STUDIES ON THE EFFECT OF TEMPERATURE ON THE CATALASE REACTION.

II. LOSS OF CATALASE ACTIVITY.

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.)

Inactivation of Catalase by Heat.

In the preceding paper (1) the possibility of spontaneous in- activation of the enzyme by heat has been mentioned as a fac- tor responsible for the diminished catalase activity, especially at temperatures above 20°C. To determine to what extent this may have played a r61e in the experiments at different temperatures, the activity of catalase subjected for various lengths of time to the influence of different temperatures has been investigated under standard conditions. Definite amounts of the catalase extract, measured into a series of crucibles, were quickly heated or cooled to the desired temperature and kept at that temperature for a certain period of time, usually an hour; the material was then quickly brought to room temperature and the amount of catalase present was determined by the oxygen set free from a known quantity of hydrogen peroxide. This was compared with the oxygen set free by the same amount of untreated catalase ex- tract, the determinations being carried out under similar condi- tions (cf. Paper I) as far as pH and concentrations are concerned.

In the first experiments we studied the effect of temperature on the catalase at 10” intervals and the results, representing the final volumes of oxygen liberated in two series of experiments, are recorded in Table I.

Obviously catalase exposed for 1 hour to temperatures below 40°C. undergoes little or no inactivation, whereas at higher temperatures there is definite inactivation which increases with

535

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536 Catalase Reaction. II

an increase in temperature. The inactivation at 60°C. is prac- tically complete, the greatest loss of activity occurring by ex- posure at temperatures between 40 and 60°C. We studied the

TABLE I.

Experi- Series. mental

temperature

“C.

I 8.8 20.0 29.6

II 9.2 21.0 31.1

.-

-

35.7 35.5 25.5

56.2 39.0 22.3

Cat&se previously heated for 1 hr. to:

309 400 -~

34.8 35.1 34.0 34.4 24.9 24.6

54.4 48.6 40.0 37.4 20.6 21.2

TABLE II.

500

32.9 0 27.3 0 21.3 2.6

28.5 0 20.4 0 16.8 2.6

- -- 60"

-

Experimental temperature.

“C.

8.9 20.2 29.5

200

50.7 41.2 23.3

- Catalase previously heated for 1 hr. to:

Catalase Activity Determined at 20°C.

60”

0 0 0

TABLE IV.

Catalase Activity Determined at 19°C.

Catalase previously exposed for 1 hr. to:

temperature at which the maximum inactivation takes place within 1 hour by limiting the intervals to only 5” with the result shown in Table II.

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S. Morgulis, M. Beber, and I. Rabkin 537

We can see from this experiment that complete inactivation must take place by an hour’s exposure at temperatures between 55 and 6O”C., since at 55°C. in the same length of time the in- activation is only partial. With the facilities at our disposal we could not determine the exact temperature causing the com- plete loss of activity in 1 hour but we performed a series of experi- ments wherein the effect of heating to temperatures 2” apart was studied. These experiments (Table III) show that maximum inactivation is really associated with a very narrow range of temperature change.

It is well to point out in this connection that we also studied the effect of low temperatures on the activity of the catalase, and found little or practically no alteration in this activity until the enzyme had been exposed for 1 hour to a temperature 15” below zero. This experiment is important as it demonst,rates that the reduced activity observed in experiments carried out at 0” as compared with the activity of the same catalase concentration tested at temperatures between 0 and 10°C. cannot possibly be due to a spontaneous inactivation of the catalase (cj. Paper I). Even at -15°C. the observed loss of activity is not greater than that caused by exposure for 1 hour at 50°C. as can be seen from the experimental results in Table IV.

We next determined the rate of inactivation of catalase at 50, 55, and 60°C. We have shown already that exposure for an hour at the latter temperature is sufficient to cause complete inactivation, while at 55” for a similar period the inactivation is about two-thirds complete. Since, however, a certain amount of inactivation must already occur during the brief interval while the temperature is being brought up to the desired level we used in these studies of the rate of inactivation a different standard from that employed in the previous tests. The degree of inactivation, instead of being compared to that of catalase kept at 20”, was measured by referring to the activity of a sim- ilar sample which had been warmed to the same temperature as the other samples and then immediately cooled to room temper- ature. This procedure is safer not only because it corrects for the inactivation incident to the preliminary alteration in tempera- ture until the desired level is attained, but it also permits a more exact timing of the entire process. In the last paper of this

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538 Catalase Reaction. II

series, when we discuss the latent period of the catalase reaction, we shall come back to this matter of the inactivation process. Here we shall merely record the results of these experiments (Table V).

