Ratnam K, Hille R
Department of Medical Biochemistry, Ohio State University, Columbus 43210.
Biochem Biophys Res Commun. 1993 Aug 16;194(3):1097-102. doi: 10.1006/bbrc.1993.1934.
The pH dependence of the behavior of chicken liver xanthine dehydrogenase in the course of reductive titrations with sodium dithionite has been examined. Below pH 8.5, the behavior of xanthine dehydrogenase is similar to that of the much better understood milk xanthine oxidase, with the amount of neutral semiquinone accumulating transiently in the course of the titration increasing somewhat as the pH decreases. At pH 10, however, an anomalously large accumulation of the neutral semiquinone is observed by both UV/visible and EPR spectroscopy. Treatment of xanthine dehydrogenase with the thiol reagent iodoacetamide significantly diminishes the ability of the enzyme to stabilize the neutral flavin semiquinone at high pH. These data are consistent with the presence of a protein thiol in the immediate vicinity of the flavin, whose ionization above pH 8.5 results in thermodynamic stabilization of the neutral flavin semiquinone over the anionic form.
已对连二亚硫酸钠还原滴定过程中鸡肝黄嘌呤脱氢酶行为的pH依赖性进行了研究。在pH 8.5以下，黄嘌呤脱氢酶的行为与人们了解得更多的牛奶黄嘌呤氧化酶相似，随着pH值降低，滴定过程中瞬时积累的中性半醌数量略有增加。然而，在pH 10时，紫外/可见光谱和电子顺磁共振光谱均观察到中性半醌异常大量积累。用硫醇试剂碘乙酰胺处理黄嘌呤脱氢酶会显著降低该酶在高pH值下稳定中性黄素半醌的能力。这些数据与黄素附近存在蛋白质硫醇一致，其在pH 8.5以上的电离导致中性黄素半醌相对于阴离子形式在热力学上更稳定。
