[13] CATALASEm Vitro 121

High concentrations of polyvalent anions, which may be contained in

test samples or in the batch of glutathione reductase used, can inhibit
glutathione peroxidase.
As differences in enzymic properties, e.g., in kinetics, of glutathione
peroxidase can not be excluded in different tissues or species, optimal
substrate concentrations, if in doubt, should be reassessed.
If the activity measured in crude samples is to be assigned to glu-
tathfone peroxidase unequivocally, a separation step and characteriza-
tion, e.g., by correlating selenium content and enzymic activity during
fractionation, should be considered.

[13] Catalase in V i t r o
By HUGO AEB1

2H202 eatal,s~2H20 + 02 (1)

ROOH + AH2 catalascH20 + ROH + A (2)
Catalase exerts a dual function: (1) decomposition of H202 to give H20
and 02 [catalytic activity, Eq. (1)] and (2) oxidation of H donors, e.g.,
methanol, ethanol, formic acid, phenols, with the consumption of I mol of
peroxide [peroxidic activity, Eq. (2)].

Kinetic Properties
The predominating reaction depends on the concentration of H donor
and the steady-state concentration or rate of production of H202 in the
system. In both cases the active catalase-H202 complex I is formed first.
The decomposition of H202, in which a second molecule of H202 serves
as H donor for complex I, proceeds exceedingly rapidly (rate constant k
107 liters mol -t sec -t) whereas peroxidative reactions proceed relatively
slowly (k - 102-103). t
The kinetics of catalase do not obey the normal pattern. On the one
hand it is not possible to saturate the enzyme with "substrate" within the
feasible concentration range (up to 5 M H202), and on the other there is a
rapid inactivation of catalase at H202 concentrations above 0.1 M, when
the active enzyme-H202 complex I is converted to the inactive com-
plexes II or III. Measurements of enzyme activity at substrate saturation
1 B. Chance, Acta Chem. Scand. 1, 236 (1947).

Copyright © 1984 by Academic Press. Inc.

METHODS IN ENZYMOLOGY, VOL. 105 All rights of reproduelion in any form reserved,
ISBN 0.12-182005-X
122 FORMATION OR REMOVAL OF OXYGEN RADICALS [13]

or determination of the Ks is therefore impossible. In contrast to reactions

proceeding at substrate saturation, the enzymic decomposition of H202 is
a first-order reaction, the rate of which is always proportional to the
peroxide concentration present. Consequently, to avoid a rapid decrease
in the initial rate of the reaction, the assay must be carried out with
relatively low concentrations of H202 (about 0.01 M). 2 As the activation
energy for the decomposition of H202 catalyzed by catalase is very low
(2500-7100 kJ/mol), there is only slight dependence on temperature
(Qi0 = 1.05-1.12). The decomposition of H202 initially (approx. 0-30 sec)
follows that of a first-order reaction with H202 concentration between
0.01 and 0.05 M. The rate constant (k) for the overall reaction is given by
k = (1/At)(ln SI/S2) = (2 • 3/At)(log SI/S2) (3)
where At = t2 - t~ = measured time interval and S~ and $2 = H202
concentrations at times tl and t2. The constant k can be used as a direct
measure of the catalase concentration. In studies with purified enzyme
preparations the specific activity (k'0 is obtained by dividing k by the
molar concentration of catalase (e).
kl = k/e (liters mol -l sec -l) (4)
The value for k'~ for pure catalase from human erythrocytes is 3.4 x 10 7
(liters mol-~ sec-~). This value is used to calculate the absolute content o f
enzyme in blood and tissues. 3

