Enzymatic Assay of Acid Phosphatase (EC 3.1.3.2)

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 + P_i

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

7.1 CONDITIONS:
T=37 °C, pH=4.8, A_410nm, Light path=1 cm

7.2 METHOD:
Spectrophotometric Stop Rate Determination

7.3 REAGENTS:

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 TEST METHOD

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 37 oC. Then add:

Reagent 7.3.4 (Enzyme)

0.10

----

7.4.3 Immediately mix by inversion and incubate at 37 oC 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 absorbance at 410 nm for both the Test and the Blank.

7.5 CALCULATIONS

7.5.1	Units/mL enzyme   =	ΔA410nm Test - ΔA410nm Blank)*(5.1)*(df)
		(10)*(18.3)*(0.1)

 

where:
     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

 

7.6    FINAL ASSAY CONCENTRATION
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.

Reference

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