Phosphatase, Alkaline Assay

Assay (Chicken Intestine Enzyme)

Method: The measurement of phosphatase activity is based upon the work of Brandenberger and Hanson (1953) and Hofstee (1954). The catalytic effect of the enzyme on the initial rate of hydrolysis of o-carboxyphenyl phosphate is determined. Salicylic acid, unlike the phosphate ester, absorbs light maximally at about 300 nm; hence the rate of hydrolysis can be determined by the increase in absorbance. One unit hydrolyzes one micromole of o-carboxyphenyl phosphate per minute at 25°C and pH 8.8 under the specified conditions.

Reagents

• 0.1 M Tris⋅HCl, pH 8.5
• 0.2 M Glycine, pH 8.8
• 0.05 M Magnesium chloride
• 3.65 mM o-Carboxyphenyl phosphate (OCPP)

Enzyme

Dissolve at one mg/ml in 0.1 M Tris-HCl, pH 8.5 (Stock solution). A further dilution may be required following activation.

Procedure

Activate the enzyme by incubating the mg/ml solution for 20-30 minutes in a 25°C water bath.

Adjust spectrophotometer to 300 nm and 25°C.

Pipette into each cuvette as follows:

0.2 M Glycine, pH 8.8	2.0 ml
3.65 mM OCPP	1.0 ml
0.05 M MgCl2	0.5 ml

Incubate in spectrophotometer at 25°C for 3-4 minutes to reach temperature equilibration and establish blank rate, if any. Add 0.1 ml of stock solution and record increase A₃₀₀/minute from initial linear portion of the curve.

Calculation

Assay (E. coli Enzyme)

Method: The assay is that of Garen and Levinthal (1960) in which the reaction velocity is determined by measuring an increase in absorbance at 410 nm resulting from the hydrolysis of p-nitrophenylphosphate to p-nitrophenol. One unit releases one micromole of p-nitrophenol per minute at 25°C, pH 8, under the specified conditions.

Reagents

• 1.5 M Tris⋅HCl buffer, pH 8.0
• 0.003 M p-nitrophenylphosphate (PNP). Care must be exercised to use an analytical grade and the correct molecular weight.

Enzyme

Dilute in reagent grade water to obtain a rate of 0.02-0.04 ΔA/minute.

Procedure

Adjust the spectrophotometer to 410 nm and 25°C.

Pipette into each cuvette as follows:

0.003 M PNP	1.0 ml
1.5 M Tris⋅HCl, pH 8.0	2.0 ml

Mix well and incubate in the spectrophotometer for 4-5 minutes to achieve temperature equilibration and to establish blank rate, if any. Add 0.1 ml of diluted enzyme and record. Determine A₄₁₀ for 3-5 minutes from linear portion of the curve.

Calculation

Assay (Calf Intestine Enzyme)

Method: One unit hydrolyzes 1 umole of p-nitrophenol phosphate per minute at 37°C, pH 9.8.

Reagents

• 1.0 M Diethanolamine with 0.05 mM MgCl₂ buffer, pH 9.8: Dilute 12.4 gm diethanolamine (85%) wtih reagent grade water. Add 0.05 ml MgCl₂ solution (see below) and adjust the pH to 9.8 (at 37°C) with HCl. Adjust to 100 ml with reagent grade water.
• MgCl₂ solution: Dissolve 20.3 gm MgCl2°6 H2O in 100 ml reagent grade water.
• 0.67 M p-Nitrophenyl phosphate solution: Dissolve 250 mg p-nitrophenyl phosphate, Na salt in 1.0 ml reagent grade water.
• Diluent: 0.1 M TEA⋅HCl. Dissolve 1.86 gm TEA⋅HCl in reagent grade water, add 0.1 ml MgCl₂ solution and 0.1 ml 0.1 M ZnCl₂, adjust the pH to 7.6 with NaOH and adjust to 100 ml with reagent grade water.

Enzyme

Use diluent to obtain approximately 0.05 - 0.06 u/ml. Let stand 15-20 minutes at room temperature.

Procedure

Adjust the spectrophotometer to 405 nm and 37°C.

Pipette into cuvettes:

	Test	Blank
Buffer	3.00 ml	3.00 ml
4-nitrophenyl phosphate	0.050 ml	0.050 ml
Mix and incubate to achieve temperature equilibration.
Diluent	------	0.050 ml
Sample	0.050 ml	------

Mix. Measure the change in absorbance and calculate ΔA/min based on the linear range of the curve.

Calculation