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M Vitro: 1 B. C H A N C E, Acta Chem. Scand. 1, 236 (1947)
M Vitro: 1 B. C H A N C E, Acta Chem. Scand. 1, 236 (1947)
[13] Catalase in V i t r o
By HUGO AEB1
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).
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
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.
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]