It is clear from these experiments that at 50°C. the inactiva- tion of the catalase is only partial even after heating for 2 hours and 20 minutes and does not apparently extend beyond about 25 per cent of the initial strength of the preparation. At 55°C. about 60 per cent of the catalase is inactivated in 13 hours, but since the same degree of inactivation is attained within 1 hour it seems that this is practically the maximum inactivation at

TABLE V.

0 min.

39.6 40.7

-

-

20 min.

38.1 35.8

-

-

Catalase heated at 50°C. for:

40 min. 60 min. 80 min.

33.6 34.0 30.3 33.2

Catalase heated at 55°C. for:

100 min. 140 min.

34.7 30.6

0 min. 5 min. 15 min. 30 min. 35 min. 45 min. 60 min. 65 min. 90 min. -- ~~______

38.3 36.0 23.6 21.9 15.8 35.9 26.7 18.7 18.7 16.0

Cat&se heated at 60°C. for:

this temperature. At 6O”C., on the contrary, the inactivation progresses very rapidly, being very nearly completed in 30 minutes, and entirely completed after 45 minutes of heating.

Fig. 1 represents a series of curves of the reaction obtained with unheated catalase and with catalase heated to 60°C. for 5, 15, and 30 minutes. This shows very striking changes not only in the total catalase activity but also in the character of the curves under these different conditions. An analysis of the curves in Fig. 2 shows that the inactivation of the catalase at 55 and 60°C. follows a definite course. We have attempted to fit these curves to various types of conical sections without much success; however, the experimental curves seem to correspond

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S. Morgulis, M. Beber, and I. Rabkin 539

closely to a theoretical curve calculated from the experimental data on the assumption that they represent points of a bimolecu- lar isotherm.

We do not know what particular significance this may have, but it seemed worth calling attention to this fact, since the bimolecular formula gives the only mathematical expression which fits the experimental data, as can be seen from Fig. 2 where

Rctlnty of Catalase

heated at 60-C. 0 Control (Unheated)

. ” 3OMm

FIG. 1. FIG. 2.

FIG. 1. Curves showirrg the course of the reaction with unheated catalase and with catalase samples heated for various lengths of time to 60°C. The concentrations and other experimental conditions are the same in all four experiments.

FIG. 2. Curves showing the inactivation of catalase heated for various lengths of time at 55 and 60°C. The ordinates give the percent of activity lost, and the abscissa the duration of heating in minutes.

the experimental points are placed in relation to the theoretical curve drawn from calculated values. Northrop (2) found that the spontaneous inactivation of trypsin follows the monomolecular isotherm if the enzyme is highly purified. With less pure prepara- tions the course of inactivation is bimolecular. Since our prepara- tion of catalase is only partially purified, this may perhaps ac- count for the circumstance that the heat inactivation follows a bimolecular course.

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540 Catalase Reaction. II

Injluence of Tempera,ture on the Destruction of Catalase by Iiydrogen Peroxide.

We have learned from the previous paper that catalase is destroyed by hydrogen peroxide, the destruction increasing with temperature. It is not possible to measure directly the catalase destruction in any one single experiment, but this can be esti- mated from a series of experiments. The differential temperature effect furnishes us the means of measuring, at least indirectly, the rate of the catalase destruction. The rate of oxygen evolu- tion, by which the catalase reaction is measured, really repre- sents the resultant of two distinct phenomena: (1) the catalytic decomposition of hydrogen peroxide, and (2) the destruction of catalase by the hydrogen peroxide. The equilibrium between the two processes is determined by temperature.

It has been shown by Morgulis (3) and by Northrop (4) that the oxygen set free is a measure of the active catalase. A study of the reaction at different temperatures gives us, therefore, a method of attacking the problem of the catalase destruction in the course of the reaction. In a series of experiments, where the only variable is the temperature factor, the final amount of oxygen liberated is a measure of the relative concentration of active catalase under these conditions. It has been shown that the higher the temperature the smaller the active concentration owing to the more rapid destruction by the peroxide of the catalase initially present.

