ANALYTICAL BIOCIJEMISTRY 47, 389-394 (1972)

Calorimetric Assay of Catalase

ASRU K. SINHA
Dwnrtment of Microbiology, Miami L:niversity, Oxford, Ohio 45056

Rcwived August 4, 1971

Various methods are available for the determination of catalase ac-

tivity (l-7). Of all the methods, the ultraviolet spectroscopy ( 1) and
permanganate titration method of von Euler (2) are reliable for de-
termining catalase of moderate or high degree of purity. In the case of
crude enzyme preparation, the iodometric assay of Jolles (3) and mano-
metric method of Perlman and Lipmann (4j are generally believed to be
reliable. However, the determination of end point in the iodometric titra-
tion is sometimes difficult and the manometric method apart from being
more cumbersome than the titrimetric or photometric assay is not without
objection (8).
In this paper a simple calorimetric nictjhod for rapid assay of catalase
is described.

METHODS AKD MATERIALS

Principle. The method is based on the fact that clichromate in acetic

acid is reduced to chromic acetate when heated in the presence of H,O,,
with the formation of perchromic acid as an unstable intermediate. The
chromic acetate thus produced i s measured calorimetrically at 570-610
mp. Since dichromate has no abaorbancy in this region, the presence of
the compound in the assay mixture dots not interfere at all with the
calorimetric determination of chromic acetate. The catalase preparation
is allowed to split H,O, for different periods of time. The reaction is
stopped at a particular time by the addition of dichromate/acetic acid
mixture and the remaining H& is determined by measuring chromic
acetate calorimetrically after heating the reaction mixture.
Reagents. (1) Dichromate/acetic acid: this reagent is prepared by
mixing a 5% solution of K,Cr,07 with glacial acetic acid (1:3, by
volume) and can be used indefinitely. (2) Hydrogen peroxide (0.2 M).
(5) Phosphate buffer (0.01 M), pI!I 7.0.
Colorinretric determination of H,O,. Different amounts of H,O,, rang-
ing from 10 to 160 pmoles, were taken in small test tubes (6 ml) and
3x9
@ 1972 hy Academic Prow. Inc.
390 ASRU K. SIKHA

2 ml of dichromate/acetic was added to each. Addition of the reagent to

H,O, instantaneously produced an unstable blue precipitate of perchro-
mic acid. On subsequent heating for 10 min in a boiling water bath, the
color of the solution changed to st.able green due to the formation of
chromic acetate. After cooling at room temperature the volume of the
reaction mixture was made 3 ml and the optical density was measured
at 570 mp in a Hitachi Perkin-Elmer spectrophotomcter. The linear rela-
tionship between the optical density and the amount of H,O, used is
shown in Fig. 1.
Determination of catalase actiuity by dichrovnate/acetic acid reagent.
Unless otherwise stated, the assay mixture contained 4 ml of H,O, solu-
tion (800 pmoles) and 5 ml of phosphate buffer in a small Erlenmeyer
flask (50 ml). 1 ml of properly diluted enzyme preparation was rapidly
mixed with the reaction mixture by a gentle swirling motion. The reac-
tion was run at, room temperature. A 1.0 ml portion of the reaction mix-
ture was withdrawn and blown into 2 ml of dichromate/acetic acid re-
agent at 60 see intervals. The hydrogen peroxide contents of the
withdrawn samples were determined by the method described above.
Determkation of catnlase activity b,q persvzanganate and iodometric

0 40 ~%o/es 120 160

of ,‘-j 0
22
FIG. 1. Reduction of K&LO, in acetic acid to chromic acetate by Hz02. The
color intensity of chromic acetate was measured at 570 mp (1 cm light path). For
details of the experimental procedure see text.
 COLOHIMETRIC ASSAY OF CATALASE 391

titrations. Catalase activity of the enzyme preparations was also deter-

mined by the permanganatc and iodomctric titration methods as de-
scribed by Sumner and Dounce (2).
Preparation of e?zzUwe snnbples. Crystalline beef-liver catalase (Sigma
Chemical C0.j suspension was centrifuged to isolate the crystals of the
enzyme, which was clissolred in phosphate buffer, l)H 7.0, 0.01 31, to
give a final concentration of 1.0 mg protein/ml. Btforc assay the cntalase
solution was properly diluted \Tith water.
P~repamtion of sheep erythroqte lysctte. Sheep blood corpuscles were
isolated by centrifugation of commercial sheep blood cells (The Brown
Laboratory, Topeka, Kan. J.
The isolated blood corpuscles were washed twice with 0.9% NaCl
solution. They were then lywd with 20 parts of cold water. The lysate
was used as such or the hemoglobins were removed beforehand. Removal
of the hemoglobins was by the method of Tsuchihasi (9). The lysate was
properly diluted with water for the assay of its catalaee content.
Preparation of yeast extract. A cell-free extract of commercial Fleisch-
mann yeast was prcparcd according to the method described earlier (10).
Prepmntion of rut liuer homoyemte. A normal male albino rat was
killed by decapitation. The liver was remorcd and homogenizcrl in cold
0.1 M phosphate buffer, pH 7.0, using a gla ss homogenizer. The homoge-
nat’e was centrifuged at 700~ for 5 min to remove debris. The protein
content, was adjusted to 80 mg/ml.
The protein content of the enzyme lwparation was determined accord-
ing to Lowry et al. (11‘) Bovine serum albumin used in this study was a
product of Pentex, Kankakee, Ill.
Calculation of results. The monomolecular velocity constant K for the
decomposition of H,O, by catalase was determined by using the equation
for a first-order reaction:

