, must be recognized as essentially correct in principle. We are led to conclude from our studies of the com- bined effect of temperature and pH variations that catalase is active probably only in the form of anions. Plotting the results of several experiments in such a way as to represent relative activity at different pH and temperatures (the maximum activity of the catalase observed at pH 7 or 8 at each experimental tem- perature being taken as 1) we note, in Fig. 3, first of all, that the curves thus obtained bear unmistakable resemblance to typical dissociation curves. This is only true for the acid range of pH. The pH at which the relative catalase activity becomes one- half varies according to the experimental temperature. On the average this occurs at pH 4, but at high temperatures (above 20°C.) this shifts definitely to the acid side. Since we cannot assign any isoelectric point to catalase, the marked repression of its activity with increasing hydrogen ion concentration suggests that we may possibly be dealing with a weak acid whose disso- ciation constant is 1 X 10-4. Michaelis and Pechstein (1) give to catalase a dissociation constant of 2.88 X 10-5, which
would correspond approximately to the one we get for ZO”C., namely 6.6 X 10-5. The first value 1 X 1O-4 is based on the average found over a large range of temperature variations. Biechy (5) states that for potato catalase he found that K = 1.42 X lo-‘.
Since at pH less than 5 catalase is rapidly inactivated, losing its activity entirely at pH 2, we conclude contrary to Michaelis and Pechstein that the molecule must be inactive. The fact that catalase activity is maintained at a high level over an extensive range of pH 5 to pH 9 likewise fails to support the contention of these authors that the catalase molecule as such is the active substance. On the contrary, it seems more reasonable to believe
pH3 4 5 6 I8 3 4 5 6 7 8 9 3 4 5 6 7 8 9
FIG. 3. A series of curves showing the relative catalase activity at differ- ent temperatures and pH. The maximum activity at pH 7 or 8 is taken as I.
that if catalase ionizes the anion is the only active part. It also appears probable from our experimental results that the anion is very readily destroyed by the excess of hydrogen peroxide, the destruction increasing progressively with rising temperature. This is suggested by our observations already mentioned that the effect of varying temperatures gradually diminishes, ulti- mately to vanish almost completely, in the region below pH 4, where the concentration of anions must be very low and where a distinct inhibition of the catalase reaction takes place. It is likewise a striking fact, brought out by the curves in Fig. 3, that the temperature effect on the acid and alkaline side of the series is reversed. Thus, the relative catalase activity at pH 9 is
smaller at high than at low temperature, while at pH 3 the relative activity is greater at high than at low temperature. It is possible that this may be due to a shifting of the dissociation constant with a change in temperature, as was noted above. However, this fact bears out our contention further that the anion is the only catalytically active part of the catalase mole- cule, since the increased ionization, due to the higher tempera- ture, evidently tends to offset the depression of ionization due to the large hydrogen ion concentration, and we should naturally expect this relative increase in catalase activity. On the alkaline side, however, due to the fact that the anion is readily attacked by peroxide and destroyed, the rise in tempera- ture will tend to produce just the opposite effect, because owing to the greater concentration of the anion the rate of destruction of the catalytic agent will likewise increase. Furthermore, the high OH ion concentration may perhaps favor the destruction by oxidation.
From all our experimental results we are led to conclude that catalase may be a weakly acid substance, the catalytic activity of which is a function of its anions. Hence the rather abrupt and rapid onset of inactivation at pH less than 5. It is also clear that the destruction of the catalase, as is shown to occur by loss of its activity with rising temperature, is favored by in- creased alkalinity, and we are, therefore, led to conclude that very probably the anion is also the part most easily oxidized by the peroxide, this process increasing with the pH.
Latent Period of the Catalase Reaction.
During the first few minutes of the catalase reaction there are occurrences of far reaching importance which ordinarily escape attention unless the reaction is studied gasometrically whereby it may be observed continuously. We refer to the latent period of the reaction. Under ordinary experimental conditions (generally a pH of 7 and a temperature of about 20°C.) the latent period is so negligible that we have repeatedly over- looked it in our experiments, or were inclined to attribute the loss of a few seconds in the initiation of the reaction to purely extraneous influences. Under certain conditions, however, the latent period has attained such magnitude as to command our
attention very strongly, and this stimulated us to study this matter more closely. As was pointed out previously (4) under the combined influence of high hydrogen peroxide concentra- tions and low temperatures the catalase reaction undergoes an interesting modification: following an immediate spurt of cata- lase activity a quiescent period sets in, which is longer the higher the hydrogen peroxide concentration. This quiescent period is, of course, distinct from a latent period of reaction. There is no variation in the latent period in these experiments with varying peroxide concentrations at the same temperature. This fact is clearly brought out by the series of curves in Fig. 4 of Paper I, all curves issuing practically from the zero point.
