Holzman T F, Brems D N, Dougherty J J
Biochemistry. 1986 Nov 4;25(22):6907-17. doi: 10.1021/bi00370a026.
In order to determine solution conditions appropriate for reoxidizing reduced bovine growth hormone (bGH), we have examined the possibility of using a particular denaturant concentration to poise the secondary and tertiary structure of the reduced protein in a stable, nativelike state. It was envisioned that the structure of the reduced molecule would differ from that of the final oxidized molecule solely by the absence of disulfide bonds. Dilution of concentrated samples of reduced and unfolded protein from 6.0 M guanidine into 4.5 M urea followed by air oxidation indicated it was possible to induce refolding and reoxidation to an oxidized monomeric species in high yield (approximately 90%). The choice of solution conditions was based on comparison of urea equilibrium denaturation data for native oxidized protein to those for completely reduced protein and to protein in which sulfhydryl groups had been either partially or completely reduced and subjected to modification with iodoacetamide or methyl methanethiolsulfonate. The denaturation behavior of these species supports the existence of equilibrium folding intermediates for bovine growth hormone and demonstrates that chemical modification of the protein is capable of inducing differences in the denaturation behavior of these intermediates. The changes in the protein absorption spectrum and helix-related circular dichroism signal, along with direct titration of protein sulfhydryl groups, indicated that the refolding/reoxidation of bGH is a multistate process. The ordered nature of the kinetic changes in these probes during reoxidation indicates that disulfide formation is a sequential process, with little mispairing in 4.5 M urea, and that it proceeds through one or more obligatory kinetic folding events. The equilibrium denaturation behavior of the oxidized molecule and the various chemically modified forms, together with the reoxidation data, indicated that the protein maintains a high degree of secondary structure without intrachain disulfide bonds. The formation of these disulfide bonds is a discrete process which occurs after a framework of protein secondary structure is established.
为了确定适合使还原型牛生长激素（bGH）重新氧化的溶液条件，我们研究了使用特定变性剂浓度使还原型蛋白质的二级和三级结构保持在稳定的、类似天然状态的可能性。据设想，还原型分子的结构与最终氧化型分子的结构差异仅在于不存在二硫键。将浓缩的还原型和未折叠型蛋白质样品从6.0 M胍稀释到4.5 M尿素中，然后进行空气氧化，结果表明有可能以高产率（约90%）诱导其重新折叠和重新氧化为氧化型单体形式。溶液条件的选择基于对天然氧化型蛋白质、完全还原型蛋白质以及巯基部分或完全还原并经碘乙酰胺或甲硫醇磺酸甲酯修饰的蛋白质的尿素平衡变性数据的比较。这些蛋白质的变性行为支持了牛生长激素存在平衡折叠中间体，并表明蛋白质的化学修饰能够诱导这些中间体变性行为的差异。蛋白质吸收光谱和与螺旋相关的圆二色性信号的变化，以及蛋白质巯基的直接滴定，表明bGH的重新折叠/重新氧化是一个多态过程。这些探针在重新氧化过程中动力学变化的有序性质表明，二硫键的形成是一个顺序过程，在4.5 M尿素中错配很少，并且它通过一个或多个强制性的动力学折叠事件进行。氧化型分子和各种化学修饰形式的平衡变性行为以及重新氧化数据表明，该蛋白质在没有链内二硫键的情况下保持高度的二级结构。这些二硫键的形成是一个离散过程，发生在蛋白质二级结构框架建立之后。
