Enzymatic Assay of 5’-Nucleotidase

1. Objective

To standardize a procedure for determining the enzymatic activity of 5’-Nucleotidase.

2. Scope

This procedure applies to all products that have a specification for 5’-nucleotidase. This procedure is NOT to be used to assay 5^’-nucleotidase from bovine liver (N2779), or 5^’-nucleotidase from Crotalus adamanteus, insoluble (N3264) or 5’-nucleotidase as an impurity.

3. Definitions

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

3.2 Unit Definition = One unit will hydrolyze 1.0 μmol of inorganic phosphorus from adenosine 5’-monophosphate per minute at pH 9.0 at 37 ºC.

3.3 STD = Phosphorus Standard

3.4 5’-AMP = Adenosine 5’-Monophosphate

3.5 P_i = Inorganic Phosphate

3.6 TSCR = Taussky-Shorr Color Reagent

4. Discussion

5^’-AMP + H₂O ^{5'- Nucleotidase} > Adenosine + P_i

5. Responsibilities

Analytical Services laboratory personnel should follow this procedure 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, A_660nm, Light path = 1 cm

7.2 METHOD:
Spectrophotometric Stop Reaction

7.3 REAGENTS:

7.3.1 200 mM Glyicine Buffer, pH 9.0 at 37 ºC (Buffer) Prepare in purified water at 15 mg/mL using Glycine (G7126). Adjust to pH 9.0 at 37 ºC with 1 M NaOH.

7.3.2 66 mM Adenosine 5’-Monophosphate Solution (5'-AMP)

7.3.2.1 Prepare in purified water at 22.9 mg/mL using Adenosine 5’-Monphosphate, Sodium Salt, from Yeast (A1752).

7.3.2.2 Correct the Adenosine 5’-Monophosphate concentration for percent water, percent sodium, and percent purity by high pressure liquid chromatography.

7.3.3 200 mM Magnesium Sulfate Solution (MgSO₄) Prepare in purified water at 49.3 mg/mL using magnesium sulfate, heptahydrate (M1880).

7.3.4 Phosphorus Standard (STD) Use Phosphorus Standard Solution, 0.645 μ moles/ mL (P3869).

7.3.5 10 N Sulfuric Acid Solution (H₂SO₄) Prepare in cold purified water at 0.278 mL/mL using sulfuric acid, 95 –98%, A.C.S. Reagent (258105).

7.3.6 10% (w/v) Ammonium Molybdate Solution [(NH₄)₆MO₇O₂₄]

7.3.6.1 Prepare in Reagent 7.3.5 (H₂SO₄) at 100 mg/mL using ammonium molybdate, tetrahydrate (A7302). This may require more than 8 hours of stirring for dissolution.

7.3.6.2 If Sigma-Aldrich Product No. A7302 is not available, Ammonium Molybdate, Tetrahydrate (M0878) may be used as a replacement.

7.3.6.3 The 10%(w/v) Ammonium Molybdate Solution is stable in the dark for 6 months at room temperature.

7.3.7 Taussky-Shorr Color Reagent (TSCR)

7.3.7.1 Prepare 100 mL by adding 10mLs of Reagent 7.3.6 [(NH₄)₆MO₇O₂₄] to 70 mL of purified water.

7.3.7.2 Then add 5 g of Ferrous Sulfate, Heptahydrate (F7002).

7.3.7.3 Mix until dissolved and dilute to a final volume of 100 mL with purified water.

7.3.8 5’-Nucleotidase Solution (Enzyme)

7.3.8.1 Immediately before use, prepare a solution containing approximately 100 units/mL in cold purified water and swirl until dissolution.

7.3.8.2 Immediately dilute Reagent 7.3.8.1 to 40 – 60 units/mL in cold purified water. Make a fresh dilution for each assay run.

