Systematic re-evaluation of the bis(2-hydroxyethyl)disulfide (HEDS) assay reveals an alternative mechanism and activity of glutaredoxins.

ABSTRACT: The reduction of bis(2-hydroxyethyl)disulfide (HEDS) by reduced glutathione (GSH) is the most commonly used assay to analyze the presence and properties of enzymatically active glutaredoxins (Grx), a family of central redox proteins in eukaryotes and glutathione-utilizing prokaryotes. Enzymatically active Grx usually prefer glutathionylated disulfide substrates. These are converted via a ping-pong mechanism. Sequential kinetic patterns for the HEDS assay have therefore been puzzling since 1991. Here we established a novel assay and used the model enzyme ScGrx7 from yeast and PfGrx from Plasmodium falciparum to test several possible causes for the sequential kinetics such as pre-enzymatic GSH depletion, simultaneous binding of a glutathionylated substrate and GSH, as well as substrate or pr

SUBMITTER: Begas P 

PROVIDER: S-EPMC5707495 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature