STUDIES ON THE EFFECT OF TEMPERATURE ON THE CATALASE REACTION.

1. EFFECT OF DlFFERENT HYDROGEN PEROXIDE CONCENTRATIONS.

BY SERGIUS RIORGULIS, M. BEBER, AND I. RABKIN,

(From the Department of Biochemistry, University of Xebraska, College of Medicine, Omaha.)

(Received for publication, March 23, 1926.)

In a recent. investigation of the catalase activity of the blood (1) it is stated that the outstanding difference between catalase and other enzymes is “its remarkable activity at lower temperatures.” It is well known, of course, that enzymes generally act best within a temperature range of 3550°C. The great activity of catalase at low temperature is, therefore, a very striking phe- nomenon which merits closer study. Does the increased activity at low temperatures indicate an anomaly in the behavior of catalase as compared to other enzymes? Does catalase possess an optimum at the lower end of the temperature scale? In the case of blood catalase this apparent anomaly may perhaps be easily explained, as was done by Bach and Zubkowa (2), on the basis of the digestion of the catalase by blood proteases whose activity increases as the temperature rises up to 47°C. It seemed desirable to study this problem, using for this purpose a catalase preparation rather than whole blood in order to avoid complicating factors. Our experiments were all made with a catalase extract which is obviously free from proteases since it retained its activity for over 15 months without any observable deterioration.

Method.

The experiments recorded in this series of papers have all been made with catalase extracted from beef kidney. The cortex of a large number of kidneys was first finely chopped, then ground with sand in a mortar, and

521

by guest on May 12, 2018

http://ww

w.jbc.org/

Dow

nloaded from

522 Catalase Reaction. I

extracted with an equal weight of water saturated with chloroform. The extraction proceeded for several days with frequent shaking, after which the watery extract was filtered through muslin, then through fluted papers. A clear, brown solution was obtained which was then treated with one-fifth its volume of chloroform. This treatment caused a precipitation of a large amount of protein and of dissolved hemoglobin, but the activity of the extract was not appreciably altered. The material was finally filtered once more and kept in a dark place in a glass-stoppered bottle, A small quantity of chloroform was added as a preservative. We performedexperiments with this preparation for a period of over 15 months and during this time no material loss of the original catalase strength was found.’

FIG. 1. Thermostat in which the catalase reaction takes place under constant shaking.

The different experimental temperatures were secured by means of a specially constructed thermostat shaking machine shown in Fig. 1.

The thermostat consists of a well insulated double walled box, the space between the inner and outer walls of which is lined with two layers of “in- sulatin,” each 1 inch thick, separated by a 2 inch cushion of horse hair. The inside of the box is lined with zinc. The box is fastened to a shelf which slides freely in grooves and is attached by means of an arm to a set of wheels operated by an electrically driven motor. The entire thermostat with its contents is thus shaken at a fixed rate of about 100 strokes per minute.

Six determinations could be made simultaneously, the interior of the

‘At the present time the preparation is more than 2 years old and still possesses its full strength.

by guest on May 12, 2018

http://ww

w.jbc.org/

Dow

nloaded from

S. Morgulis, M. Beber, and I. Rabkin 523

box being provided with this number of holders for bottles in which the reaction took place. These are ordinary wide mouth glass bottles of about 500 cc. capacity, to which the various reagents were transferred directly. The catalase solution was measured into nickel crucibles, provided with a round knob on the bottom, which when lowered carefully into the bottle would remain in an upright position until the shaking began. The least jar sufficed to cause the crucibles to capsize and empty their contents into the bottle where the catalase solution immediately mixed with the other reagents.

The thermostat was filled with water of the desired temperature (or with ice) to such a depth that the six bottles were submerged up to the shoulder when placed in the holders. The latter were extensively perforated, thus permitting the water to flow freely about the bottles. The thermostat with the bottles in position was now closed, first by a zinc tray fitting snug- ly into the box but allowing the necks of the bottles to protrude freely. A layer of insulatin and a thick felt mat came on top of this and, finally, the thermostat was closed with a wooden cover. All these covers were so made that they fitted neatly around the neck of the bottles which alone remained exposed. The temperature of the water was determined by means of a thermometer inserted through the several covers to the bottom of the box.

