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):3519-26. doi: 10.1021/bi00360a007.

Abstract

Trypanothione reductase from Crithidia fasciculata has been purified ca. 1400-fold to homogeneity in an overall yield of 60%. The pure enzyme showed a pH optimum of 7.5-8.0 and was highly specific for its physiological substrates NADPH and trypanothione that had Km values of 7 and 53 microM, respectively. Trypanothione reductase was found to be a dimer of identical subunits with Mr 53 800 each. The enzyme displayed a visible absorption spectrum that was indicative of a flavoprotein with a lambda max at 464 nm. The flavin was liberated by thermal denaturation of the protein and identified, both by high-performance liquid chromatography (HPLC) and by fluorescence studies, as FAD. The extinction coefficient of pure enzyme at 464 nm was determined to be 11.3 mM-1 cm-1. Upon titration with 5,5'-dithiobis(2-nitrobenzoic acid), oxidized enzyme was found to contain 2.2 (+/- 0.1) free thiols, whereas NADPH-reduced enzyme showed 3.9 (+/- 0.3). Furthermore, whereas oxidized enzyme was stable toward inactivating alkylation by 2.0 mM iodoacetamide, NADPH-reduced enzyme was inactivated with a half-life of 14 min. These data suggested that a redox-active cystine residue was present at the enzyme active site. Upon reduction of the enzyme with 2 electron equiv of dithionite, a new peak in the absorption spectrum was observed at 530 nm, thus indicating that a charge-transfer complex between one of the newly reduced thiols and the oxidized FAD had formed.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acids / analysis
  • Animals
  • Binding Sites
  • Crithidia / enzymology*
  • Dithionite / pharmacology
  • Kinetics
  • Macromolecular Substances
  • Molecular Weight
  • NADH, NADPH Oxidoreductases / isolation & purification*
  • NADH, NADPH Oxidoreductases / metabolism

Substances

  • Amino Acids
  • Macromolecular Substances
  • Dithionite
  • NADH, NADPH Oxidoreductases
  • trypanothione reductase