Enzymatic Assay of Acid Phosphatase (EC 3.1.3.2)

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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 @ 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

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

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 26.5mg/mL solution in purified water using Citric Acid, Trisodium, Dihydrate, Product Number C7254. Adjust to pH 4.8 at 37 °C 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, our Stock Number 104-0, also known as Product Number S6750.

7.3.3 100mM Sodium Hydroxide Solution (NaOH)
Prepare 200 mL by diluting 20 mL of 1.0 N Sodium Hydroxide, 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 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 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 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..