The crucibles with the catalase solution having been lowered into the bottles, the latter were closed tightly with soft rubber stoppers carrying two tubes: one straight tube to which a piece of pressure tubing was attached and opened or closed by means of a screw clamp; and a bent tube provided with a bulb by which the evolved gas was conveyed from the reaction bottles into eudiometer tubes. The former served to keep the interior of the bottles at atmospheric pressure, alloying the free expansion or contraction of the air to take place during the preliminary period, while the temperature was equalized, without affecting the volume of the system. After the tempera- ture of the entire system had been equalized, the bottles were closed off by tightening the screw clamps and the thermostat was now set in motion by turning the switch of the motor. The gas generated in the course of the reaction was collected over water in eudiometer tubes of different capacities -according to the amount of oxygen expected in the particular experiment. The volumes of gas recorded in the tables have been corrected for tempera- ture, barometric pressure, and aqueous tension, so that the data invariably represent volumes at standard conditions of 0°C. and 760 mm. of Hg.

In experiments of relatively short duration the temperature could be maintained generally within IV., though in experiments lasting several hours, except those at O”C!., a somewhat greater variation between the be- ginning and end temperature would take place. In this case, however, we -could still secure a more or less definite temperature by leaving one reagent bottle vacant and packing ice into this to keep the temperature of the box from rising.

The total volume of the reaction mixture in our experiments was either .50 or 20 cc., the smaller volume being selected as our preference in all later experiments. The hydrogen ion concentration was also maintained con-

by guest on May 12, 2018

http://ww

w.jbc.org/

Dow

nloaded from

524 Catalase Reaction. I

stant in all our experiments except where the variation in pH was the pri- mary objective of the tests. This will be discussed in the last paper of this series, but it will be stated here that unless otherwise specified the reaction was always studied at a pH of 7.0, this being maintained with the aid of a phosphate buffer, the concentration of which in the total volume was l/60 molar. We used Merck’s Superoxol as the source of hydrogen peroxide. This is to be preferred to other grades because it is neutral and is not con- taminated by various preservatives, it keeps well in a cool dark place, and the required concentration can be quickly prepared by appropriate dilution. We always checked up the peroxide content of the diluted material by direct titration with standard KMnOa.

E$ect of Temperature on the Catalase Reaction at Dij’erent Hydrogen Peroxide Concentrations.

We performed many experiments in which the catalase reac- tion was studied under varying hydrogen peroxide concentrations and temperature conditions. What impresses one at once in these experiments is the fact that at temperatures above 30°C. the reaction tends to become explosive, running a short but very vigorous course. At temperatures around 35°C. the reaction proceeds with great force and generally is completed in a minute or two. At temperatures of 30°C. or less the reaction velocity is very markedly affected, many hours being required for the reaction to run its full course below 10°C. As the velocity diminishes with a lowering of the temperature the extent of the reaction, as measured by the amount of oxygen set free, in- creases progressively up to a certain point, the maximum reac- tion, however, occurring above 0°C.

The marked difference in catalase activity under circumstances where temperature is the only variable factor may be due, of course, to the fact that its optimum, unlike that of other enzymes, is between 0 and 10°C. Issajew (3) claims that yeast catalase has an optimum of 40°C. but his experiments have not been properly controlled to determine the degree of spontaneous decomposition of the peroxide at high temperatures. In our experience this is so serious a factor that we could not rely on the results obtained at ,temperatures of 40’ and over, and even ex- periments at 35-40°C. could be made only with the utmost precautions.