Thus, in one set of experiments we were able to calculate that, compared to the activity of the catalase at IO%., there has been destroyed in the very first few minutes of the reaction 20 per cent at 16”C., 36 per cent at 2O”C., 39 per cent at 25”C., and 41.6 per cent at 30°C. The total loss of catalase activity by the conclusion of the reaction at these four different tem- peratures was 25, 39.5, 46.3, and 55.6 per cent respectively. The very great destruction of the catalase at the very beginning of the reaction is, therefore, evident. This, of course, is not surprising since the hydrogen peroxide also disappears during the reaction, the greatest concentration being present during the early stages.

As the destruction at temperatures somewhat below 10°C. is

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S. Morgulis, M. Beber, and I. Rabkin 541

negligible or practically nil, we may use this active catalase con- centration as a standard for comparison and thus obtain the relative catalase activity at various other temperatures. We can illustrate this with a few specific examples, taking the maximum results obtained for each separate series (at about 10°C.) as representing a unit of catalase activity. The catalase activities under various conditions are represented in Table VI. Although each of the series reported in Table VI is an independent set of experiments, there is remarkable agreement in the results, be-

N

0.2 0.3 0.4 0.5

0.98 0.83 0.89 0.57 0.72 0.43 0.68 0.42

.- 20°C. 30°C.

0.3 0.4 0.5

0.75 0.66

0.68 0.46 0.38

19.4v. 30°C.

0.36 1.00 0.47 0.54 0.74 0.48 0.72 0.67 0.45 0.90 0.58 0.43 1.08 0.50 0.39

i

- I

TABLE VI.

Relative cat&se activity. Cat&se destroyed.

20%. 27°C. 20°C.

per cent

2 11 28 32

20°C.

25 34

19.4”C.

26 33 42 50

27°C.

per cent 17 43 57 58

30°C.

32 54 62

30°C.

53 52 55 57 61

traying a definite regularity in the extent of the catalase destruc- tion in relation both to the experimental temperature and to the peroxide concentration.

But these results tell us as yet nothing of the course of the catalase destruction. We may, however, get light upon this process by extending the principle of the proportionality of the catalase to the oxygen liberated (the principle which has been defined by Northrop (4) mathematically) to other stages of the reaction as well as to the terminal stage. Since the velocity of the reaction at different temperatures varies greatly, such a

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542 Catalase Reaction. II

direct comparison of corresponding stages meets with con- siderable difficulty. This, however, can be overcome by super- imposing the curves of the reaction at different temperatures in such a manner that the beginning and end of each curve exactly coincide. This can be done by plotting the curves with dif- ferent time units on the abscissa, these units being proportional to the respective reaction velocities. Referring now to the curve of the experiment at the low temperature as the standard, the relative catalase activity at different times can be calcu- lated from the heights of the ordinates at corresponding points, and thus the degree of destruction at those times determined for

FIG. 3.

Msn 0 t 4 6 B 10 U I5 Mn 0 IO 10 30 A(

FIG. 4. FIG. 5.

FIG. 3. Curves showing the rate of catalase destruction at 20, 25, and 30°C. The ordinates represent catalase concentration.

FIGS. 4 and 5. Curves showing the rate of inactivation of catalase at 20 and 30X, respectively, and with 0.3,0.4, and 0.5 N hydrogen peroxide.

the different temperatures. Although such indirect procedures are generally fraught with obvious dangers, the strikingly sim- ilar results obtained by this method under a great variety of experimental conditions lends considerable assurance to this as a valuable aid for the study of this problem.

The data presented graphically in Figs. 3, 4, and 5, show that the catalase destruction during the reaction follows a very definite course, the rate varying with temperature. In one series of experiments the velocity of the catalase destruction was two to three times as great at 25°C. as at 2O”C., while at 30°C. it was eight to ten times as great. The data fit the monomolecular

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S. Morgulis, M. Beber, and I. Rabkin

TABLE VII.

