Enzymatic Assay of Acid Phosphatase (EC

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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 @ 25ºC

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

It is the responsibility of all trained Analytical Services personnel to 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 26.5mg/ml solution in purified water using Citric Acid, Trisodium, Dihydrate, Sigma-Aldrich Product Number C7254. Adjust to pH 4.8 at 37oC with 1M 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, Sigma-Aldrich Stock Number 104-0, also known as Sigma-Aldrich Product Number S6750.

7.3.3     100mM Sodium Hydroxide Solution (NaOH)
Prepare 200 ml by diluting 20 ml of 1.0 N Sodium Hydroxide, Sigma-Aldrich Product Number 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:

  Test Blank
Reagent 7.3.1 (Buffer) 0.50 0.50
Reagent 7.3.2 (PNPP) 0.50 0.50


7.4.2    Mix by inversion and equilibrate to 37oC. Then add:

Reagent 7.3.4 (Enzyme) 0.10 ----


7.4.3    Immediately mix by inversion and incubate at 37oC for exactly 10 minutes. Then add:

Reagent 7.3.3 (NaOH) 4.00 4.00
Reagent 7.3.4 (Enzyme) ----- 0.10


7.4.4    Mix by inversion and using a suitable spectrophotometer record the A410nm 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 protein = Units/ml enzyme
mg protein/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.

8. References & Attachments

Bergmeyer, H.U., Gawehn, K., and Grassl, M. (1974) in Methods of Enzymatic Analysis (Bergmeyer H.U.) Volume I, 2nd ed., 495-496, Academic Press, Inc., New York, NY

Replacing OP SSPNPP02.

9. Approval

Review, approvals and signatures for this document will be generated electronically using EDMS. Print a “For Use” copy if hardcopy with signature verification is required.


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