Glucose-6-Phosphate Dehydrogenase Assay
Method: This dehydrogenase is rather unique in that it possesses dual coenzyme specificity. When assayed under conditions that are optimal for the particular coenzyme, the ratio of observed catalytic activity is NAD/NADP = 1.8. The reaction velocity is determined by measuring the increase in absorbance at 340 nm resulting from the reduction of NAD or NADP. One unit reduces one micromole of pyridine nucleotide per minute at 30°C and pH 7.8 under the specified conditions.
Dissolve lyophilized enzyme or dilute enzyme suspension to 1.0 mg/ml in 5 mM glycine buffer, pH 8.0. Care should be taken not to shake the solution as precipitation may result. Dilute further immediately before use in 5 mM glycine buffer, containing 0.1% albumin, to obtain a rate of 0.02 - 0.045 ΔA/min.
Adjust spectrophotometer to 340 and 30°C.
Pipette into each cuvette as follows:
Incubate in spectrophotometer at 30deg.C for 7 - 8 minutes to achieve temperature equilibration and establish blank rate, if any. Add 0.1 ml diluted enzyme and record increase in A340/min for 4 to 5 minutes. Calculate �A340/minute from the initial linear portion of the curve.