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 Pi = Inorganic Phosphate

3.6 TSCR = Taussky-Shorr Color Reagent

4. Discussion

5-AMP + H2O 5'- Nucleotidase > Adenosine + Pi

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, A660nm, 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:

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

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:

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:

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

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:

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:

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

ΔA660nm Standard = A660nm Std - A660nm Std Blank

Plot the ΔA660nm of the Standards vs μmoles of Phosphorus. Calculate and record the slope, y-intercept, and linear regression(r-square).

7.6.2 Sample Determination

ΔA660nm Sample = A660nmTest-1 or Test-2 - A660nmTest Blank-1

ΔA660nm Sample = A660nm Test-3 or Test-4 - A660nmTest Blank-2

Determine the micromoles of phosphorus liberated using the Standard curve.

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

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.

Materials
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Reference

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

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