The observed difference in activity under different tempera-

by guest on May 12, 2018

http://ww

w.jbc.org/

Dow

nloaded from

S. Morgulis, M. Beber, and I. Rabkin 525

tures, however, may also be due to other factors which either jointly or separately alter the catalase reaction. Thus, the loss of activity with rising temperature might be caused by a progres- sive spontaneous inactivation of the catalase, or it might result from an actual destruction of catalase by the excess of hydrogen peroxide. WTe shall attempt to analyze these factors with the view of gaining an insight into the correct interpretation of this peculiarity of the behavior of catalase at different temperatures. It has been already pointed out by one of us (4) that the catalase activity is depressed by increasing the hydrogen peroxide con- centration. In fact, it is possible by rendering the concentra- tion of the hydrogen peroxide sufficiently high to stop the reac- tion almost completely. This loss of activity is not a linear function of the peroxide concentration. In one experiment where we tested the effect of concentrations from 0.36 to 18.8 N,

99 per cent of the activity was lost through a 52-fold increase in the concentration; 76 per cent through a 13-fold increase, and 16 per cent through a 1.3-fold increase in the concentration. How does temperature affect the catalase reaction with varying hy- drogen peroxide concentrations?

In Table I the results obtained in a number of experiments, wherein the combined influence of the two factors was studied, are summarized. It is quite obvious that the effect is cumulative, as is also illustrated by the curves in Fig. 2. The depressing effect of the hydrogen peroxide is intensified by a rise in tempera- ture, but to varying degrees. From the series of curves in Fig. 2 it is seen that the greatest contrast in the catalase activity is manifested at 3” and the smallest at 24.5”C. In other ex- periments at still higher temperatures we succeeded in abolishing entirely the difference in activity with varying hydrogen peroxide concentrations. Working with a range of concentrations of 0.2, 0.3, 0.4, and 0.5 N 29 cc. of oxygen were given off at 34°C. in each instance, whereas at 0.5”C. 52, 64, 90, and 80 cc. of oxygen respectively were given off. The catalase concentra- tion, of course, was the same in all experiments.

In the next paper it will be shown that spontaneous inactiva- tion of catalase apparently does not take place under the condi- tions of our experiments. We are bound to conclude, therefore, that catalase must be destroyed by the excess of hydrogen

by guest on May 12, 2018

http://ww

w.jbc.org/

Dow

nloaded from

526 Catalase Reaction. I

peroxide in the system. Thus there is in the catalase reaction an antagonism between the decomposition of hydrogen peroxide by the catalase with the resulting liberation of oxygen and the destruction of the catalase by the hydrogen peroxide which causes a diminution in the oxygen set free. The balance between

TABLE I.

Relative Catalase Activity.

Temperature.

“C.

0 8

19.4 30

3 11.5 15.8 19.8 24.5

0 10 16.1 20.2 24.7 29.5

15.1 20 25.4 30

.

-

_

-_

_ _

-

0.36 N 0.54 N 0.72 N 0.90 N

55.5

68.2 46.5

0.36 N

100.0 95.5 90.7 73.4 56.9

0.37 N

-

.-

.-

_- -

76.0 40.5 29.8 13.0 100.0 88.2 61.4 62.8

73.5 66.6 57.7 49.5 48.0 44.7 42.7 38.5

0.72 N 1.08 N 1.44 N .- 82.6 70.9

55.6 49.6

53.0 49.1 49.5 42.6

50.4 37.3 42.6 33.9 39.6 32.6

0.55 N 0.74 ii

100.0 96.2 71.5 59.8 49.0

0.22 i-4

41.7 21.4 82.5 56.0 75.0 59.5 60.5 52.4 53.9 49.5 44.5 41.2

0.36 N

57.9 48.2

97.0 78.0 58.6 44.8

0.50 N

100.0 70.0 59.0 44.8

HnOz concentration.

