Purification and characterization of trypanothione reductase from Crithidia fasciculata, a newly discovered member of the family of disulfide-containing flavoprotein reductases.
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)
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D003421:Crithidia / Q000201:enzymology*; D004227:Dithionite / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D046911:Macromolecular Substances; D004227:Dithionite; D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase