Diethanolamine Assay for the Enzymatic Assay of Alkaline Phosphatase (EC 3.1.3.1)

1. Objective

To standardize a procedure for the enzymatic assay of alkaline phosphatase, diethanolamine assay.

2. Scope

2.1 This procedure applies to all products that have a specification for alkaline phosphatase utilizing the diethanolamine assay system.

2.2 This enzyme assay is not to be used to assay alkaline phosphatase in which the specific activity is cited only in glycine units.

3. Definitions

3.1 Purified Water - Water from a deionizing system, resistivity ~18MΩּcm @25 °C

3.2 Unit Definition - One DEA unit will hydrolyze 1.0 µmole of p-nitrophenyl phosphate per minute at pH 9.8 at 37 °C.

3.2 Pi – Inorganic Phosphate

4. Discussion

p-Nitrophenyl Phosphate + H₂O ^{Alkaline 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 Safety Data Sheets (SDS) for hazards and appropriate handling precautions.

7. Procedure

7.1 CONDITIONS:

T = 37 °C, pH = 9.8, A_750nm, Light path = 1 cm

7.2 METHOD:

Continuous Spectrophotometric Rate Determination

7.3 REAGENTS:

7.3.1 1.0 M Diethanolamine Buffer with 0.50 mM Magnesium Chloride, pH 9.8 at 37 °C (Buffer)
Prepare a 140 mg/mL solution in purified water using diethanolamine, such as product number D8885. Adjust the pH of the diethanolamine to 9.8 at 37 °C with 5 M HCl. Dilute pH adjusted solution to 105 mg/mL Diethanolamine final concentration with purified water and add 0.5 mL/L of 1M magnesium chloride solution, such as product number M1028.
Prepare fresh and protect from light.

7.3.2 0.67 M p-Nitrophenyl Phosphate Solution (PNPP)
Prepare a 247 mg/mL solution in purified water using Phosphatase Substrate, such as product number P4744. Prepare fresh and protect from light.

7.3.3 Alkaline Phosphatase Enzyme Solution (Enzyme)

7.3.3.1 Immediately before use, prepare a solution containing approximately 0.15 units/mL of alkaline phosphatase in cold Buffer.

7.3.3.2 For liquid samples, prepare a concentrated (2000 unit/mL or greater) stock solution in cold buffer immediately before use. Perform subsequent dilutions to achieve final solution containing approximately 0.15 unit/mL in cold Buffer.

7.4 TEST METHOD:

7.4.1 Pipette (in milliliters) the following reagents in the following sequence into the appropriate cuvette:

	Test 1	Test 2	Test 3	Blank
Buffer (Reagent 7.3.1)	2.91	2.905	2.90	2.95
PNPP (Reagent 7.3.2)	0.05	0.05	0.05	0.05

7.4.2 Mix by inversion and equilibrate to 37 ^oC. Monitor the A405nm until constant using a thermostatted spectrophotometer. Then add:

	Test 1	Test 2	Test 3	Blank
Enzyme (Reagent 7.3.3)	0.04	0.045	0.05	-----

7.4.3 Immediately mix by inversion and record the increase in A405nm for approximately 5 minutes. Obtain the ΔA405nm / minute using the maximum linear rate for both the test and blank using a minimum of four data points over a one minute time interval.

7.5 CALCULATIONS

7.5.1	Units/mL enzyme =	(ΔA405nm/min Test – ΔA405nm/min Blank) (VF)
		(18.5) (VE)

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

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

7.5.4 V_F = Volume (in milliliters) of assay
df = Dilution factor
18.5 = Millimolar extinction coefficient of p-Nitrophenol at 405 nm
V_E = Volume (in milliliters) of enzyme used

7.6 FINAL ASSAY CONCENTRATIONS
In a 3.00 mL reaction mix, the final concentrations are 983 mM diethanolamine, 0.49 mM magnesium chloride, 11.2 mM p-nitrophenyl phosphate and approximately 0.0075 unit alkaline phosphatase.

8. Approval

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