Methodology for Enzymes

(Hydrogen peroxide: Hydrogen peroxide oxidoreductase EC

Catalase has a double function as it catalyses the following reactions:
1.  Decomposition of hydrogen peroxide to give water and oxygen
2H2O + O2

2.  Oxidation of H donors for example methanol, formic acid, phenol with the consumption of one mole of peroxide
H2O + ROH + A

The UV light absorption of hydrogen peroxide solution can be easily measured between 230 and 250nm. On decomposition of hydrogen peroxide by catalase, the absorption decreases with time. The enzyme activity could be arrived at from this decrease. But this method is applicable only with enzyme solutions which do not absorb strongly at 230-250nm.

» Phosphate Buffer, 0.067M (pH 7.0). Dissolve 3.522g KH2PO4 and 7.268g Na2HPO4.2 H2O in distilled water and make up the volume to one liter.
» Hydrogen Peroxide-Phosphate Buffer. Dilute 0.16mL of H2O2 (10% w/v) to 100mL with phosphate buffer. Prepare fresh. The absorbance of the solution should be about 0.5 at 240nm with a lcm light path.
» Enzyme Extract
   Homogenize plant tissue in a blender with M/150 phosphate buffer (assay buffer diluted 10 times) at 1 — 4°C and centrifuge. Stir the sediment with cold phosphate buffer, allow to stand in the cold with occasional shaking and then repeat the extraction once or twice. The extraction should not take longer than 24h. Use the combined supernatants (sometimes opales­cent) for the assay. The catalase activity can change considerably on storage of the tissue. In comparative studies, therefore, always use the same conditions of extraction, storage and temperature.

» Wavelength: 240nm
» Final Vol : 3mL approximately
» Room Temperature
» Read against a control cuvette containing enzyme solution as in the ex­perimental cuvette, but containing H2O2-free PO4 buffer (M/15)
» Pipette into the experimental cuvette
» 3mL H2O2-PO4 buffer. Mix in 0.01-0.04mL sample with a glass or plastic rod flattened at one end. Note the time At required for a decrease in absorbance from 0.45 to 0.4. This value is used for calculations. If t is greater than 60sec repeat the measurements with a more concentrated solu­tion of the sample.

One g tissue is homogenized in a total volume of 20mL, diluted 1 to 10 volume with water and taken 0.01mL for assay. The absorbance at 240nm decreased from 0.45 to 0.4 in 13.9 sec.
= 1.22 units in the assay mixture (or)

1.22 x 10
= 1220 units/mL extract i.e., 2.44 X 104 units/g tissue

Calculate the concentration of H2O2 using the extinction coefficient 0.036 per mmole per mL.

1.  Luck, H, (1974) In: Methods in Enzymatic Analysis 2 (Ed Bergmeyer) Academic Press New York p 885.