-

1.08 N

1.80 N

49.0

these opposed reactions is determined by temperature, on one hand, and by the relative peroxide concentration, on the other. At low temperatures the balance shifts in favor of the catalytic reaction, and at high temperatures, on the contrary, the destruc-- tion of the catalase by the peroxide is favored, leading to rapid

by guest on May 12, 2018

http://ww

w.jbc.org/

Dow

nloaded from

S. Morgulis, M. Beber, and I. Rabkin 527

cessation of the catalytic reaction. The destruction of the cata- lase is presumably an oxidation phenomenon, and this would seem to be supported by the peculiarity of the temperature curves (Fig. 2). Greater catalytic activity in the cold is, there- fore, due to less enzyme destruction through oxidation. At temperatures below 10°C. such destruction either does not occur

Catoh fict,v,ty at d,fferent Catoh fict,v,ty at d,fferent

Temperatuves and dtffarent Temperatuves and dtffarent

tiydmqw Peroxidr Cm tiydmqw Peroxidr Cm 0 0 018N 018N 0 0 014N 014N V V 090N 090N

FIG. 2. FIG. 3.

FIG. 2. Relative catalase activity at different Hz02 concentrations.

FIG. 3. Catalase activity at different temperatures and different hydro- gen peroxide concentrations.

or else is very negligible; it is moderate between 10 and 2O”C., but becomes much more extensive with further rise in the ex- perimental temperature. At 3540°C. the destruction is prac- tically instantaneous in the presence of an excess of hydrogen peroxide. We are led to conclude, therefore, and this will find corroboration later on, that the rate of oxidation of the catalase increases more with temperature than does the catalytic reaction.

by guest on May 12, 2018

http://ww

w.jbc.org/

Dow

nloaded from

528 Catalase Reaction. I

Although the catalase destruction below 10°C. no longer plays a significant role, the catalase reaction is definitely inhibited at 0°C. This is shown in Fig. 3, where the curves obtained at different hydrogen peroxide concentrations attain their maximum peak between 0 and 10°C. We maintain that it is erroneous to assume that the maximum activity occurs at 0°C. The reduced catalase activity at or near 0°C. is, of course, entirely different from the loss of activity sustained at high temperatures. As will be shown in the next paper, it is not due to inactivation of the enzyme by the low temperature.

A detailed study of the curves of the catalase reaction under different temperature conditions is very instructive, and some interesting things are brought out particularly in the initial stages, when the reaction is followed from minute to minute. At a definite hydrogen ion concentration (a pH of 7 in our ex- periments) and catalase concentration, the reaction commences practically at once upon mixing the reagents and there is usually an appreciable evolution of oxygen in the first few minutes no matter what the hydrogen peroxide concentration or the experi- mental temperature may be. At temperatures of 15 to 30°C. this liberation of gas continues until the reaction is completed. At lower temperatures, however, following the quick initial evolution of gas for a minute or two, the reaction stops for longer or shorter time, depending upon the temperature and the perox- ide concentration. When again resumed, the reaction proceeds with interruptions which are longer the higher the hydrogen peroxide concentration and the lower the temperature. Thus, in one series of experiments at 3°C. and 0.36 N hydrogen peroxide the catalase reaction proceeded uninterruptedly and was completed in 190 minutes. At twice that concentration the reaction, after the first 2 minutes, remained inhibited for 15 minutes and, when it started up once more, only about one-fifth of the initial volume of gas was set free in the next 15 minutes. The reaction then proceeded slowly and was completed in 370 minutes. With still higher hydrogen peroxide concentrations (1.08 and 1.44 N) the immediate reaction was followed by a quiescent period of 25 minutes, while with 1.80 N, after the initial evolution of gas lasting 1 minute, the reaction remained in abeyance for 35 min- utes, then it started up and continued uninterruptedly to the end.

by guest on May 12, 2018

http://ww

w.jbc.org/

Dow

nloaded from

S. Morgulis, .M. Beber, and I. Rabkin 529

0 10 to 30 40 50 60 70 eo 90 100 110 110 130 M,n

140

FIG. 4. Curves of four series of experiments with varying hydrogen peroxide concentrations and at different temperatures. The maximum activity has been found invariably with the 0.54 N HzOz. The curves at the lower temperatures are not completed, as the time at 0°C. extends to 270 minutes, but the direction of the curves is clearly indicated from the first half of the period. The curves at 8°C. with the greater peroxide concentra- tion also extend beyond the limits of the figure, the reaction being com- pleted after 195 minutes.

