Enzymatic Activity of Glucose-6-Phosphatase [EC 3.1.3.9] | Sigma-Alrich.com

Document History

Replaces enzyme assay procedure SSGLUC02. Document number and formatting changed to reflect current numbering and formatting guidelines. The procedure was modified to include a standard curve that replaces a single-point calibration standard. The buffer has also been changed from 0.1M Cacodylate, pH 6.5 @37°C to eliminate the use of arsenic. Refer to CR SOP DEK-ENZ40.

1. Objective

To standardize a procedure for the enzymatic determination of Glucose-6-phosphatase activity at Sigma-Aldrich St. Louis.

2. Scope

This procedure applies to the enzymatic assay for Sigma-Aldrich Product Number G5758, Glucose-6-Phosphatase from Rabbit Liver, Crude.

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
Pi - Inorganic Phosphate
PPi - Inorganic Pyrophosphate

4. Discussion

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

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 = 6.5, A660nm, 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, Sigma-Aldrich Product Number 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, Sigma-Aldrich Product Number G7879 .

7.3.3.    20% Trichloroacetic Acid (TCA)
Prepare 10 ml in purified water using Trichloroacetic Acid Solution, 6.1 N, Sigma-Aldrich Product Number T0699 .

7.3.4.    Phosphorus Standard Solution, 20 μg/ml (Std)
Use neat, Sigma-Aldrich Product Number P3869 .

7.3.5.    5M Sulfuric Acid Solution
Prepare 50 ml in purified water using Sulfuric Acid, Aldrich Product Number 258105 .

7.3.6.    10% Ammonium Molybdate Solution
Prepare 10 ml in Reagent 7.3.5 using Ammonium Molybdate Tetrahydrate, Sigma-Aldrich Product Number A7302.

7.3.7.    Taussky-Shorr Color Reagent (TSCR)
Add 10mls of Reagent 7.3.6 to 70 ml of purified water with stirring. Then add 5 g of Ferrous Sulfate Heptahydrate, Sigma-Aldrich Product Number Sigma-Aldrich Product Number F7002. Stir this solution until completely dissolved and bring to a final volume of 100ml using 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.    Let all samples, blanks and standards incubate 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.    Calculations:

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 μmoles 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 μmoles of Pi liberated as follows:

μmoles Pi =

(ΔA660nm Test - b)

m

 

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

Units/mg S=

(μmoles 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

8. References & Attachments

8.1. Nordlie, R.C. and Arion, W.J. (1966) Methods in Enzymology 9, 619-625

8.2. Taussky, H.H. and Shorr, E. (1953) J. Biol. Chem. 202, 675-685.

9. Approval

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

Materials

     
Related Links