In our experience, a distinct latent period becomes manifest when the catalase reaction is carried out under two particular experimental conditions. First, it becomes very prominent when catalase inactivated by heating for various lengths of time at 60°C. is studied. It was found then that not only did the activity diminish with an increase in the time of heating up to 45 minutes, at which time the enzyme was completely inactivated, but that a definite latent period appeared which became longer as the time of inactivation increased (see Fig. 1, Paper II (8)). Thus, after 5 minutes heating of the catalase sample 30 seconds were required before the reaction started; after 15 minutes heating, 90 seconds, and after 30 minutes heating the latent period increased to 270 seconds. It will also be noted from the Fig. 1 already referred to that there is a direct relationship between the degree of inactivation of the catalase material and the latent period of the catalase reaction.
The other experimental condition under which the latent period attains considerable importance is at pH less than 5, especially when the experiments are made at temperatures below 20°C. In fact, under such experimental conditions the latent period may become extremely prolonged (see Fig. 2). In Table I we record a number of observations on the latent period at different. temperatures and hydrogen ion concentrations of the mixtures.
We do not record the latent periods observed at greater alkalinity since, on account of the use of different buffers, the results obtained at pH above 9 do not properly belong in the above series. However, we can say that over the entire range of
pH 9 to pH 13 we have failed to note any significant change from the magnitude of the latent period recorded in the table for pH 8 or 9. In fact, at pH 12 and 13 the latent period tends to become distinctly shorter than at lower pH values. The point we wish to emphasize in this connection is that in the case of high hydrogen ion concentrations the latent period also varies directly with the loss of activity of the catalase (at pH less than 5) and inversely with the reaction velocity. The increase in the magnitude of the latent period on the acid side with lowering of the experimental temperature is very striking, indeed.
Considering the great increase in the latent period of the cata- lase reaction under conditions whose principal effect is the in- hibition of the activity of the catalase, we venture to suggest that there are two ways in which this enzyme may be altered, each manifesting itself in a reduction of the catalytic decomposi- tion of hydrogen peroxide, but involving entirely distinct mecha- nisms. One type of alteration, we feel reasonably certain in assuming, is an actual oxidation of the catalase by the excess of peroxide, and we propose to designate this as the destruction process. This destruction increases with temperature as well as with the concentration of the hydrogen peroxide, and is also apparently dependent upon the OH ion concentration. This type of loss of activity has no effect upon the latent period of the catalase reaction. The other type of alteration manifests itself not alone in a reduction of catalase activity but also, and in a very striking manner, in a lengthening of the latent period of the reaction. We propose to differentiate this type of change as the inactivation phenomenon. It may be produced either through heating the enzyme, or it may result from an increase in hydrogen ion concentration beyond the pH of 5. The latent period becomes especially marked under the additional influence of low temperature. Assuming, as we obviously must, that at pH 4 or less the catalase is present principally as inactive mole- cules, we can readily understand why, especially when disso- ciation is reduced still further by lowering the temperature, the catalytic activity under such conditions is slow in getting under way. It is not improbable that a sufficient concentration of the active particles is required for the reaction to take place, and the more the dissociation of catalase is depressed by the combined
action of acidity (pH below 5) and lowering of the experimental temperature the longer will be the time necessary to reach that point. This interpretation is in exact agreement with the ob- served facts. We may likewise conclude that heat inactivation is a phenomenon involving some alteration of the catalase mole- cule which interferes with its dissociat,ion. It is interesting to point out in this connection that material which was found com- pletely inactivated by heating for 1 hour at 6O”C., according to tests made at 20°C. or below that, would, in several instances, still produce a slight reaction when tested at 30°C. (cf. Table I, Paper II). Evidently, even after an exposure to heat for 1 hour
TABLE I.
