Ellman's-reagent-mediated regeneration of trypanothione in situ: substrate-economical microplate and time-dependent inhibition assays for trypanothione reductase.
Trypanothione reductase (TryR) is a key enzyme involved in the oxidative stress management of the Trypanosoma and Leishmania parasites, which helps to maintain an intracellular reducing environment by reduction of the small-molecular-mass disulphide trypanothione (T[S](2)) to its di-thiol derivative dihydrotrypanothione (T[SH](2)). TryR inhibition studies are currently impaired by the prohibitive costs of the native enzyme substrate T[S](2). Such costs are particularly notable in time-dependent and high-throughput inhibition assays. In the present study we report a protocol that greatly decreases the substrate quantities needed for such assays. This is achieved by coupling the assay with the chemical oxidant 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), which can rapidly re-oxidize the T[SH](2) product back into the disulphide substrate T[S](2), thereby maintaining constant substrate concentrations and avoiding deviations from rate linearity due to substrate depletion. This has enabled the development of a continuous microplate assay for both classical and time-dependent TryR inhibition in which linear reaction rates can be maintained for 60 min or more using minimal substrate concentrations (<1 microM, compared with a substrate K (m) value of 30 microM) that would normally be completely consumed within seconds. In this manner, substrate requirements are decreased by orders of magnitude. The characterization of a novel time-dependent inhibitor, cis -3-oxo-8,9b-bis-(N(1)-acrylamidospermidyl)-1,2,3,4,4a,9b-hexahydrobenzofuran (PK43), is also described using these procedures.
Hamilton
Chris J
CJ
Division of Biological Chemistry & Molecular Microbiology, School of Life Sciences, Carnelley Building, University of Dundee, Dundee DD1 4HN, UK.
Saravanamuthu
Ahilan
A
Eggleston
Ian M
IM
Fairlamb
Alan H
AH
D001152:Arsenicals / Q000494:pharmacology; D001671:Biochemistry / Q000295:instrumentation / Q000379:methods*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000737:chemistry*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005978:Glutathione / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D016014:Linear Models; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013095:Spermidine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D013997:Time Factors; D014966:Xanthenes / Q000494:pharmacology
C472028:3-oxo-8,9b-bis(N(1)-acrylamidospermidyl)-1,2,3,4,4a,9b-hexahydroxanthene; D001152:Arsenicals; D004791:Enzyme Inhibitors; D014966:Xanthenes; C044719:melarsen oxide; C044809:trypanothione; D004228:Dithionitrobenzoic Acid; D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase; D005978:Glutathione; D013095:Spermidine; D004121:Dimethyl Sulfoxide