Enzymatic Activity of Glucose-6-Phosphatase [EC 3.1.3.9]

1. Objective

To standardize a procedure for the enzymatic determination of glucose-6-phosphatase activity

2. Scope

This procedure applies to the enzymatic assay for glucose-6-phosphatase from rabbit liver.

3. Definitions

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
P_i - Inorganic Phosphate
PP_i - Inorganic Pyrophosphate

4. Discussion

4.1.    Enzymatic reaction:
G6 - P + H₂O    ^G-6-Pase   > Glucose + P_i
Liberated inorganic phosphate is quantified by the method of Taussky-Shorr.

4.2.    Glucose-6-Phosphatase also catalyzes the following reactions:

4.2.1.    (PP_i or Nucleoside di - or tripos phate) + Glucose    ^G-6-Pase   > G6 - P + (P_i or Nucleoside momo or diphosphate)

4.2.2.    PP_i + H₂O    ^G-6-Pase   > 2P_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 = 6.5, A660 nm, Light path = 1 cm

7.2.    Method: Spectrophotometric Stop Rate Determination

7.3.    Reagents:

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:

	Test	Blank
Buffer (7.3.1.)	3.00	3.00
Substrate (7.3.2.)	1.00	1.00

7.4.2.    Mix by swirling and equilibrate at 37 °C for a minimum of 5 minutes, then add:

Enzyme (7.3.8.)

0.10

-----

7.4.3.    Immediately mix by swirling and incubate at 37 °C for exactly 5 minutes, then add:

TCA (7.3.3.)	0.90	0.90
Enzyme (7.3.8.)	-----	0.10

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:

		Test						Std
	Test	Blank	Std 1	Std 2	Std 3	Std 4	Std 5	Blank
Purified Water	-----	-----	1.80	1.60	1.40	1.20	1.00	2.00
Standard (7.3.4.)	-----	-----	0.20	0.40	0.60	0.80	1.00	-----
Test (7.4.4.)	2.00	-----	-----	-----	-----	-----	-----	-----
Blank (7.4.4.)	-----	2.00	-----	-----	-----	-----	-----	-----

7.5.2.    Mix all samples, blanks and standards by swirling and then add:

TSCR (7.3.7.)

2.00

2.00

2.00

2.00

2.00

2.00

2.00

2.00

7.5.3.    Incubate samples, blanks and standards at 25 °C for 5-6 minutes. Transfer all solutions to appropriate cuvettes and record the A_660nm 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.    Calculations:

7.6.1.    Calculate the ΔA_660nm of the standards as follows:
ΔA_660nm = (A_660nm Std - A_660nm Std Blank)

7.6.2.    Plot ΔA_660nm 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 ΔA_660nm of each test solution as follows: 
ΔA_660nm = (A_660nm Test - A_660nm Test Blank)

7.6.4.    Calculate the micromoles of P_i liberated as follows:

μmol Pi =	(ΔA660nm Test - b)
	m

7.6.5.     Calculate the units per mg of enzyme as follows:

Units/mg S=	(μmol Pi * 5.0 * df)
	(T * 0.1 * 2.0)

Where:
    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