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

C. J. Hamilton, A. Saravanamuthu, I. M. Eggleston, A. H. Fairlamb

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    84 Citations (Scopus)

    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.
    Original languageEnglish
    Pages (from-to)529-537
    Number of pages9
    JournalBiochemical Journal
    Volume369
    DOIs
    Publication statusPublished - 2003

    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

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