Assay Method

Principle
In the ultraviolet range H202 shows a continual increase in absorption
with decreasing wavelength. The decomposition of H202 can be followed
directly by the decrease in absorbance at 240 nm (e240 = 0.00394 --- 0.0002
liters mmol -I mm-I). 4 The difference in absorbance (AA240) per unit time
is a measure of the catalase activity.
To avoid inactivation of the enzyme during the assay (usually 30 sec)
or formation of bubbles in the cuvette due to the liberation of 02, it is
necessary to use a relatively low H202 concentration (10 mM). The H~O2
concentration is critical inasmuch as there is direct proportionality be-
tween the substrate concentration and the rate of decomposition. Due to
the special situation in catalase the dependence of the H202 decomposi-
R. K. Bonnichsen, B. Chance, and H. Theorell, Acta Chem. Scand. 1, 685 (1947).
R. K. Bonnichsen, this series, Vol. II, p. 781.
D. P. Nelson and L. A. Kiesow, A n a l Biochem. 49, 474 (1972).
[13] CATALASEin Vitro 123

tion on the temperature is small (Qi0 - 1.1) so that measurements can be

carried out between 0 and 37°; however, 20° is recommended. The pH
activity curve relative to V0 has a fairly broad pH optimum (pH 6.8-7.5):
measurements are made at pH 7.0. 5

Reagents
Phosphate buffer 50 mM, pH 7.0: dissolve (a) 6.81 g KH2POa, and (b)
8.90 g Na2HPO4 • 2H20 in distilled water and make up to 1000 ml
each. Mix solutions (a) and (b) in the proportion 1:1.5 (v/v)
Hydrogen peroxide 30 mM: dilute 0.34 ml 30% hydrogen peroxide
with phosphate buffer to I00 ml

Procedure
Measurement in Blood. Venous blood containing heparin or citrate is
centrifuged and the plasma and leukocyte layers are removed. The eryth-
rocyte sediment is washed three times with isotonic NaCI. A stock hemo-
lysate is prepared containing - 5 g Hb/100 ml by the addition of four parts
by volume of distilled water. A 1 • 500 dilution of this concentrated hemo-
lysate is prepared with phosphate buffer immediately before the assay is
performed and Hb (hemoglobin) content is determined in duplicate (e.g.,
by the method of Drabkin). For capillary blood, 0. I or 0.02 ml is hemo-
lyzed in 250 or 50 ml distilled water. If the hemoglobin content of the
blood is required as reference point, it must be determined in a separate
sample of blood. 6-8
M e a s u r e m e n t in Tissues. Catalase in tissues with relatively high activ-
ity, such as liver and kidney, can be determined spectrophotometrically if
complete lysis of all organelles and clear (or only slightly colored) solu-
tions or extracts can be obtained. A detergent (e.g., 1% Triton X-100)
must be used in the preparation of the stock homogenate (1 + 9 or 1 + 19),
otherwise too low values will result. Further dilutions can be made with
phosphate buffer, pH 7.0 (I : 100 to 1 • 500, depending on tissue and spe-
cies). However, if the sample after lysis of the organelles cannot be di-
luted to this extent, the considerable UV absorption of Triton X-I00 must
be kept in mind. As an alternative digitonin (0.01%) or sodium cholate
(0.25%) can be used. Normally, catalase activity of tissue samples is
expressed on a milligram wet weight or milligram total N basis. A conven-
B. Chance, H. Sies, and A. Boveris, Physiol. Rev. 59, 527 (1979).
6 H. Aebi, #~ "Exposes Annuels de Biochimie M~dicale," 29ieme s6rie, p. 139. Masson,
Paris, 1969.
7 H. Aebi and H. Suter, hi "BiochemicalMethods in Red Cell Genetics" (J. J. Yunis, ed,),
p. 255. Academic Press, New York, 1969.
8 H. Aebi, S. R. Wyss, B. Scherz, and J. Gross, Biochem. Genet. 14, 791 (1976).
 124 FORMATION OR REMOVAL OF OXYGEN RADICALS [13]

ient method for the measurement of catalase activity in tissue extracts has
been described by Cohen et al. 9

Assay Conditions
Wavelength, 240 nm; light path, 10 mm; final volume, 3.00 mi. Read
the sample containing, 2.00 ml enzyme solution or hemolysate and 1 ml
H202 at 20° ( - room temperature) against a blank containing, 1 ml phos-
phate buffer instead of substrate and 2 ml enzyme solution or hemolysate.
The reaction is started by addition of H202. The initial absorbance should
be approximately A = 0.500. Mix well with a plastic paddle and follow the
decrease in absorbance with a recorder for about 30 sec.

