An elevated concentration of plasma total homocysteine is an independent risk factor for cardiovascular disease. Greater than 80% of circulating homocysteine is covalently bound to plasma protein by disulfide bonds. It is known that albumin combines with cysteine in circulation to form albumin-Cys34-S-S-Cys. Studies are now presented to show that the formation of albumin-bound homocysteine proceeds through the generation of an albumin thiolate anion. Incubation of human plasma with L- 35S-homocysteine results in the association of >90% of the protein-bound 35S-homocysteine with albumin as shown by nonreduced SDS-polyacrylamide gel electrophoresis. Treatment of the complex with β-mercaptoethanol results in near quantitative release of the bound L- 35S-homocysteine, demonstrating that the binding of homocysteine to albumin is through a disulfide bond. Furthermore, using an in vitro model system to study the mechanisms of this disulfide bond formation, we show that homocysteine binds to albumin in two steps. In the first step homocysteine rapidly displaces cysteine from albumin-Cys34-S-S-Cys, forming albumin-Cys34 thiolate anion and homocysteine-cysteine mixed disulfide. In the second step, albumin thiolate anion attacks homocysteine-cysteine mixed disulfide to yield primarily albumin-Cys 34-S-S-Hcy and to a much lesser extent albumin-Cys 34-S-S-Cys. The results clearly suggest that when reduced homocysteine enters circulation, it attacks albumin-Cys34-S-S-Cys to form albumin-Cys34 thiolate anion, which in turn, reacts with homocysteine-cysteine mixed disulfide or homocystine to form albumin-bound homocysteine.
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