To standardize a procedure for the enzymatic determination of glucose-6-phosphatase activity
This procedure applies to the enzymatic assay for glucose-6-phosphatase from rabbit liver.
Purified Water - Water from a deionizing system, resistivity > or = 18MΩ.cm @ 25 °C.
G 6-P - Glucose 6-Phosphate
G-6-Pase - Glucose-6-Phosphatase
Pi - Inorganic Phosphate
PPi - Inorganic Pyrophosphate
4.1. Enzymatic reaction:
G6 - P + H2O G-6-Pase > Glucose + Pi
Liberated inorganic phosphate is quantified by the method of Taussky-Shorr.
4.2. Glucose-6-Phosphatase also catalyzes the following reactions:
4.2.1. (PPi or Nucleoside di - or tripos phate) + Glucose G-6-Pase > G6 - P + (Pi or Nucleoside momo or diphosphate)
4.2.2. PPi + H2O G-6-Pase > 2Pi
Analytical services laboratory personnel should follow this procedure as written.
Refer to Safety Data Sheets (SDS) for hazards and appropriate handling precautions.
7.1. Conditions: T = 37 °C, pH = 6.5, A660 nm, Light path = 1 cm
7.2. Method: Spectrophotometric Stop Rate Determination
7.3.1. 100 mM Bis-Tris Buffer, pH 6.5 at 37 °C (Buffer)
Prepare 200 mL in purified water using Bis-Tris, Product No. B9754 . Adjust the pH to 6.5 at 37 °C.
7.3.2. 200 mM Glucose 6-phosphate (Sub)
Prepare 10 mL in purified water using D-glucose 6-phosphate sodium salt, Product No. G7879.
7.3.3. 20% Trichloroacetic Acid (TCA)
Prepare 10 mL in purified water using trichloroacetic acid solution, 6.1 N, Product No. T0699.
7.3.4. Phosphorus Standard Solution, 20 μg/mL (Std)
Use neat, Product No. P3869.
7.3.5. 5 M Sulfuric Acid Solution
Prepare 50 mL in purified water using Sulfuric Acid, Product No. 258105.
7.3.6. 10% Ammonium Molybdate Solution
Prepare 10 mL in Reagent 7.3.5 using Ammonium Molybdate Tetrahydrate, Product No. A7302.
7.3.7. Taussky-Shorr Color Reagent (TSCR)
Add 10 mL of Reagent 7.3.6 to 70 mL of purified water with stirring. Then add 5 g of ferrous sulfate heptahydrate, Product No. F7002. Stir this solution until completely dissolved and adjust to a final volume of 100 mL with purified water.
7.3.8. Glucose-6-Phosphatase Enzyme Solution (Enz)
Immediately before use, prepare a solution containing 1.0 – 2.0 units/mL in cold purified water.
7.4. Enzymatic Assay:
7.4.1. Pipette the following (in milliliters) into suitable containers:
7.4.2. Mix by swirling and equilibrate at 37 °C for a minimum of 5 minutes, then add:
7.4.3. Immediately mix by swirling and incubate at 37 °C for exactly 5 minutes, then add:
7.4.4. Tightly cap and mix by inversion. Incubate for 5 minutes at 25 °C and centrifuge all sample and blank reaction mixtures at 4,000 rpm for 10 minutes. Use the supernatant in the color development step.
7.5. Color Development:
7.5.1. Prepare a standard curve by pipetting (in milliliters) the following into suitable containers:
7.5.2. Mix all samples, blanks and standards by swirling and then add:
7.5.3. Incubate samples, blanks and standards at 25 °C for 5-6 minutes. Transfer all solutions to appropriate cuvettes and record the A660nm for each. It is important to let all color development reaction mixtures incubate for the same period of time as the color complex will continue to develop over time.
7.6.1. Calculate the ΔA660nm of the standards as follows:
ΔA660nm = (A660nm Std - A660nm Std Blank)
7.6.2. Plot ΔA660nm of the standards versus micromoles of phosphorus and obtain the slope (m) and y-intercept (b) of the linear regression. Use these in the calculations for the test reaction mixtures.
7.6.3. Calculate the ΔA660nm of each test solution as follows:
ΔA660nm = (A660nm Test - A660nm Test Blank)
7.6.4. Calculate the micromoles of Pi liberated as follows:
7.6.5. Calculate the units per mg of enzyme as follows:
5.0 = the final volume (in milliliters) of the enzymatic reaction
0.10 = volume (in milliliters) of the enzyme solution used
2.0 = volume (in milliliters) of enzyme assay used in color development
df = dilution factor of the enzyme solution