Ellman's-reagent-mediated regeneration of trypanothione in situ: substrate economical microplate and time-dependent inhibition assays for trypanothione reductase

Hamilton, C. J., Saravanamuthu, A., Eggleston, I. M. and Fairlamb, A. H. (2003) Ellman's-reagent-mediated regeneration of trypanothione in situ: substrate economical microplate and time-dependent inhibition assays for trypanothione reductase. Biochemical Journal, 369. pp. 529-537. ISSN 0264-6021

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Abstract

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], 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 muM, compared with a substrate K-m value of 30 muM) 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.

Item Type: Article
Uncontrolled Keywords: purification,5 '-dithio-bis-(2-nitrobenzoic acid),disulfide reductase,crithidia-fasciculata,enzyme,disulphide recycling,5,trypanosoma-cruzi,leishmania-donovani,oxidative stress,high-throughput screening,expression,specificity,glutathione-reductase
Faculty \ School: Faculty of Science > School of Pharmacy
Depositing User: Rachel Smith
Date Deposited: 18 May 2011 09:41
Last Modified: 21 Apr 2020 20:32
URI: https://ueaeprints.uea.ac.uk/id/eprint/30699
DOI: 10.1042/bj20021298

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