An enzymatic colorimetric assay for glucose-6-phosphate

Abstract

A specific colorimetric assay for the determination of glucose-6-phosphate (G6P) was developed. This assay is based on the oxidation of G6P in the presence of glucose-6-phosphate dehydrogenase (G6PD) and nicotinamide adenine dinucleotide phosphate (NADP(+)); the NADPH thereby generated reduces the tetrazolium salt WST-1 [2-(4-indophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium, monosodium salt] to water-soluble yellow-colored formazan with 1-methoxy-5-methylphenazium methylsulfate (1-mPMS) as an electron carrier. The assay is optimized for reaction buffer pH, enzyme/dye concentration, and reaction time course. The limit of detection of the assay is 0.15 μM (15 pmol/well). The usefulness of the assay is demonstrated by the accurate measurement of the G6P concentration in fetal bovine serum (FBS).

Fig. 1

Optimization of G6P assay. The G6P concentration was 10 μM. A: pH effect on the reaction; B: dye effect on the assay; C: Electron carrier effect on the assay; D: NADP⁺ effect on the assay; E: effect of G6PD concentration on absorbance; F: reaction time course of the assay. Unless otherwise noted, each point in the figure is a mean of three replicates; error bars represent the standard deviation. In some panels, error bars are invisible because of the symbols are larger than the error bars.

Fig. 2

Validation of the assay. A: The G6P assay was tested using a panel of compounds (G6P, F6P, G1P, ribulose-5P and ribose-5P at 100 μM and glucose at 100 mM); B: the relationship of absorbance with G6P concentration; C: linear range of the assay (from 1.0 to 200 μM).

Fig. 3

A typical calibration curve for G6P measurement in FBS samples. Each datum point is a mean of triplicate measurements). y = 0.001169 x, n = 8.

Scheme1

Mechanism of the chemical reactions for detection of G6P.