Abstract

Although many salts denature proteins (e.g. guanidine hydrochloride and guanidine thiocyanate) or stabilize them (e.g. guanidine sulfate), not much is known about salt-protein interactions at the molecular level. Using Fourier transform infrared spectroscopy (FTIR) and molecular modeling, the interaction of guanidinium salts with homopolypeptides was investigated. Evidence is presented in this thesis that these salts interact with the polypeptide side chains and not with the backbone and a model of this interaction is proposed. The temperature-dependent unfolding of horseradish peroxidase (HRPC) in the ferric and ferrous states is monitored through FTIR, two dimensional-FTIR (2D-FTIR), circular dichroism (CD) and fluorescence spectroscopies. Reduction of Fe(III) to Fe(II) in HRPC results in the appearance of high intensity aggregation bands (1616cm -1 and 1684cm -1 ), which disappear at higher temperatures. High-resolution 2D-FTIR spectroscopy shows aggregation bands in ferric HRPC not previously observed by 1D-FTIR spectroscopy. The unfolding sequence of HRPC in both states is described and a mechanism underlying the reversible aggregation phenomena is proposed. This illustrates the usefulness of combining spectroscopy and molecular modeling in gaining molecular level information.