Peroxidase Assay

Method: Peroxidase activities have traditionally been expressed in units based upon the rate of oxidation of pyrogallol, a method introduced by Willstalter and Stoll in 1917, and which more recent studies have shown to be somewhat inadequate. (Maehly and Chance 1954.) A wide variety of hydrogen donors have been utilized in peroxidase assay systems including potentially carcinogenic compounds such as o-dianisidine. An improved assay has been adopted using 4-aminoantipyrine as hydrogen donor (Trinder 1966). The reaction rate is determined by measuring an increase in absorbance at 510 nm resulting from the decomposition of hydrogen peroxide. One unit results in the decomposition of one micromole of hydrogen peroxide per minute at 25°C and pH 7.0 under the specified conditions.

Reagents

• 0.2 M Potassium phosphate buffer pH 7.0
• 0.0017 M Hydrogen peroxide. Prepare by diluting 1 ml of 30% hydrogen peroxide (Merck Superoxol or equivalent) to 100 ml with reagent grade water. Further dilute 1 ml of this solution to 50 ml with 0.2 M potassium phosphate buffer pH 7.0. Prepare fresh daily.
• 0.0025 M 4-Aminoantipyrine with 0.17 M Phenol: Prepare by dissolving 810 mg phenol in 40 ml reagent grade water. Add 25 mg 4-aminoantipyrine and dilute to a final volume of 50 ml with reagent grade water.

Enzyme

Dissolve at one mg/ml in reagent grade water. Immediately prior to use, dilute further to obtain a rate of 0.02-0.04 ΔA/min.

Procedure

Adjust spectrophotometer to 510 nm and 25°C.

Pipette into each cuvette as follows:

Phenol/aminoantipyrine solution	1.4 ml
0.0017 M Hydrogen peroxide	1.5 ml

Incubate in spectrophotometer at 25°C for 3-4 minutes to achieve temperature equilibration and establish blank rate, if any. Add 0.1 ml of diluted enzyme and record the increase in A₅₁₀ for 4-5 minutes. Calculate ΔA₅₁₀/minute from linear portion of the curve.

Calculation

Note: Although the reaction rate obtained with the phenolantipyrine method is 4.5-4.7 times less than previous methods, the peroxidase preparations are the same.

The RZ (Reinheitzahl) which is the absorbance ratio, A₄₀₃/A₂₇₅, (RZ) has been used as an indication of purity. However, Shannon et al. (1966) report that this ratio for the isozymes varies from 2.50 to 4.19. This, together with the influence exerted by buffer and pH, would seem to render questionable the preciseness of this ratio as a criterion of purity.