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Enzymatic Assay of Glucose-6-Phosphate Dehydrogenase (EC 1.1.1.49)

1. Objective

To standardize a procedure for the enzymatic determination of Glucose-6-Phosphate Dehydrogenase.

2. Scope

This procedure applies to most products that have a specification for Glucose-6-Phosphate Dehydrogenase by enzymatic determination. This assay is not to be used for Glucose-6-Phosphate Dehydrogenase from Leuconostoc Mesenteroides, Sigma-Aldrich Product Numbers G5760, G5885, G8404, and G8529.

3. Definitions

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

3.2. Unit Definition - One unit will oxidize 1.0 μmole of D-glucose-6-phosphate to 6-phospho-D-gluconate per minute in the presence of β-NADP at pH 7.4 at 25°C.

3.3. G-6-PDH - Glucose-6-Phosphate Dehydrogenase

3.4. β-NADP - β-Nicotinamide Adenine Dinucleotide Phosphate, Oxidized Form

3.5. β-NADPH - β-Nicotinamide Adenine Dinucleotide Phosphate, Reduced Form

4. Discussion

D-Glucose-6-Phosphate + β-NADP    G-6-PDH   > 6-Phospho-D-gluconate + β-NADPH
  Mg2+  

5. Responsibilities

It is the responsibility of all Analytical Services laboratory trained personnel to follow this protocol as written.

6. Safety

Refer to the Safety Data Sheet (SDS) for hazards and appropriate handling precautions.

7. Procedure

7.1. CONDITIONS
T = 25°C, pH = 7.4, A340nm, Light path = 1 cm

7.2. METHOD
Spectrophotometric Rate Determination

7.3. REAGENTS

7.3.1.   250 mM Glycylglycine (Buffer)
Prepare a 33 mg/ml solution in purified water using Glycylglycine, Free Base, Sigma-Aldrich Product Number G1002. Adjust to pH 7.4 at 25°C with 1 M NaOH or 1 M HCl.

7.3.2.   60 mM D-Glucose 6-Phosphate Solution (G-6-P)
Prepare a 17 mg/ml solution in purified water using D-Glucose-6 Phosphate, Monosodium Salt, Sigma-Aldrich Product Number G7879.

7.3.3.   20 mM β-Nicotinamide Adenine Dinucleotide Phosphate Solution (β-NADP)
Prepare a 15.4 mg/ml solution in purified water using β-Nicotinamide Adenine Dinucleotide Phosphate, Sodium Salt, Sigma-Aldrich Product Number N0505.

7.3.4.   300 mM Magnesium Chloride Solution (MgCl2)
Prepare a 0.3 ml/ml solution in purified water using 1.0 M Magnesium Chloride Solution, Sigma-Aldrich Product Number M1028.

7.3.5.   Glucose-6-Phosphate Dehydrogenase Enzyme Solution (Enzyme)
Immediately before use, prepare at solution containing 0.3-0.6 units/ml in cold buffer (Reagent 7.3.1).

7.4. PROCEDURE

7.4.1.   Prepare a reaction cocktail by pipetting (in milliliters) the following reagents into a suitable container:

Purified Water 21.0
Buffer (Reagent 7.3.1) 5.0
G-6-P (Reagent 7.3.2) 1.0
β-NADP (Reagent 7.3.3) 1.0
MgCl2 (Reagent 7.3.4) 1.0

7.4.2.   Mix and equilibrate to 25°C. Adjust the pH of the reaction cocktail to 7.4 with 1 M NaOH or 1 M HCl.

7.4.3.   Pipette (in milliliters) the following reagents into suitable cuvettes:

  Test Blank
Reaction Cocktail (Reagent 7.4.1) 2.90 2.90

7.4.4.   Equilibrate to 25°C. Monitor the A340nm until constant, using a suitably thermostatted spectrophotometer. Then add:

Buffer (Reagent 7.3.1) ------ 0.10
Enzyme (Reagent 7.3.5) 0.10 ------

7.4.5.   Immediately mix by inversion and record the increase in A340nm for approximately 10 minutes. Obtain the ΔA340nm/minute using the maximum linear rate for both the Test and Blank using a minimum of 4 data points over a one minute time interval.

7.5. CALCULATIONS

7.5.1. Units/mL enzyme = (A340nm/min Test - A340nm/min Blank)(3)(df)
    (6.22)(0.1)


Where:
3 = Total volume (in milliliters) of assay
df = Dilution factor
6.22 = Millimolar extinction coefficient of β-NADPH at 340 nm
0.1 = Volume (in milliliters) of enzyme used

7.5.2. Units/mg solid = Units/mL enzyme
    mg solid/mL enzyme



7.5.3. Units/mg protein = Units/mL enzyme
    mg protein/mL enzyme

7.6. FINAL ASSAY CONCENTRATION
In a 3.00 ml reaction mix, the final concentrations are 50 mM glycylglycine, 2 mM D-glucose-6-phosphate, 0.67 mM β-nicotinamide adenine dinucleotide phosphate,10 mM magnesium chloride, and 0.03 - 0.06 units glucose 6 phosphate dehydrogenase.

8. References & Attachments

Noltmann, E.A., Gubler, C.J., and Kuby, S.A. (1961) Journal of Biological Chemistry 236, 1225- 1230.

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

     
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