Phosphatase, Acid Assay

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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

\frac{\text{Units}}{\text{mg}}\text{ = } \frac{\frac{\Delta \text{A}_{300}}{\text{min}}\text{ x 1000}}{\text{3500 x }\frac{\text{mg enzyme}}{\text{ml reaction mixture}}}\text{where 3500 is the molar extinction coefficient of salicylic acid at 300 nm}

Phosphatase, Acid Products

Description

Phosphatase, Acid

Source:

Wheat Germ (Triticum vulgare)

A non-specific esterase partially purified to the 0.35-0.55 fraction by the method described by Singer, JBC, 174, 11 (1948). Also active as a lipase. A lyophilized powder.

Store at -20°C.

…

Activity

≥0.15 units per mg dry weight

Certificate of Analysis

Code

Product details

LS001141

1 gm

$120.00

LS001144

Bulk