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

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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.