Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2003 Feb 1;369(Pt 3):529–537. doi: 10.1042/BJ20021298

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

Chris J Hamilton 1, Ahilan Saravanamuthu 1, Ian M Eggleston 1, Alan H Fairlamb 1
PMCID: PMC1223126  PMID: 12416994

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](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.

Full Text

The Full Text of this article is available as a PDF (226.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aumercier M., Meziane-Cherif D., Moutiez M., Tartar A., Sergheraert C. A microplate assay for trypanothione reductase inhibitors. Anal Biochem. 1994 Nov 15;223(1):161–164. doi: 10.1006/abio.1994.1563. [DOI] [PubMed] [Google Scholar]
  2. Bailey S., Smith K., Fairlamb A. H., Hunter W. N. Substrate interactions between trypanothione reductase and N1-glutathionylspermidine disulphide at 0.28-nm resolution. Eur J Biochem. 1993 Apr 1;213(1):67–75. doi: 10.1111/j.1432-1033.1993.tb17734.x. [DOI] [PubMed] [Google Scholar]
  3. Bond C. S., Zhang Y., Berriman M., Cunningham M. L., Fairlamb A. H., Hunter W. N. Crystal structure of Trypanosoma cruzi trypanothione reductase in complex with trypanothione, and the structure-based discovery of new natural product inhibitors. Structure. 1999 Jan 15;7(1):81–89. doi: 10.1016/s0969-2126(99)80011-2. [DOI] [PubMed] [Google Scholar]
  4. Borges A., Cunningham M. L., Tovar J., Fairlamb A. H. Site-directed mutagenesis of the redox-active cysteines of Trypanosoma cruzi trypanothione reductase. Eur J Biochem. 1995 Mar 15;228(3):745–752. doi: 10.1111/j.1432-1033.1995.tb20319.x. [DOI] [PubMed] [Google Scholar]
  5. Cunningham M. L., Fairlamb A. H. Trypanothione reductase from Leishmania donovani. Purification, characterisation and inhibition by trivalent antimonials. Eur J Biochem. 1995 Jun 1;230(2):460–468. doi: 10.1111/j.1432-1033.1995.tb20583.x. [DOI] [PubMed] [Google Scholar]
  6. Cunningham M. L., Zvelebil M. J., Fairlamb A. H. Mechanism of inhibition of trypanothione reductase and glutathione reductase by trivalent organic arsenicals. Eur J Biochem. 1994 Apr 1;221(1):285–295. doi: 10.1111/j.1432-1033.1994.tb18740.x. [DOI] [PubMed] [Google Scholar]
  7. Dumas C., Ouellette M., Tovar J., Cunningham M. L., Fairlamb A. H., Tamar S., Olivier M., Papadopoulou B. Disruption of the trypanothione reductase gene of Leishmania decreases its ability to survive oxidative stress in macrophages. EMBO J. 1997 May 15;16(10):2590–2598. doi: 10.1093/emboj/16.10.2590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. FitzGerald G. B., Bauman C., Hussoin M. S., Wick M. M. 2,4-Dihydroxybenzylamine: a specific inhibitor of glutathione reductase. Biochem Pharmacol. 1991 Jan 15;41(2):185–190. doi: 10.1016/0006-2952(91)90475-k. [DOI] [PubMed] [Google Scholar]
  9. Henderson G. B., Fairlamb A. H., Ulrich P., Cerami A. Substrate specificity of the flavoprotein trypanothione disulfide reductase from Crithidia fasciculata. Biochemistry. 1987 Jun 2;26(11):3023–3027. doi: 10.1021/bi00385a011. [DOI] [PubMed] [Google Scholar]
  10. Krieger S., Schwarz W., Ariyanayagam M. R., Fairlamb A. H., Krauth-Siegel R. L., Clayton C. Trypanosomes lacking trypanothione reductase are avirulent and show increased sensitivity to oxidative stress. Mol Microbiol. 2000 Feb;35(3):542–552. doi: 10.1046/j.1365-2958.2000.01721.x. [DOI] [PubMed] [Google Scholar]
