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 = 18 MΩ•cm @ 25 ºC
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 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
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 37 ºC (Buffer) Prepare in purified water at 15.0 mg/mL using Glycine, Sigma-Aldrich Product Number 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, 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:
7.4.1.2 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/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:
7.4.2.2 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/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:
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.
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 2.00 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. 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.
References
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