INFLUENCE OF THE ZN(II) COFACTOR ON THE REFOLDING OF BOVINE CARBONICANHYDRASE

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。该项目使用毛细管电泳跟踪球形金属蛋白牛碳酸酐酶II （BCA， E.C. 4.2.1.1）在十二烷基硫酸钠（SDS）处理下展开，在去除SDS后重新折叠，无论是否存在Zn（II）辅助因子。这项研究表明，锌（II）辅因子不是重新折叠成原生构象所必需的，它不与未折叠的蛋白质保持关联，并且不会显著改变重新折叠的速率。然而，锌（II）辅助因子的存在确实使回收蛋白质的总量增加了两倍。毛细管电泳（CE）可以根据天然蛋白和变性蛋白与SDS聚集的电泳迁移率（¿）的差异来区分天然蛋白和变性蛋白。此外，通过亲和毛细管电泳（ACE）观察BCA与带电芳基磺酰胺的结合来探测活性位点。这些研究为未来使用BCA作为模型来研究蛋白质与SDS之间相互作用的物理有机研究奠定了基础。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project used capillary electrophoresis to follow a globular metalloprotein, bovine carbonic anhydrase II (BCA, E.C. 4.2.1.1), on unfolding upon treatment with sodium dodecyl sulfate (SDS), and refolding upon removal of SDS, both in the presence and the absence of its Zn(II) cofactor. This research demonstrated that the Zn(II) cofactor is not required for refolding into a native-like conformation, does not remain associated with the unfolded protein, and does not significantly change the rate of refolding. The presence of the Zn(II) cofactor, however, did increase the total amount of recovered protein by a factor of two. Capillary electrophoresis (CE) could distinguish between native and denatured protein, based on the difference in electrophoretic mobility (¿) between the native protein and the aggregate of denatured protein and SDS. In addition, the active site was probed by observing binding of BCA to a charged arylsulfonamide using affinity capillary electrophoresis (ACE). These studies provide a foundation for future physical-organic studies using BCA as a model to examine interactions between proteins and SDS.