543

0.3 N HzOs, 27°C. 0.4 N HzOz, 27°C.

K A--2 K A--2

0.0355 0.85 0.0330 0.83 0.0300 0.80 0.0317 0.70 0.0315 0.63 0.0296 0.56 0.0301 0.45

t

2 2.5 3.5 5 6.5 8.5

10

t

5 10 13 17 20

-

. _

.-

-

0.0084 0.91 0.0058 0.88 0.0065 0.83 0.0050 0.80 0.0052 0.79

0.4 N H202,2O”C. 0.5 N H202,2O”C.

4 0.0064 0.94 8.5 0.0054 0.90

12.8 0.0045 0.88 17 0.0040 0.86 21.3 0.0046 0.80 25 0.0048 0.75

2 0.0060 0.98 9 0.0055 0.89

14 0.0056 0.84 19 0.0057 0.78 24 0.0056 0.73 29 0.0054 0.70 34 0.0050 0.68 39 0.0045 0.67

- 0.5 N HeOz, 20°C. 0.5 N HzOz, 27°C.

5 0.0087 0.91 8 0.0156 0.75

10 0.0140 0.73 12 0.0110 0.71 15 0.0109 0.69 18 0.0106 0.66

5 0.0078 0.92 13 0.0058 0.84 20 0.0059 0.78 26 o.qo50 0.74

TABLE VIII.

Value of p for cat&se destruction.

Series. Temperature range.

HsO2 concentration.

0.3 N I 0.4 N I 0.5 N

“C.

20-27 20-30

I II

45,000 41,600 1 57,800 33,700 1 40,400

0.37 N 0.55 N

40,500 28,400 41,600 42,400 41,000 35,000

III 20-25 25-30 20-30

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544 Catalase Reaction. II

equation (in a number of instances with remarkable precision) so that the values calculated from the average velocity constant agree very well with the values obtained by the method de- scribed above. A few examples will suffice to demonstrate the magnitude of the values of K from the familiar equation K =

l log -4- t A - x’

where A = 1 represents the initial concentration

of active catalase and A - x represents the catalase concen- tration at various times t during the reaction (Table VII).

The rate of destruction of catalase by the hydrogen peroxide depends upon temperature. The Arrhenius factor, ,u, for the effect of temperature has been calculated for several series of experiments with the results shown in Table VIII.

A comparison of these p values with those recorded in Table V, Paper I, for the catalase reaction indicates a much greater uniformity in t$he case of the catalase destruction at different temperatures; it is also clear that in the latter instance these values are of an entirely different magnitude, even the smallest value being much greater than the largest p value observed for the catalytic decomposition of hydrogen peroxide by catalase.

SUMMARY.

1. Exposure of catalase to temperatures up to 40°C. for 1 hour causes practically no inactivation. Inactivation begins at about that temperature reaching a maximum at about 58°C.

2. The rate of inactivation at 55 and at 60°C. seems to follow the course of a bimolecular reaction.

3. The inactivation at 50°C. even over a long period does not exceed 25 per cent of the catalase, at 55°C. 60 per cent, whereas at 60°C. the inactivation is complete in less than 45 minutes.

4. Cooling the catalase does not reduce its activity until -10°C. is reached. Rut even at -15°C. an hour’s exposure occasions only about 12 per cent loss of activity.

5. Hydrogen peroxide destroys catalase, the rate of destruc- tion increasing with an increase in experimental temperature and in hydrogen peroxide concentration.

6. The destruction of catalase follows the course of a mono- molecular reaction.

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S. Morgulis, M. Beber, and I. Rabkin 545

7. The value of the Arrhenius factor p is very large and quite constant as compared to those values obtained for the catalytic decomposition of hydrogen peroxide by catalase.

BIBLIOGRAPHY.

1. Morgulis, S., Beber, M., and Rabkin, I., J. Biol. Chem., 1926, lxviii, 521. 2. Northrop, J. H., J. Gen. Physiol., 1922, iv, 261-274. 3. Morgulis, S., Ergebn. Physiol., 1 Abt., 1924, xxiii, 308-367. 4. Northrop, J. H., J. Gen. Physiol., 1925, vii, 373-387.

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Sergius Morgulis, M. Beber and I. RabkinACTIVITY

REACTION: II. LOSS OF CATALASETEMPERATURE ON THE CATALASE

STUDIES ON THE EFFECT OF

1926, 68:535-545.J. Biol. Chem. 

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