Nitric oxide-related species-induced protein oxidation: reversible, irreversible, and protective effects on enzyme function of papain.
Protein oxidation, irreversible modification, and inactivation may play key roles in various neurodegenerative disorders. Therefore, we studied the effects of the potentially in vivo occurring nitric oxide-related species on two different markers of protein oxidation: protein carbonyl generation on bovine serum albumine (BSA) and loss of activity of a cysteine-dependent protease, papain, in vitro by using Angeli's salt, papanonoate, SIN-1, and S-nitrosoglutathione (GSNO) as donors of nitroxyl, nitric oxide, peroxynitrite, and nitrosonium ions, respectively. Angeli's salt, SIN-1, and papanonoate (0-1000 microM) all generated a concentration-dependent increase in carbonyl formation on BSA (107, 60, and 45%, respectively). GSNO did not affect carbonyl formation. Papain was inhibited by Angeli's salt, SIN-1, papanonoate, and GSNO with IC50 values of 0.62, 2.3, 54, and 80 microM, respectively. Angeli's salt (3.16 microM)-induced papain inactivation was only partially reversible, while the effects of GSNO (316 microM) and papanonoate (316 microM) were reversible upon addition of excess DTT. The Angeli's salt-mediated DTT-irreversible inhibition of papain was prevented by GSNO or papanonoate pretreatment, hypothetically through mixed disulfide formation or S-nitrosylation of the catalytically critical thiol group of papain. These results, for the first time, compare the generation of carbonyls in proteins by Angeli's salt, papanonoate, and SIN-1. Furthermore, these results suggest that S-nitrosothiols may have a novel function in protecting critical thiols from irreversible oxidative damage.
Väänänen
Antti J
AJ
Institute of Biomedicine (Pharmacology), Biomedicum Helsinki, P.O. Box 63, University of Helsinki 00014, Finland.
Kankuri
Esko
E
Rauhala
Pekka
P
D000975:Antioxidants / Q000494:pharmacology; D009569:Nitric Oxide / Q000502:physiology*; D010084:Oxidation-Reduction; D010206:Papain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism
D000975:Antioxidants; D013438:Sulfhydryl Compounds; D009569:Nitric Oxide; D010206:Papain