  11. Marsh I. R., Bradley M. Substrate specificity of trypanothione reductase. Eur J Biochem. 1997 Feb 1;243(3):690–694. doi: 10.1111/j.1432-1033.1997.00690.x. [DOI] [PubMed] [Google Scholar]
  12. Morrison J. F., Walsh C. T. The behavior and significance of slow-binding enzyme inhibitors. Adv Enzymol Relat Areas Mol Biol. 1988;61:201–301. doi: 10.1002/9780470123072.ch5. [DOI] [PubMed] [Google Scholar]
  13. Patel M. P., Blanchard J. S. Expression, purification, and characterization of Mycobacterium tuberculosis mycothione reductase. Biochemistry. 1999 Sep 7;38(36):11827–11833. doi: 10.1021/bi991025h. [DOI] [PubMed] [Google Scholar]
  14. Riddles P. W., Blakeley R. L., Zerner B. Ellman's reagent: 5,5'-dithiobis(2-nitrobenzoic acid)--a reexamination. Anal Biochem. 1979 Apr 1;94(1):75–81. doi: 10.1016/0003-2697(79)90792-9. [DOI] [PubMed] [Google Scholar]
  15. Salmon-Chemin L., Buisine E., Yardley V., Kohler S., Debreu M. A., Landry V., Sergheraert C., Croft S. L., Krauth-Siegel R. L., Davioud-Charvet E. 2- and 3-substituted 1,4-naphthoquinone derivatives as subversive substrates of trypanothione reductase and lipoamide dehydrogenase from Trypanosoma cruzi: synthesis and correlation between redox cycling activities and in vitro cytotoxicity. J Med Chem. 2001 Feb 15;44(4):548–565. doi: 10.1021/jm001079l. [DOI] [PubMed] [Google Scholar]
  16. Shames S. L., Fairlamb A. H., Cerami A., Walsh C. T. Purification and characterization of trypanothione reductase from Crithidia fasciculata, a newly discovered member of the family of disulfide-containing flavoprotein reductases. Biochemistry. 1986 Jun 17;25(12):3519–3526. doi: 10.1021/bi00360a007. [DOI] [PubMed] [Google Scholar]
  17. Tovar J., Cunningham M. L., Smith A. C., Croft S. L., Fairlamb A. H. Down-regulation of Leishmania donovani trypanothione reductase by heterologous expression of a trans-dominant mutant homologue: effect on parasite intracellular survival. Proc Natl Acad Sci U S A. 1998 Apr 28;95(9):5311–5316. doi: 10.1073/pnas.95.9.5311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Tovar J., Wilkinson S., Mottram J. C., Fairlamb A. H. Evidence that trypanothione reductase is an essential enzyme in Leishmania by targeted replacement of the tryA gene locus. Mol Microbiol. 1998 Jul;29(2):653–660. doi: 10.1046/j.1365-2958.1998.00968.x. [DOI] [PubMed] [Google Scholar]
  19. delCardayre S. B., Stock K. P., Newton G. L., Fahey R. C., Davies J. E. Coenzyme A disulfide reductase, the primary low molecular weight disulfide reductase from Staphylococcus aureus. Purification and characterization of the native enzyme. J Biol Chem. 1998 Mar 6;273(10):5744–5751. doi: 10.1074/jbc.273.10.5744. [DOI] [PubMed] [Google Scholar]
  20. el-Waer A. F., Benson T., Douglas K. T. Synthesis of substrate analogues for trypanothione reductase. Int J Pept Protein Res. 1993 Feb;41(2):141–146. doi: 10.1111/j.1399-3011.1993.tb00124.x. [DOI] [PubMed] [Google Scholar]
  21. el-Waer A., Douglas K. T., Smith K., Fairlamb A. H. Synthesis of N-benzyloxycarbonyl-L-cysteinylglycine 3-dimethylaminopropylamide disulfide: a cheap and convenient new assay for trypanothione reductase. Anal Biochem. 1991 Oct;198(1):212–216. doi: 10.1016/0003-2697(91)90531-w. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES