Albumin thiolate anion is an intermediate in the formation of albumin-S-S-homocysteine
Albumin thiolate anion is an intermediate in the formation of albumin-S-S-homocysteine
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DOI：
10.1074/jbc.m104324200
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发表时间：
2001-08-10
影响因子：
4.8
通讯作者：
Jacobsen, DW
中科院分区：
文献类型：
--
作者：
Sengupta, S;Chen, H;Jacobsen, DW
血浆总同型半胱氨酸浓度升高是心血管疾病的独立危险因素。超过80%的循环同型半胱氨酸通过二硫键与血浆蛋白共价结合。已知白蛋白在循环中与半胱氨酸结合形成白蛋白- cys (34)-S-S-Cys。现在的研究表明，白蛋白结合的同型半胱氨酸的形成是通过白蛋白硫代阴离子的产生进行的。非还原sds -聚丙烯酰胺凝胶电泳显示，l - s -35-同型半胱氨酸在人血浆中孵卵，可使90%的蛋白质结合的s -35-同型半胱氨酸与白蛋白相关联。用-巯基乙醇处理络合物导致结合的l - s -35同型半胱氨酸的近定量释放，表明同型半胱氨酸与白蛋白的结合是通过二硫键。此外，利用体外模型系统研究这种二硫键形成的机制，我们发现同型半胱氨酸与白蛋白的结合分两个步骤。在第一步，同型半胱氨酸迅速取代白蛋白-胱氨酸(34)- s - s -胱氨酸中的半胱氨酸，形成白蛋白-胱氨酸（34）硫酸阴离子和同型半胱氨酸-半胱氨酸混合二硫化物。第二步，白蛋白硫酸盐阴离子攻击同型半胱氨酸-半胱氨酸混合二硫，主要生成白蛋白-胱氨酸(34)-S-S-Hcy和少量白蛋白-胱氨酸(34)-S-S-Cys。结果清楚地表明，当还原的同型半胱氨酸进入循环时，它攻击白蛋白-半胱氨酸(34)- s - s -半胱氨酸形成白蛋白-半胱氨酸（34）硫代阴离子，该阴离子又与同型半胱氨酸-半胱氨酸混合二硫或同型半胱氨酸反应形成白蛋白结合的同型半胱氨酸。
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-Cys(34)-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-S-35-homocysteine results in the association of > 90% of the protein-bound S-35-homocysteine with albumin as shown by nonreduced SDS-polyacrylamide gel electrophoresis. Treatment of the complex with beta -mercaptoethanol results in near quantitative release of the bound L-S-35-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-Cys(34)-S-S-Cys, forming albumin-Cys(34) 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-Cys(34)-S-S-Cys to form albumin-Cys(34) thiolate anion, which in turn, reacts with homocysteine-cysteine mixed disulfide or homocystine to form albumin-bound homocysteine.