Phosphatase, Acid Assay

Method: Based on the work of Brandenberger and Hanson (1953) and Hofstee (1954). The initial rate of hydrolysis of o-carboxyphenyl phosphate is determined by following the increase in absorbance at 300 nm resulting from the release of salicylic acid. One unit hydrolyzes one micromole of o-carboxyphenyl phosphate per minute at 25°C, pH 5.0 under the specified conditions. (An equivalent specific activity is obtained under above conditions using p-nitrophenyl-phosphate substrate).

Reagents 

• 0.15 M Sodium acetate buffer, pH 5.0
• 3.65 mM o-Carboxyphenyl-phosphate

Enzyme

Dissolve at a concentration of 1.0 mg/ml in reagent grade water.

Procedure

Adjust the spectrophotometer to 300 nm and 25°C.

Pipette into each cuvette as follows:

0.15 M Acetate buffer, pH 5.0	2.0 ml
3.65 mM o-Carboxyphenyl-phosphate	0.5 ml

Incubate in spectrophotometer for 3-4 minutes to reach temperature equilibration and establish blank rate, if any. Add 0.5 ml enzyme and record increase in A₃₀₀ for 4-5 minutes. Calculate ΔA₃₀₀/minute from the initial linear portion of the curve.

Calculation