where S, is the initial concentration of H,O, and S is the concentration of

the peroxide at t min. The values of K were plotted against time in
minutes and the velocity constant of catalase K(0) at 0 min was dcter-
mined by extrapolation.
The catalase contents of the enzyme preparations were expressed in
terms of Katalusefaiihigkeit or “Kat. f” according to yen Euler and
Josephson (12) :

Kat. f = k’(O)
gm protein/ml
392 ASRU K. SINHA

RESULTS AND DISCUSSIOK

The catalase contents of different enzyme preparations were assayed by

the permanganate titration method and Kat. f values obtained by this
method were compared with those of the values determined by the colori-
metric method using dichromate/acetic acid reagent. It was found that
the Kat. f values obtained from both methods were comparable (Table 1).
Determination of the catalase activity of the erythrocyte lysate contain-
ing hemoglobin was difficult, due to the presence of heme protein in the
assay mixture for permanganate titrat,ion. The extent, of interference
caused by hemoglobin was small in the calorimetric assay of the enzyme
and was overcome easily wit,h the help of an appropriate control
experiment.
Although dichromatc in acetic acid is reduced by various types of
oxidizable compounds including free sugars and basic amino acids, the
concentration of such compounds in the assay system is usually t,oo low to
produce any appreciable interference in the calorimetric determination
of H,O? as described above. In this context it is interesting to note that
protein itself does not reduce dichromate in acetic acid. If excess amount
of protein is present in the assay systetn, it is usually precipitated out
after addition of the reagent.
Sometimes it was necessary to dilute an enzyme preparation in the
range of 1: 1000 before the catalase activity of the preparation could be
determined. Dilution of the preparation in this range usually results in
rapid loss of enzymic activity. As protein does not interfere with the
calorimetric determination of H?O, by dichromatc reagent,, bovine serum
albumin was added to the assay misture to prevent the destruction of
catalase activity caused by dilution. In a typical experiment, the dilution
(1: 1000) of the rat, liver homogenate was done in 0.5% bovine serum
albumin. Assay of the catalase activity was made in an identical reaction

TABLE 1
Comparison of Kat. f Values of Different Enzyme Sources by Calorimetric and
Permanganate Titration Methods

Kat. f

Calorimetric Permanganate
Catalase source assay t,itration

Beef-liver cry&. catalase 165 X 103 161 X 10”

Sheep erythrocyte lysate
(with hemoglobin) 98 Not detd.
(without hemoglobin) 72 64
Yeast extract 108 101
 COLORIMETRIC ASSAY OF CATALASE 393

TABLE 2
Comparison of Cat,alase Activity (K) of Rat Liver Homogenate in Presence and
Absence of Added Bovine Serum hlbumin

Catalnse activity (K)

Calorimetric
Time Calorimetric N&Or assay
(min) assay titration (with BSA)
~-___
1 0.060'2 0.0612 0.0711
2 0.0480 0.0467 0.0710
3 0. oml 0.0403 0.0701

mixture described under “Methods and Materials” escept that 50 mg of

bovine serunl albumin was added to it. Aft.er addition of the dichromate
reagent the reaction mixture was heated for 10 min, and after cooling at,
room temperature the precipitated protein was removed by centrifugation
and the optical density measured as described earlier. The presence of
bovine serum albumin (BSAj in the process of dilution and assay of
catalase activity of rat liver homogenate was found to prevent destruc-
tion of the enzymic activity effected by dilution (Table 2).
The assay mixture should preferably contain no mineral or trichloro-
acetic acid, since the sensitivity of the calorimetric assay of H,O, by
the above method is greatly rcducccl in the presence of these acids.

A simple calorimetric assay for ratalasc activity has been described

using K,@r,O,/acetic acid reagent. Kat. f values of different enzyme
sources were determined by the calorimetric method and compared with
the values obtained by titrimetrir methods.

ACKNOWLEDGMENTS
The author thanks Dr. J. Ii. Bhattacharjee for providing facilities for this work.
The work was partly financed by a grant from the National Science Foundation
( GB 28558X).

REFERENCES
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394 ASRLJ K. SINHA

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