Polyphenol Oxidase Assay

Method: Polyphenol oxidase oxidizes tyrosine to dihydroxyphenylalanine which in turn is oxidized to o-quinone. The latter is accompanied by an increase in absorbance at 280 nm. The rate of increase is proportional to enzyme concentration and linear during a period of 5-10 minutes after an initial lag. One unit causes a change in absorbance at 280 nm of 0.001 per minute at 25°C, pH 6.5 under the specified conditions.

Reagents

• 0.5 M Potassium phosphate buffer, pH 6.5
• 0.001 M L-Tyrosine

Enzyme

Dissolve at a concentration of 1 mg/ml in reagent grade water. Dilute further in reagent grade water to a concentration of 200-400 units/ml.

Procedure

Adjust the spectrophotometer to 280 nm and 25°C. Pipette into each cuvette as follows:

0.5 M Phosphate buffer, pH 6.5	1.0 ml
0.001 M L-Tyrosine	1.0 ml
Reagent grade water	0.9 ml

Oxygenate this reaction mixture by bubbling oxygen into cuvettes through a capillary tube for 4-5 minutes. Transfer cuvettes to the spectrophotometer and record A₂₈₀ for 4-5 minutes to achieve temperature equilibration and to establish blank rate, if any. Add 0.1 ml of appropriately diluted enzyme and record A₂₈₀ for 10-12 minutes. Determine ΔA₂₈₀ from the linear portion of the curve. A non-linear "lag" of 2-3 minutes can be expected.

Calculation