7.4 PROCEDURE

7.4.1 Assay-1

7.4.1.1 Pipette (in milliliters) the following reagents into suitable containers:

	Test-1	Test-2	Blank-1
Reagent 7.3.1 (Buffer)	1.50	1.50	1.50
Purified water	0.24	0.24	0.24
Reagent 7.3.3 (MgSO4)	0.10	0.10	0.10
Reagent 7.3.2 (5’-AMP)	0.15	0.15	0.15

7.4.1.2 Mix by swirling and equilibrate to 37 ºC. Then add:

Reagent 7.3.8 (Enzyme)

0.005

0.005

-----

7.4.1.3 Mix by swirling and incubate Test-1 for exactly 1.0 minute at 37 ºC and incubate Test-2 for exactly 2.0 minutes. Then add:

Reagent 7.3.7 (TSCR)	4.00	4.00	4.00
Reagent 7.3.8 (Enzyme)	-----	-----	0.005
Purified Water	2.00	2.00	2.00

7.4.1.4 Mix by inversion and incubate for five minutes at room temperature. Transfer to suitable cuvettes and measure the A660 nm for Test-1, Test-2, and Blank-1 versus purified water using a suitable spectrophotometer.

7.4.1.5 If results appear inconsistent, then repeat Steps 7.4.1.1 to 7.4.1.4.

7.4.2 Assay-2

7.4.2.1 Pipette (in milliliters) the following reagents into suitable containers:

	Test-3	Test-4	Blank-2
Reagent 7.3.1 (Buffer)	1.50	1.50	1.50
Purified water	0.24	0.24	0.24
Reagent 7.3.3 (MgSO4)	0.10	0.10	0.10
Reagent 7.3.2 (5’-AMP)	0.15	0.15	0.15

7.4.2.2 Mix by swirling and equilibrate to 37 ºC. Then add:

Reagent 7.3.8 (Enzyme)

0.010

0.010

-----

7.4.2.3 Mix by swirling and incubate Test-1 for exactly 1.0 minute at 37 ºC and incubate Test-2 for exactly 2.0 minutes. Then add:

Reagent 7.3.7 (TSCR)	4.00	4.00	4.00
Reagent 7.3.8 (Enzyme)	-----	-----	0.010
Purified Water	2.00	2.00	2.00

7.4.2.4 Mix by inversion and incubate for five minutes at room temperature. Transfer to suitable cuvettes and measure at 660 nm for Test-3, Test-4, and Blank-2 versus purified water using a suitable spectrophotometer.

7.4.2.5 If results appear inconsistent, then repeat Steps 7.4.2.1 to 7.4.2.4.

7.5 STANDARD CURVE

7.5.1 A standard curve is made by pipetting (in milliliters) the following reagents into suitable containers with vented caps:

	Std 1	Std 2	Std 3	Std 4	Std 5	Std 6	Std 7	Std Blank
Reagent 7.3.4 (STD)	0.05	0.20	0.40	0.60	0.80	1.00	2.00	-----
Purified Water	3.95	3.80	3.60	3.40	3.20	3.00	2.00	4.00
Reagent 7.3.7 (TSCR)	4.00	4.00	4.00	4.00	4.00	4.00	4.00	4.00

7.5.2 Mix by inversion and incubate for five minutes at room temperature. Transfer to suitable cuvette and measure at A660 nm for standards and standard-blank versus purified water using a suitable spectrophotometer.

7.6 CALCULATIONS

7.6.1 Standard Curve

ΔA_660nm Standard = A_660nm Std - A_660nm Std Blank

Plot the ΔA_660nm of the Standards vs μmoles of Phosphorus. Calculate and record the slope, y-intercept, and linear regression(r-square).

7.6.2 Sample Determination

ΔA_660nm Sample = A_660nmTest-1 or Test-2 - A_660nmTest Blank-1

ΔA_660nm Sample = A_660nm Test-3 or Test-4 - A_660nmTest Blank-2

Determine the micromoles of phosphorus liberated using the Standard curve.

	Units/mL enzyme =	(µmol of phosphorus released)(df)
		(0.5)(10)(2.5)

df = Dilution Factor
0.005= Volume (in milliliter) of enzyme used or
0.010= Volume (in milliliter) of enzyme used
T= Time (in minutes) of assay per unit definition

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

Units/mg protein =	Units/mL enzyme
	mg protein/mL enzyme

7.7 FINAL ASSAY CONCENTRATION :
In a 2 mL reaction mix, the final concentrations are 150 mM glycine, 10 mM magnesium sulfate, 5.0 mM adenosine 5’-monophosphate, and 0.2 – 0.6 units of 5’-nucleotidase.

Reference

1. Heppel LA, Hilmoe R. 1951. PURIFICATION AND PROPERTIES OF 5-NUCLEOTIDASE. Journal of Biological Chemistry. 188(2):665-676. https://doi.org/10.1016/s0021-9258(19)77739-4