Stability of Enzyme
Catalase in intact erythrocytes and in concentrated hemolysates is
stable up to 6 days when kept at 2°. However, there is a relatively rapid
decline of activity in dilute hemolysates which is more likely due to de-
composition of the enzyme into subunits than to proteolytic changes. At a
concentration of 1.2 mg Hb/ml the activity decreases by 10-15% within 24
hr; at a concentration of 0.06 mg Hb/ml the loss of activity is 10% after 1
hr and 80-90% after 24 hr. Consequently, hemolysate samples should be
analyzed within 5-10 min after dilution.

Definition of Units and Specific Activity

It is not possible to define international catalase units (U) according, to
IUB recommendations due to the abnormal kinetics. Therefore, the use of
a number of differently defined units and different methods of evaluation
is acceptable for this enzyme (a selection of methods is listed in the
table).~° Use of the rate constant of a first-order reaction (k) is recom-
mended. The rate constant related to the hemoglobin content (k/g,Hb) can
serve as a measure of the specific activity of erythrocyte catalase. Equa-
tion (3) applies in this case. If the decrease in absorbance is recorded, the
value of log A tlA2 for a measured time interval or the time required for a
certain decrease in absorbance can be determined.
For a time interval of 15 sec the following, relationship is obtained
according, to Eq. (3):
k = (2.3/15)(1og AI/A2) = 0.153(1og, ALIA2) (sec -I) (5)
To calculate k/mi or k/g, Hb proceed as follows:

9 G. Cohen, D. Dembiec, and J. Marcus, Anal. Biochem. 34, 30 (1970).

~o H. Aebi, in "Methods of Enzymatic Analysis" (H. U. Bergmeyer, ed.), 3rd Ed. Verlag
Chemic, Weinheim, F. R. G., in preparation, 1983.
[13] CATALASE in Vio'o 125

OTHER METHODS OF DETERMINATION

Method/technique References Material

Determination of H202 removal

Titrimetric methods
Iodometric Bonnichsen et al. ~ Tissues, blood
Permanganometric Bonnichsen3 Tissues, blood
Spectrophotometry
Substrate: H202 (E240) Bergmeyer" Purified preparations
Cohen et al. 9 Tissues, organelles
Substrate: perborate (E220) Thomson et al. ~2 Tissue fractions
Photometry (E405-E~15)
Vanadic acid Warburg and KrippahP Cell cultures
Titanium tetrachloride Pilz and Johannb Blood
Fluorimetry
Scopoletin Perschke and Brodac Aqueous
Diacetyldichlorofluorescein Keston and Brandta solutions
Determination of 02 production
Oxygen electrode Ogata I~ Blood
Del Rio et al.'4 Plant material
Meerhof and Roos j~ Blood
Polarography
Catalase and SOD Rigo and RotiliC 6 Tissue homogenate
hemolysate
Immunoprecipitation Higashi et al." Blood, tissues
(with anti-catalase) Ben-Yoseph and Shapira t~ Blood
Screening techniques
Capillary tube Fung and Petrishko f Microbial cultures
"Siebtest" Gross et al. 18 Blood
Automated procedure
Technicon AutoAnalyzer Lamy et al. 19 Blood
Leighton et al. 2° Tissue fractions

° O. Warburg and G. Krippahl, Z. Naturforschung lllb, 340 (1963). b W. Pilz and J.

