Srivastava S K, Beutler E
Biochem J. 1970 Sep;119(3):353-7. doi: 10.1042/bj1190353.
A complex of haemoglobin and GSH was prepared by incubating haemoglobin with GSH and acetylphenylhydrazine. GSH could be released from the crude preparation by incubation with NADPH. However, when the haemoglobin preparation was separated from glutathione reductase by DEAE-Sephadex chromatography, NADPH no longer released GSH. Rather, the addition of a combination of either partially purified human erythrocyte or crystalline glutathione reductase and NADPH was required to release GSH from the haemoglobin-GSH complex. This complex is commonly believed to represent a mixed disulphide of GSH and the cysteine-beta-93 thiol group. This interpretation was supported by the finding that prior alkylation of available haemoglobin thiol groups prevented the formation of the complex. By using haemoglobin-[(35)S]GSH complex as a substrate, it was shown that GSH itself released the radioactivity from the complex only very slowly. In contrast, the release of [(35)S]GSH was very rapid in the presence of NADPH and glutathione reductase. This suggests that the cleavage of the haemoglobin-GSH complex is not mediated by GSH with cyclic reduction of GSSG formed, but rather proceeds enzymically through glutathione reductase.
通过将血红蛋白与谷胱甘肽（GSH）和乙酰苯肼孵育制备血红蛋白与GSH的复合物。通过与烟酰胺腺嘌呤二核苷酸磷酸（NADPH）孵育，可从粗制品中释放出GSH。然而，当通过二乙氨基乙基葡聚糖（DEAE-Sephadex）色谱法将血红蛋白制剂与谷胱甘肽还原酶分离时，NADPH不再释放GSH。相反，需要添加部分纯化的人红细胞或结晶谷胱甘肽还原酶与NADPH的组合，才能从血红蛋白-GSH复合物中释放GSH。通常认为该复合物代表GSH与半胱氨酸-β-93巯基的混合二硫化物。这一解释得到以下发现的支持：对可用的血红蛋白巯基进行预先烷基化可阻止复合物的形成。通过使用血红蛋白-[（35）S]GSH复合物作为底物，结果表明GSH本身从复合物中释放放射性的速度非常缓慢。相反，在NADPH和谷胱甘肽还原酶存在下，[（35）S]GSH的释放非常迅速。这表明血红蛋白-GSH复合物的裂解不是由GSH通过形成的氧化型谷胱甘肽（GSSG）的循环还原介导的，而是通过谷胱甘肽还原酶进行酶促反应。
