Investigations of S-transnitrosylation reactions between low- and high-molecular-weight S-nitroso compounds and their thiols by high-performance liquid chromatography and gas chromatography-mass spectrometry.
S-Transnitrosylation reactions are supposed to be the basic principle by which nitric oxide-related biological activities are regulated in vivo. Mechanisms of S-transnitrosylation reactions are poorly understood and equilibria constants for physiological S-nitroso compounds and thiols are rare. In the present study we investigated S-transnitrosylation reactions of the thiols homocysteine, cysteine, glutathione, N-acetylcysteine, N-acetylpenicillamine, and human plasma albumin and their corresponding S-nitroso compounds SNhC, SNC, GSNO, SNAC, SNAP, and SNALB utilizing high-performance liquid chromatographic and gas chromatographic-mass spectrometric techniques. These methods allowed to study S-transnitrosylation reactions in mixtures of several S-nitroso compound/thiol pairs, to determine equilibria constants, and to elucidate the mechanism of S-transnitrosylation reactions. We obtained the following order for the equilibria constants in aqueous buffered solution at pH 7.4: SNhC approximately SNAC > GSNO approximately SNALB > SNAP > SNC. Our results suggest that the mechanism of S-transnitrosylation reactions of these S-nitroso compounds and their thiols involve heterolytic cleavage of the S&sbond;N bond. Incubation of SNC with human red blood cells resulted in a dose-dependent formation of GSNO in the cytosol through S-transnitrosylation of intracellular GSH by the SNC transported into the cells. This reaction was accompanied with an almost complete disappearance of the SNC fraction transported into the cells. This finding is in full agreement with the equilibrium constant Keq of 1.9 for the reaction SNC + GSH <--> Cys + GSNO in aqueous buffer.
Tsikas
D
D
Institute of Clinical Pharmacology, Hannover Medical School, Hannover, 30623, Germany. tsikas.dimitros@mh-hannover.de
Sandmann
J
J
Rossa
S
S
Gutzki
F M
FM
Frölich
J C
JC
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D005978:Glutathione / Q000378:metabolism; D006710:Homocysteine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D019811:Hydroxylamine / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008970:Molecular Weight; D009569:Nitric Oxide / Q000096:biosynthesis; D009603:Nitroso Compounds / Q000737:chemistry* / Q000378:metabolism*; D010396:Penicillamine / Q000031:analogs & derivatives / Q000378:metabolism; D026423:S-Nitroso-N-Acetylpenicillamine; D026403:S-Nitrosothiols; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism*
D009603:Nitroso Compounds; D026403:S-Nitrosothiols; D013438:Sulfhydryl Compounds; D006710:Homocysteine; C079202:S-nitrosohomocysteine; D019811:Hydroxylamine; D009569:Nitric Oxide; C024940:S-nitroso-N-acetylcysteine; D026423:S-Nitroso-N-Acetylpenicillamine; C021315:S-nitrosocysteine; D005978:Glutathione; D010396:Penicillamine; D003545:Cysteine; D000111:Acetylcysteine