Johann, Z. Anal. Claem. 210, 358 (1965). ~ H. Perschke and E. Broda, N a t u r e {London) 190,
257 (1961). d A. S. Keston and R. Brandt, Anal. Biochem. 11, I (1965). "T. Higashi, M.
Yagi, and H. Hirai, J. Biochem. (Tokyo) 49, 707 (1961).f D. Y. C. Fung and D. T. Petrishko,
Appl. Microbiol. 26, 631 (1973).

k/ml = ka (6)
k/g Hb = k/ml(lOOO/b) = (2.3/15)(a/b)(Iog AI/A2) (sec -~) (7)
where A~ is A240 at t = 0, A2 is A240 at t = 15 sec, a is the dilution factor [Hb
concentration in blood or erythrocyte sediment (mg Hb/ml)/Hb concen-
tration in cuvette (rag Hb/ml)], and b is the Hb content of blood or eryth-
rocyte sediment (grams/liter).
For the difference in absorbance of 0.450-0.400 (log AI/A2 = 0.05115)
the following relation holds:
k = (2.3/At)(log Aj/A2) = 0.1175/At (sec -I) (8)
126 FORMATION OR REMOVAL OF OXYGEN RADICALS [14]

Other Methods of Determination

This contribution deals only with the catalytic (not the peroxidic) ac-
tivity of catalase. It can be measured by following either the decomposi-
tion of H202 or the liberation of 02. Accordingly, the remaining substrate
concentration at a given moment of the reaction can be determined by UV
spectrophotometry9,~'.~2 or--at the end of the reaction period--by simple
titration. 2,3 On the other hand, O~ production can be followed with the
oxygen electrode ~3-~5 or by polarography. ~6 Alternatively, catalase can
also be measured by immunoprecipitation. ~7 The method of choice for
biological material, however, is UV spectrophotometry. Titrimetric meth-
ods are suitable for comparative studies. For large series of measure-
ments there are either simple screening tests which give a quick indication
of the approximative catalase activity, '8 or automated methods (e.g., us-
ing the Technicon AutoAnalyzer) available 19,2°(see the table).
u H. U. Bergmeyer, Biochem. Z. 327~ 255 (1955).
t2 j. F. Thomson, S. L. Nance, and S. L. Tollaksen, Proc. Soc. Exp. Biol. Med. 157, 33
(1978).
,3 M. Ogata, in "Symposium on Genetics and Biochemistry of Acatalasemia" (IX Annual
Meeting of the Japan Society of Human Genetics), at Wakayama Medical College, 1964.
14 L. A. Del Rio et al., Anal. Biochem. 80, 409 (1977).
15 L. J. Meerhof and D. Roos, J. Reticuloendothel. Soc. 28, 419 (1980).
,6 A. Rigo and G. Rotilio, Anal. Biochem. 81, 157 (1977).
~7 y. Ben-Yoseph and E. Shapira, J. Lab. Clin. Med. 81, 133 (1973).
,8 j. Gross, A. Hartwig, and A. Golding, Z. Med. Labortech. 16, 336 (1975).
L9j. N. Lamy et al., Bull. Soc. Chim. Biol. 49, 1167 (1967).
20 F. Leighton et al., J. Cell Biol. 37, 482 (1968).

[14] Assays of L i p o x y g e n a s e , 1 , 4 - P e n t a d i e n e F a t t y Acids,

a n d 02 C o n c e n t r a t i o n s : C h e m i l u m i n e s c e n c e M e t h o d s
By SIMO LAAKSO, ESA-MATTI LILIUS, and PEKKATURUNEN

Lipoxygenases are iron-containing enzymes that catalyze the dioxy-

genation, by molecular oxygen, of cis,cis-1,4-pentadiene fatty acids. The
reaction is a source of weak chemiluminescence due to dissociation of
free radicals from the main path of hydroperoxidation.~ Under alkaline
conditions the intensity of light emission can be drastically amplified by
the presence of luminol? The kinetics of luminol chemiluminescence in
' A. Boveris, E. Cadenas, and B. Chance, Photobiochem. Photobiophys. 1, 175 0980).
2 E.-M. Lilius and S. Laakso, Anal. Biochem. 119, 135 (1982).

Copyright© 1984by AcademicPress, Inc.

METHODS IN ENZYMOLOGY,VOL. 105 All rightsof reproductionin any form reserved.
ISBN 0-12-182005-X