O’C

by guest on May 12, 2018

http://ww

w.jbc.org/

Dow

nloaded from

530 Catalase Reaction. I

This condition has not been observed except at temperatures below 10°C. As can be seen from Fig. 4 the curves become markedly altered tending more and more to approach straight lines as the experimental temperature is lowered. The thing we wish to emphasize, however, is that even with high hydrogen peroxide concentrations and at very low temperatures there is almost invariably an immediate reaction upon mixing the re- agents, the latent period of the reaction being relatively insig- nificant as may be shown, for instance, by the following records from two separate experimental series:

Temperature. Latent period.

“C.

9.2- 9.8 21.0-21.4 31.1-31.4

sec.

10-15 8 o-3

Van? Hoff’s temperature coefficient for a 10” temperature interval calculated from our experimental results is rather variable, though the variations are not haphazard but regular and depend upon both factors discussed in this section. There are obviously conditions of both temperature and peroxide con- centration when the coefficient is quite constant. Some calcula- tions are given in Table II.

The average temperature coefficient for this series is 4, which corresponds closely to the value found by Bohnson and Robert- son (5) in their study of the catalytic decomposition of hydrogen peroxide by iron and copper salts. This uniformity of the coefficients, however, disappears in experiments where a greater range of peroxide concentrations has been tested, especially at temperatures below 10°C. (see Table III).

The tendency of the coefficients to greater variation at ex- treme HzOz concentrations and especially with increasing tem- perature is shown by Table III. The least variability in this series as in the previous was obtained with a 0.72 N con- centration, the average value in this case being 4.33. This is shown even more strikingly when the coefficients are calculated over different temperature ranges, as is done in Table IV.

by guest on May 12, 2018

http://ww

w.jbc.org/

Dow

nloaded from

S. lMorgulis, M. Beber, and 1. Rabkin 531

From Issajew’s data (3) the temperature coefficient for yeast catalase can be calculated for a range of temperature of 0-10°C.

TABLE II.

“C.

10-20 10-30 20-30

HzOz concentration.

0.37 N

3.19 3.69 4.32

0.56 N 0.74 N

4.76 3.98 4.03 3.98 4.28 3.82

TABLE III.

Temperature range.

“C.

3.0-24.5 11.5-24.5 19.8-24.5

Hz02 concentration.

0.36 N 0.72 N 1.44 N

2.69 4.45 4.67 3.53 4.28 5.89 4.36 5.01

TABLE IV.

1 80 N

5.36 5.75 6.46

Temperature mnge. H202 concentration.

0.36 N 0.72 N 1.44 N 1.80 N

1.76 4.73 4.28 2.08 4.39 4.85 4.31 2.15 4.51 4.64 5.05 2.69 4.45 4.72 5.62

TABLE V.

Temperature range.

“C.

l-10 10-20 20-30

Hz02 concentration.

0.3 N 0.4 N 0.5 N

0 2,100 4,200 2,400 9,100 13,200

19,500 25,000 25,000

and is found to vary from 1.50 to 1.42, depending upon the peroxide concentration. His findings, thus, correspond more or

by guest on May 12, 2018

http://ww

w.jbc.org/

Dow

nloaded from

532 Catalase Reaction. I

less to the value we obtained for a temperature range of 3-11.5”C. with 0.36 N hydrogen peroxide, which furthermore corresponds to the value (1.5) given by Senter (6) who experimented ex- clusively with small peroxide concentrations. It is clear, how- ever, that the values recorded by either Issajew or Senter can- not represent anything but a very special condition and have no general significance. Except when a definite amount of hydrogen peroxide reacts with a definite quantity of catalase, the values of the van’t Hoff coefficient vary considerably with increasing temperature. We regard, therefore, our value of about 4 as representing more nearly the average temperature coefficient.

