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Glucose-6-Phosphate Dehydrogenase (G6PDH)

(D-Glucose-6-phosphate:NADP+ 1-oxidoreductase; EC

Glucose-6-phosphate dehydrogenase (G-6-PDH) catalyzes the oxidation of glucose 6-phosphate as shown in the following reaction:

D-Glucose-6-phosphate + NADP+ ------> D-Gluconate-6-phosphate + NADPH + H+ 
The enzyme has been purified from yeasts, E. coli and various mammalian tissues. Glucose-6-phosphate dehydrogenase activity has been found to be deficient in 10-13% of American black males. As a result of this genetic defect, this population is susceptible to severe hemolytic anemia unless the condition is diagnosed (Orten, J.M. and O.W. Neuhans, Human Biochemistry, 10th Edition, p. 817, 1982, Mosby, St. Louis).

1.Source: Leuconostoc Mesenteroides
   Form: Freeze-dried powder 
   Solubility: Distilled water or dilute buffer 
   Stability: Store at -20 C (-4 F) 
   Activity (approx.): 20 U/mg material 
   Protein (approx.): 50% (biuret) 
   Contaminants (approx.): 6-PGDH 0.01%; Hexokinase 0.002%; Creatine kinase 0.001%; Phosphoglucomutase 0.01% 
   Catalog No.: 078A0020 

The amount of enzyme which will reduce one micromole of NADP per minute at pH 7.6 and 25C.

The increase in absorbance at 340 nm, caused by the reduction of NADP is a measure of the catalytic activity of glucose-6-phosphate dehydrogenase.

  1. 0.1 M Triethanolamine (TEA) buffer, pH 7.6 (1.86g TEA-HCl in about 80 ml distilled water, adjust pH to 7.6 with NaOH and make up the volume to 100 ml with distilled water).
  2. 0.1 M magnesium chloride solution (2.05g MgCl2&6H2O/100 ml, in distilled water).
  3. 0.03 M glucose-6-phosphate solution (10 mg/ml) in distilled water.
  4. 13 mM NADP (10 mg NADP-Na2/ml in distilled water).
  5. G-6-PDH solution: For freeze-dried enzyme, dissolve 10 mg enzyme in 1 ml distilled water and dilute 1:500 with cold TEA buffer. Prepare just prior to use. For enzyme in suspension, dilute suspension 1:2500 with cold TEA buffer, just prior to use.

  1. Set spectrophotometer (equipped with strip chart recorder and temperature control) at 340 nm and 25C.
  2. Into the cuvette, pipette the following:

    TEA buffer 2.50 ml
    MgCl2 0.20 ml
    Glucose-6-Phosphate 0.10 ml
    NADP 0.10 ml

  3. Mix and incubate at 25C for 5 minutes in the spectrophotometer.
  4. Record blank rate at 340 nm for 2-3 minutes.
  5. Pipette 0.05 ml fresh enzyme solution into the cuvette. Mix and record increase in absorbance at 340 nm for 5 minutes.
  6. Calculate (delta)E340 nm/min from the linear portion of the curve.

Activity (U/mg) = (ΔE340nm/min)(Total Vol.)(Enz. Diln.)
(6.22)(Enz. Vol.)(mg Enz./ml)

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