1. Objective

To standardize a procedure for the enzymatic assay of Acid Phosphatase.

2. Scope

This procedure applies to all products that have a specification for Acid Phosphatase.

3. Definitions

3.1 Purified Water – water from a deionizing system, resistivity > or = 18MΩ•cm @ 25 ºC

3.2. Unit Definition – One unit will hydrolyze 1.0 μmole of p-nitrophenyl phosphate per minute at pH 4.8 at 37 °C.

4. Discussion

p-Nitrophenyl Phosphate + H2O Acid Phosphatase > p-Nitrophenol + Pi

5. Responsibilities

Analytical services personnel should follow this protocol as written.

6. Safety

Refer to the Safety Data Sheet (SDS) for hazards and appropriate handling precautions.

7. Procedure

T=37 °C, pH=4.8, A410nm, Light path=1 cm

Spectrophotometric Stop Rate Determination


7.3.1 90 mM Citrate Buffer, pH 4.8 at 37 °C (Buffer)
Prepare a 26.5 mg/mL solution in purified water using Citric Acid, Trisodium, Dihydrate, Product No C7254. Adjust to pH 4.8 at 37 °C with 1 M NaOH/HCL.

7.3.2 15.2 mM P-Nitrophenyl Phosphate (PNPP)
Prepare a 5.64 mg /mL solution in purified water using p-Nitrophenyl Phosphate.

7.3.3 100 mM Sodium Hydroxide Solution (NaOH)
Prepare 200 mL by diluting 20 mL of 1.0 N Sodium Hydroxide, Product No. S2567 in purified water, using a 200 mL volumetric flask.

7.3.4 Acid Phosphatase Enzyme Solution (Enzyme)
Immediately before use, prepare a solution containing 0.15-0.25 un/mL of Acid Phosphatase in cold purified water.


7.4.1 Pipette (in milliliters) the following reagents into suitable containers:
7.4.2 Mix by inversion and equilibrate to 37 oC. Then add:
7.4.3 Immediately mix by inversion and incubate at 37 oC for exactly 10 minutes. Then add:

7.4.4 Mix by inversion and using a suitable spectrophotometer record the absorbance at 410 nm for both the Test and the Blank.


7.5.1 Units/mL enzyme = ΔA410nm Test - ΔA410nm Blank)*(5.1)*(df)

5.1 = Total volume (in milliliters) of solution
df = Dilution factor
10 = Time of assay (in milliliters) as per the Unit Definition
18.3 = Millimolar extinction coefficient of p-Nitrophenol
0.1 = Volume (in milliliter) of enzyme used

7.5.2 Units/mg solid = Units/mL enzyme
mg solid/mL enzyme

7.5.3 Units/mg solid = Units/mL enzyme
mg solid/mL enzyme

In a 1.10 mL reaction mix, the final concentrations are 41 mM citric acid, 6.9 mM p-nitrophenyl phosphate, and 0.015-0.025 units of acid phosphatase.



Bergmeyer H, Gawehn K, Grassl M. 1974. Methods of Enzymatic Analysis. Volume I, 2nd ed.. New York, NY: Academic Press, Inc..

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