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

Research output: Contribution to journalArticlepeer-review

82 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

Cite this