CATALASE from Microorganism

CAO-519

Hydrogen-peroxide: hydrogen-peroxide oxidoreductase (EC 1. 11. 1. 6)
2H₂O₂ ⇆ O₂ + 2H₂O

                                                                                            

 

PREPARATION and SPECIFICATION
Appearance:  Orive green solution
Activity:  Grade V 150,000 U/ml or more
PROPERTIES
Stability:  Stable at 4°C (Fig.1)
Molecular weight:  approx. 53,000 (by mass spectrometry)
Inhibitors :  NaN₃
Optimum pH :  7.2-9.0(Fig.2)
Optimum temperature 35-40°C(Fig.3)
pH Stability
 pH 6.2-8.9 (25°C, 16hr)(Fig.4)
Thermal stability :  below 35°C (pH 7.0, 30min)(Fig.5)
Effect of various chemicals (Table 1)

APPLICATIONS

This enzyme is useful for eliminating the interference of hydrogen-peroxide in clinical analysis.

ASSAY

Principle:

Catalase

2H₂O₂                                  ►O₂+2H₂O


The disappearance of hydrogen peroxide is measured by the Titanium color method ¹).

Unit definition:

One unit causes the hydrolysis of one micromole of hydrogen peroxide per minute under the conditions described below.

Method:

Reagents
A. 10mM Phosphate buffer, pH 7.0 (at 25°C): 
B. H₂O₂ solution:  16mM [0.182ml of 30% (W/V)H₂O₂/100ml of buffer A] (Should be prepare fresh and store on ice.)
C. Titanium reagent:  (Nacalai tesque)
D. Enzyme diluent:  Buffer A

Procedure

1. Prepare 0.25ml of the substrate solution (B) into a test tube and equilibrate at 25°C for about 5 minutes

2. Add 0.25ml of the enzyme solution* and mix.

3.After exactly 5 minutes at 25°C, add 2.5ml of Titanium reagent (C) to stop the reaction and

measure the optical density at 410nm against water (OD test)
At the same time, prepare the blank by first mixing the substrate solution with 2.5ml of

Titanium reagent after 5 min-incubation at 25°C, follow by the addition of enzyme solution (OD blank)

* Dilute the enzyme preparation to 0.35-1.35U/ml with ice-cold enzyme diluent (D).

Calculation

Activity can be calculated by using the following formula :

∆OD(OD blank–OD test)×Vt×df

Volume activity (U/ml) =                                                    =∆OD×3.43×1/F×df

F×t×1.0×Vs

 

Weight activity (U/mg) = (U/ml)×1/C

Vt
: Total volume (3.0ml)
Vs
: Sample volume (0.25ml)
F
: Extinction coefficient of Titanium color product developed by the presence of 1.0mM hydrogen peroxide (F should be determined in each lot of Titanium reagent by using a known concentration of hydrogen peroxide. F is usually around 0.7.)
t
: Reaction time (5 minutes)
1.0
: Light path length (cm)
df
: Dilution factor
C
: Enzyme concentration in dissolution (c mg/ml)

REFERENCES

  1. F.Patti and P.B.-Maury; Bull.Soc.Chem.Biol.,35, 1177 (1953)

Table 1. Effect of Various Chemicals on D-Lactate dehydrogenase
[The enzyme dissolved in 10mM K-phosphate buffer, pH 7.0 (200U/ml) was incubated with each chemical at 25°C for 1hr.]
Chemical Conc.(mM) Residual
activity(%)		 Chemical Concn.(mM) Residual
activity(%)
None − 100 NaF 2.0 89.3
Metal salt 2.0   NaN₃ 2.0 3.8
AgNO₃   76.6 EDTA 5.0 96.6
BaCl₂   99.6 IAA 2.0 99.4
CaCl₂ 62.4 Borate 20 98.8
CoCl₂   101.2 SDS 0.05% 96.1
CuSO₄   99.7 Triton X-100 0.10% 95.7
FeSO₄   98.2 Brij 35
0.10% 97.8
MgSO₄   99.2 Span 20 0.10% 94.5
MnCl₂   28.7 Na-cholate 0.10% 95.3
NiCl₂   53.6      
ZnCl₂   96.1      

EDTA, ethylenediaminetetraacetate; IAA, iodoacetamide; SDS, sodium dodecyl sulfate;
DAC, Dimethylbenzylalkylammonium chloride

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