Latent Period of the Reaction in Seconds.
PR
3
4
5 6 7
8
9
Experimental temperatures.
29.5%. 31.1c 8.4”C. 9.8T. 19.4%. 21.5”C. - .-
1120 560 165 130 26 12 111 21
240 216 30 32 18 6 17 12
18 20 35(?) 11 12 6 15 11 7 9 7 5 15 15 7 8 7 4
8 10
7 10
-7
-
there is still enough dissociable material present in the catalase solution to produce a sufficient concentration of active particles under the proper temperature conditions to cause some catalytic decomposition of the hydrogen peroxide. We may, therefore, regard complete inactivation, either by heat or by acidity, as a condition under which the latent period of the catalase reaction has become infinitely large.
SUMMARY.
1. Catalase prepared from beef kidney shows strong activity at hydrogen ion concentrations ranging from pH 5 to pH 9, with the greatest activity being limited to the narrower range of pH 6 to pH 8.
2. At pH less than 5 the catalase activity is lost very rapidly and stops entirely at pH 2. The loss of activity between pH 9 and pH 13 is slow and much more gradual.
3. At pH 13 the catalase still has considerable activity (about one-half the original), but this is lost completely at pH 14.
4. The loss of catalytic activity with increasing temperature varies with the pH of the reaction mixture. The maximum destruction of catalase by rising temperature occurs within the range of pH of greatest catalytic activity.
5. At pH less than 4 the temperature effect almost entirely disappears, and the relative activity is greater at the higher temperatures. In mixtures with higher pH the relative activity diminishes with rising temperature.
6. Catalase seems to behave like a weak acid, the anion ap- parently being the catalytically active part.
7. A marked latent period appears when catalase has been inactivated by heating to 60°C. or when the reaction is studied at pH below 5 and at temperatures below 20°C. The latent period probably represents the time required for an accumula- tion of the active catalase dissociation product essential to start the decomposition of hydrogen peroxide, and is longer the less favorable the conditions for the dissociation.
IV. A THEORY OF THE CATALASE REACTION.
No theory of catalase activity can be built except upon the basis of an exact knowledge of the chemistry of hydrogen peroxide decomposition. Considering the paucity of this knowledge and in addition the complete ignorance of the chemical nature of catalase, the task is not materially different from that of solving an equation with two unknowns. The most that can be achieved in this state of the subject is to harmonize the known facts re- garding the catalase reaction and to search for some modes of its behavior. With the principal elements remaining unknown, the representation of the reaction must necessarily be symbolic rather than definitive.
One can entertain little doubt that the first step in the enzymic decomposition of hydrogen peroxide must be a dissociation of the catalase into an active form. The evidence obtained from our study of the influence of varying pH favors the conception
that the active form may possibly be the anion of catalase, though one must not mistake this evidence for actual proof. Without stressing this point unduly we may, however, suppose that a change like this occurs: catalase-+active catalase, which may be represented symbolically thus:
EM * -ffA (1)
We may now consider the evidence for this assumption. A8 hydrogen ion concentrations exceeding 1 X 10m5 N this change is impeded with the result that the decomposition of hydrogen peroxide is diminished until it ceases altogether at a hydrogen ion concentration of 1 X 1O-2 N. The change EMJEA is governed by temperature and pH, increasing with both. It is not unlikely that heating so alters the catalase that the change E,-+E, is impeded with the result that 25 per cent of the catalase becomes unavailable after heating at 5O”C., 60 per cent at 55”C., and at 60°C. the entire amount of catalase is thus affected. Since, however, the change EM+EA is promoted by a temperature rise we actually find that a catalase sample which, heated at 6O”C., has apparently lost its activity completely upon being tested at temperatures of about 20°C. will still display a small activity when tested at 30°C. Similarly, when the change EM+EA is de- pressed by acidity, the cataIase is relatively more effective at high than at low temperature. Finally, when the change E&~-SEA is impeded still more efficiently by a combination of factors, this manifests itself in a great prolongation of the latent period of the reaction. The occurrence of the latent period shows that a certain effective concentration of EA is necessary to initiate the reaction with hydrogen peroxide. In this respect we can produce experimentally summation or antagonism of effects by proper combination of such factors as temperature and acidity. The quiescent period which, of course, is radically different from the latent period, is another manifestation of the same phenomenon. The quiescent period is the summation effect of low temperature and high hydrogen peroxide concentration. The slow trans- formation EAv+EA in the cold makes the effective accumulation of EA difficult, especially in the presence of an excess of hydrogen peroxide, and a quiescent period sets in, which varies with the hydrogen peroxide concentration, but this will be discussed later.
