On-plate enzyme and inhibition assay of glucose-6-phosphate dehydrogenase using thin-layer chromatography

Abstract

We performed on-plate enzyme and inhibition assays of glucose 6-phosphate dehydrogenase using thin-layer chromatography. The assays were accomplished based on different retardation factors of the substrates, enzyme, and products. All the necessary steps were integrated on-plate in one developing process, including substrate/enzyme mixing, reaction starting, and quenching as well as product separation. In order to quantitatively measure the enzyme reaction, the developed plate was then densitometrically evaluated to determine the peak area of the product. Rapid and high-throughput assays were achieved by loading different substrate spots and/or enzyme (and inhibition) spots in different tracks on the plate. The on-plate enzyme assay could be finished in a developing time of only 4 min, with good track-to-track and plate-to-plate repeatability. Moreover, we determined the Km values of the enzyme reaction and Ki values of the inhibition (Pb(2+) Cd(2+) and Cu(2+) as inhibitors), as well as the corresponding kinetics using the on-plate assay. Taken together, our method expanded the application of thin-layer chromatography in enzyme assays, and it could be potentially used in research fields for rapid and quantitative measurement of enzyme activity and inhibition.

Recent Advances in Effect‐directed Enzyme Assays based on Thin‐layer Chromatography

“…Mutations in the G6PDH gene result in enzyme variants with different G6PDH activities (Cappellini and Fiorelli, ). Therefore, an efficient method to perform both G6PDH activity and inhibition assays would help to improve understanding of G6PDH regulation in the human metabolism and its potential beneficial effects as a drug target for related diseases (Tian et al , ).…”

“…Rapid, high‐throughput and quantitative enzyme and inhibition assays of G6PDH on TLC plates have been developed (Fig’ ) (Tian et al , ). The assays were based on different TLC retardation factors of the substrate and products of the G6PDH catalysed reaction.…”

“…The assay was optimised by testing different amounts of enzyme and coenzyme. Moreover, Michaelis–Menten diagrams, Km values of the enzyme reaction, and Ki values of Pb 2+ Cd 2+ and Cu 2+ as G6PDH inhibitors were determined (Tian et al , ). The G6PDH assay on TLC plates does not allow the separation of compounds from complex matrices prior to activity screening and is therefore not a bioautographic assay per se .…”