Rothwarf D M, Li Y J, Scheraga H A
Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301, USA.
Biochemistry. 1998 Mar 17;37(11):3760-6. doi: 10.1021/bi972822n.
During the regeneration of bovine pancreatic ribonuclease A (RNase A) from the reduced to the native form with mixtures of oxidized and reduced dithiothreitol at 25 degrees C, pH 8.0, the disulfide-containing protein intermediates achieve a steady-state distribution. By manipulating the redox conditions after the attainment of the steady-state condition, it has been possible to kinetically trap and, thereby, isolate and identify the disulfide-bonded species that follow the rate-determining step in the regeneration pathway. Two three-disulfide species have been identified by peptide mapping. Both species contain three native disulfide-bond pairings, one lacks the 65-72 disulfide bond (des-[65-72]), and the other lacks the 40-95 disulfide bond (des-[40-95]). These species are the same as those identified during the reduction of RNase A. By restarting the regeneration process from isolated des-[65-72] and des-[40-95], it is shown that both intermediates lie directly on regeneration pathways.
在25℃、pH 8.0的条件下，用氧化型和还原型二硫苏糖醇的混合物将牛胰核糖核酸酶A（RNase A）从还原态再生为天然态的过程中，含二硫键的蛋白质中间体达到了稳态分布。通过在达到稳态条件后操纵氧化还原条件，有可能从动力学上捕获，从而分离并鉴定在再生途径中位于速率决定步骤之后的二硫键连接的物种。通过肽图谱分析鉴定出了两种含三个二硫键的物种。这两种物种都含有三个天然二硫键配对，一种缺少65-72位的二硫键（des-[65-72]），另一种缺少40-95位的二硫键（des-[40-95]）。这些物种与在RNase A还原过程中鉴定出的物种相同。通过从分离出的des-[65-72]和des-[40-95]重新开始再生过程，结果表明这两种中间体都直接位于再生途径上。