How does the active catalase EA produce decomposition of hydrogen peroxide? What is the nature of this process? Actually the reaction is probably complex and proceeds in more than one step, but not knowing the intermediate stages, we may sim- plify this into the symbolic representation:
T EA +H202 --) ER +02 (2)
In other words, the setting free of oxygen from hydrogen peroxide involves (1) an oxidation of the HzOz, and (2) a reduc- tion of the catalase to an inactive state. The latter change is apparently irreversible (Morgulis (9)). It is, of course, a familiar fact that hydrogen peroxide is a reducing as well as an oxidizing agent, and we attribute the liberation of oxygen by catalase to the oxidation of the hydrogen peroxide for several reasons. The prototype of reaction (2) is perhaps found in the reaction
t Ag20 + Hz02 = 2Ag + HZ0 + 02, where the hydrogen peroxide is likewise oxidized with the resulting liberation of oxygen and the AgzO reduced to metallic silver. It is noteworthy that when hydrogen peroxide functions as an oxidant no evolution of oxygen gas can be detected. Thus in our studies of the oxidation of uric acid by hydrogen peroxide (9) no formation of free oxygen has been observed unless catalase was added to the system. This is also easily demonstrated by a simple experiment. If to a neutral solution of KI is added a neutral solution of hydrogen peroxide there is an immediate liberation of iodine which colors the mixture yellow, but no oxygen is given off. In this step of the reaction the HzOz is, of course, the oxidant, and 2 I- is oxidized to IZ. It is only after the iodine has been liberated, following a brief but measurable interval of time, that oxygen is now set free, while the yellow coloration fades out owing to the disappearance of the iodine which oxidizes the hydrogen peroxide and is thus changed back to the iodide. The evolution of oxygen in this typical reaction of the catalytic decomposition of hydrogen peroxide is associated with a process of oxidation of the hydrogen peroxide (by iodine), but not with the oxidation of the iodide (by the hydrogen peroxide). We believe that the setting free of oxygen in the catalase reaction-the intimate
mechanism of which we do not as yet understand-is likewise the result of oxidation of hydrogen peroxide. There is no evi- dence but the conjecture is not perhaps too far fetched that a peroxide is formed as an intermediate compound, EA+ 2Hz02 --fEp + 2Hz0, which is responsible for the oxidation of the hydrogen peroxide. But whatever the mechanism of this reac- tion may be, the oxidation of the hydrogen peroxide EA + HzOz--+
T En + 02, varies with temperature. Since it also depends upon the concentration of EA it will be affected by hydrogen ion con- centration in the sense that its course will be markedly repressed below pH 5.
Were there no complicating circumstances, the oxygen set free would be proportional to the concentration of EM, but suffi- cient evidence has been found to show that EA is being de- stroyed in the course of the reaction so that we are dealing with a changing concentration of EA. This destruction is undoubtedly a process of oxidation
EA + Hz0~ --) Eo + 11~0 (3)
and proceeds best at high temperatures, but increases also with a rise in hydrogen peroxide concentration and in OH ion concen- tration. We may now represent diagrammatically the changes in catalase incident to its reaction with’hydrogen peroxide as follows:
T EM + EA -+ ER + 02
dissociation I
reduction
Eo
Hence the obvious antagonism so far as the visible result of the reaction with the liberation of oxygen gas is concerned between the oxidation of hydrogen peroxide and the reduction of the hydrogen peroxide, the former alone resulting in oxygen evolu- tion. The reaction EA-+Eo is slower than the reaction EA-+
ER + O2 and frequently, especially at intermediate temperatures, does not become manifest until the reaction had already ad- vanced so far that the concentration of EA is very small as com- pared to the concentration of the still undecomposed hydrogen peroxide. In every reaction, therefore, there is a balance of effects which depends ultimately upon three factors-tempera- ture, pH, and hydrogen peroxide concentration. It is possible by a suitable increase in the effective concentration of catalase (or diminished concentration of hydrogen peroxide) to produce even at high temperatures (3540°C.) quick and complete de- composition of the hydrogen peroxide before any oxidation of catalase can occur. In other words, by proper adjustment of the relative enzyme and hydrogen peroxide concentrations and with the aid of temperature it is possible to make the reaction follow only one course:
t EM--,EA+',R+O,
On the other hand, by increasing the hydrogen peroxide concen- tration and the temperature it is possible to quicken the reac- tion in either direction. Since, however, the oxidation reaction has a greater critical thermal increment (cf. Table VIII, Paper II) the energy will be spent chiefly in destroying the catalase:
t EM + EA + ER +Oa
+
Eo
In this case the reaction EA+Eo will be so quickened by the high temperature that only a small part of EA will remain available to react with the hydrogen peroxide to produce oxygen. The oxygen produced under this condition is no longer affected by variations in hydrogen peroxide concentration. The same thing was shown in a less acute form in the experiments reported in Paper I (see Fig. 2). However, these striking results are only obtained at temperatures of 35°C. or above, and the reaction proceeds to completion with very great velocity (usually within 1 to 2 minutes). Owing to the distinctly explosive character of the reaction under such conditions it is not possible to measure its course.