Arrhenius’ factor for the velocity of reaction increment with temperature, P, has also been worked out for several series of experiments. The constants for the catalase reaction have been determined by Northrop’s procedure (7). The values for p obtained for different experiments, while showing considerable variation among themselves, nevertheless display great uni- formity in the general trend of the change. A single example (Table V) will suffice to demonstrate this point.

It is obvious, thus, that the value for P recorded by Senter, namely 6200, is really without significance since P increases with a rise in temperature besides being affected also by the concentra- tion of the hydrogen peroxide. The conclusion to be drawn is that the catalase reaction proceeds at an accelerated velocity as the temperature rises.

SUMMARY.

1. Increasing experimental temperature causes reduction in catalytic activity which is greater the higher the concentration of hydrogen peroxide. This reduction is the result of the de- struction of catalase by the hydrogen peroxide, hence the maxi- mum activity occurs at low temperatures where the destruction is less.

2. The catalase activity is greatest at temperatures between 0 and 10°C. The loss of activity between 10 and 20°C. is much smaller than between 20 and 3O”C., while between 30 and 40°C. the loss is so rapid that the catalase reaction is very quickly exhausted.

3. Lowering the experimental temperature and increasing the

by guest on May 12, 2018

http://ww

w.jbc.org/

Dow

nloaded from

S. Morgulis, M. Beber, and I. Rabkin 533

hydrogen peroxide concentration flattens out the curve of the catalase reaction until it becomes, at very low temperatures, a straight line. The slope of the curve varies inversely with the hydrogen peroxide concentration.

4. Under the combined action of low temperature and high peroxide concentration there appears a quiescent period which, following an immediate reaction lasting a minute or two, extends over a longer or shorter time depending upon the hydrogen peroxide concentration. Under these experimental conditions there is no appreciable latent period of reaction.

5. The van% Hoff temperature coefficient for the catalase reaction varies with the experimental temperature and hydrogen peroxide concentration. In our experiments with 0.72 N hydro- gen peroxide the coefficient seems quite constant over a con- siderable range of temperatures and has a value of 4.

6. The Arrhenius’ factor, EL, also increases with increasing temperature and with increasing hydrogen peroxide concentra- tion. An increase in temperature produces an accelerated change in the catalase reaction velocity.

BIBLIOGRAPHY.

1. Walling, L., and Stoland, 0. O., Am. J. Physiol., 1923, Ixvi, 503-518. 2. Bach, A., and Zubkowa, S., Biochem. Z., 1922, 283-291. cxxv, 3. Issajew, W., Z. physiol. Chem., 1904, xliv, 102-116. 4. Morgulis, S., J. Biol. Chem., 1921, xlvii, 341-375; Ergebn. Physiol., 1

Abt., 1924, xxiii, 308-367. 5. Bohnson, V. L., and Robertson, A. C., J. Am. Chem. Sot., 1923, xiv,

2512-2522. 6. Senter, G., Z. physik. Chem., 1903, xliv, 257-318. 7. Northrop, J. H., J. Gen. Physiol., 1925, vii, 373-387.

by guest on May 12, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Sergius Morgulis, M. Beber and I. RabkinCONCENTRATIONS

HYDROGEN PEROXIDE REACTION: I. EFFECT OF DIFFERENTTEMPERATURE ON THE CATALASE

STUDIES ON THE EFFECT OF

1926, 68:521-533.J. Biol. Chem. 

  http://www.jbc.org/content/68/3/521.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

alerts to choose from all of JBC's e-mailClick here

  ml#ref-list-1

http://www.jbc.org/content/68/3/521.citation.full.htaccessed free atThis article cites 0 references, 0 of which can be

by guest on May 12, 2018

http://ww

w.jbc.org/

Dow

nloaded from