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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,

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

(Receivedfor 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
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.’

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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.
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

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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-
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.

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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-
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-

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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
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.

HnOz concentration.
Temperature.

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0.36 N 0.54 N 0.72 N 0.90 N 1.08 N
-
“C.

0 55.5 76.0 40.5 29.8 13.0


8 100.0 88.2 61.4 62.8
19.4 68.2 73.5 66.6 57.7 49.5
30 46.5 48.0 44.7 42.7 38.5
.
0.36 N 0.72 N 1.08 N 1.44 N 1.80 N
- .- .-
3 100.0 82.6 49.0
11.5 95.5 70.9 53.0
15.8 90.7 49.1 50.4 37.3
19.8 73.4 55.6 49.5 42.6 33.9
24.5 56.9 49.6 42.6 39.6 32.6
_ .-
0.37 N 0.55 N 0.74 ii
-_
0 41.7 21.4
10 100.0 82.5 56.0
16.1 96.2 75.0 59.5
20.2 71.5 60.5 52.4
24.7 59.8 53.9 49.5
29.5 49.0 44.5 41.2
__ _-
0.22 i-4 0.36 N 0.50 N
-
15.1 97.0 100.0
20 78.0 70.0
25.4 57.9 58.6 59.0
30 48.2 44.8 44.8
- -
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
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

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Catoh fict,v,ty at d,fferent
Temperatuves and dtffarent
tiydmqw Peroxidr Cm
0 018N 0 014N V 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.
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.

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

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O’C
0 10 to 30 40 50 60 70 eo 90 100 110 110 130 140
M,n

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.
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:

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Temperature. Latent period.

“C. sec.
9.2- 9.8 10-15
21.0-21.4 8
31.1-31.4 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.
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.

HzOz concentration.

0.37 N 0.56 N 0.74 N

“C.
10-20 3.19 4.76 3.98
10-30 3.69 4.03 3.98
20-30 4.32 4.28 3.82

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TABLE III.

Hz02 concentration.
Temperature range.
0.36 N 0.72 N 1.44 N 1 80 N

“C.

3.0-24.5 2.69 4.45 4.67 5.36


11.5-24.5 3.53 4.28 5.75
19.8-24.5 5.89 4.36 5.01 6.46

TABLE IV.

H202 concentration.
Temperature mnge.
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.

Hz02 concentration.
Temperature range.
0.3 N 0.4 N 0.5 N

“C.

l-10 0 2,100 4,200


10-20 2,400 9,100 13,200
20-30 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
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

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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
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.

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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, cxxv, 283-291.
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.
STUDIES ON THE EFFECT OF
TEMPERATURE ON THE CATALASE
REACTION: I. EFFECT OF DIFFERENT
HYDROGEN PEROXIDE
CONCENTRATIONS
Sergius Morgulis, M. Beber and I. Rabkin
J. Biol. Chem. 1926, 68:521-533.

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