We are now in a position to understand better the quiescent period, which we have seen to appear in the cold in the presence of a large hydrogen peroxide excess. Given a certain catalase concentration and a favorable pH (pH 7 in our experiments) the immediate reaction with the liberation of oxygen is deter- mined by the concentration of free EA. But following this initial spurt of activity during which the available EA is all used up further dissociation EAf--+EA proceeds extremely slowly owing to the low experimental temperature, and the reaction at once slows down or, depending upon the relative concentra- tion of the hydrogen peroxide in the system, stops altogether. We have shown already how the curve of catalase activity under these conditions tends more and more to approach a straight line (see Fig. 1, Paper I) the steepness of which varies inversely with the hydrogen peroxide concentration. Where the concen- tration of hydrogen peroxide is suficiently great, the subsidiary reaction, EA + H202--+Eo + HaO, tends to reduce the con- centration of EA and thus keep this below the effective level necessary for the initiation of the catalytic reaction, EA + HzOz
--+EB + 02, and a quiescent period ensues which is longer the greater the concentration of the hydrogen peroxide. The quiescent period has therefore this in common with the latent period that both represent a lapse of time necessary for the active catalase EA to attain an effective concentration. The differences between the two, however, are radical. Apart from the fact that the latent period precedes any activity, whereas the quiescent period follows an initial reaction, the former is asso- ciated with an impeded transformation EM--+EA, while the latter results from the subsidiary reaction EA+EO, which constant- ly brings down the concentration of EA in the system and thus prolongs the time necessary for the attainment of an effec- tive concentration. By increasing sufficiently the hydrogen peroxide concentration, as was done in one experiment where this has been brought up to 18.8 N, it is possible to limit the catalase activity to just the immediate reaction of the available free EA. In this case the reaction as far as the liberation of oxygen is concerned stops during the 1st minute, and the quiescent period which then ensues becomes infinitely long. This is,
therefore, similar to the condition previously suggested to occur under certain experimental circumstances (heating the catalase to 60°C. for over 30 minutes or lowering the pH to 2) when the change EM+EA is so completely hindered that the latent period also becomes infinitely long.
BIBLIOGRAPHY.
1. Michaelis, L., and Pechstein, H., Biochem. Z., 1913, liii, 320-355. 2. Sorensen, S. P. L., Biochem. Z., 1909, xxi, 200-304. 3. Bodansky, M., J. Biol. Chem., 1919, xl, 127-130. 4. Morgulis, S., J. Biol. Chem., 1921, xlvii, 341-375. 5. Biechy, T., Fermentforschung, 1924, viii, 135-166. 6. Kolthoff, I. M., J. Biol. Chem., 1925, lxiii, 135-141. 7. Morgulis, S., Beber, M., and Rabkin, I., J. Biol. Chem., 1926, lxviii, 521. 8. Morgulis, S., Beber, M., and Rabkin, I., J. Biol. Chem., 1926, lxviii, 535. 9. Morgulis, S., Ergebn. Physiol., 1 Abt., 1924, xxiii, 308-367.
