To standardize a procedure for the enzymatic determination of glucose-6-phosphate dehydrogenase.
This procedure applies to most products that have a specification for glucose-6-phosphate dehydrogenase by enzymatic determination. This assay is not for glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides.
3.1. Purified Water - water from a deionizing system, resistivity > or = 18MΩ•cm @ 25 ºC
3.2. Unit Definition - One unit will oxidize 1.0 μmol 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
Analytical services personnel should follow this protocol as written.
Refer to the Safety Data Sheet (SDS) for hazards and appropriate handling precautions.
T = 25 °C, pH = 7.4, absorbance at 340 nm, Light path = 1 cm
Spectrophotometric Rate Determination
7.3.1. 250 mM Glycylglycine (Buffer)
Prepare a 33 mg/mL solution in purified water using Glycylglycine, Free Base, Product No. 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 17 mg/mL in purified water using D-Glucose-6 Phosphate, Monosodium Salt, Product No. G7879.
7.3.3. 20 mM β-Nicotinamide Adenine Dinucleotide Phosphate Solution (β-NADP)
Prepare 15.4 mg/mL in purified water using β-Nicotinamide Adenine Dinucleotide Phosphate, Sodium Salt, Product No. N0505.
7.3.4. 300 mM Magnesium Chloride Solution (MgCl2)
Prepare 0.3 mL/mL in purified water using 1.0 M Magnesium Chloride Solution, Product No. M1028.
7.3.5. Glucose-6-Phosphate Dehydrogenase Enzyme Solution (Enzyme)
Immediately before use, prepare 0.3-0.6 units/mL in cold buffer (Reagent 7.3.1).
7.4.1. Prepare a reaction cocktail by pipetting (in milliliters) the following reagents into a suitable container:
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:
7.4.4. Equilibrate to 25 °C. Monitor the A340nm until constant, using a suitably thermostatted spectrophotometer. Then add:
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.
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
7.6. 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.
Noltmann, E.A., Gubler, C.J., and Kuby, S.A. (1961) Journal of Biological Chemistry 236, 1225- 1230.
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