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, Sigma-Aldrich Product Number N2779, or 5-Nucleotidase from Crotalus adamanteus, Insoluble, Sigma-Aldrich Product Number N3264 or 5-Nucleotidase as an impurity.

3. Definitions

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

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

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

It is the responsibility of trained Analytical Services laboratory personnel to 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 37ºC (Buffer) Prepare in purified water at 15.0 mg/ml using Glycine, Sigma-Aldrich Product Number G7126. Adjust to pH 9.0 at 37ºC 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, Sigma-Aldrich Product Number 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, Sigma-Aldrich Product Number M1880.

7.3.4     Phosphorus Standard (STD) Use Phosphorus Standard Solution, 0.645 μ moles/ ml, Sigma-Aldrich Product Number P3869.

7.3.5    10N Sulfuric Acid Solution (H2SO4) Prepare in cold purified water at 0.278 ml/ml using SulfuricAcid, 95 –98%, A.C.S. Reagent, Sigma-Aldrich Product Number 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, Sigma-Aldrich Product Number A7302. This may require more than 8 hours of stirring for dissolution.

7.3.6.2    If Sigma-Aldrich Product Number A7302 is not available, Ammonium Molybdate,Tetrahydrate, Sigma-Aldrich Product Number M0878 can 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, Sigma-Aldrich Product Number 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    2 Mix by swirling and equilibrate to 37ºC. 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 37ºC 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/record the A660nm 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    2 Mix by swirling and equilibrate to 37ºC. 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 37ºC 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/record the A660nm 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/record the A660nm 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 µmoles of phosphorus liberated using the Standard curve.

  Units/ml enzyme = (µmoles 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 2.00 ml reaction mix, the final concentrations are 150mM Glycine, 10mM Magnesium Sulfate, 5.0mM Adenosine 5’-Monophosphate, and 0.2 – 0.6 units of 5’-Nucleotidase.

8. References & Attachments

Heppel, P. A. and Hilmoe, R. J. (1951) Journal of Biological Chemistry 188, 665-676.

9. Approval

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

Materials

     
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