EFFECTS OF PROTEIN DENATURATION ON THE ROTATIONAL REORIENTATION KINETICS OF DANSYLATED BOVINE SERUM-ALBUMIN

We have previously investigated the time-resolved intensity and anisotropy decay of dansylated bovine serum albumin (BSA) using multifrequency phase and modulation fluorescence spectroscopy (J. Phys. Chem. 1993, 97, 4231-4238). The present study focuses on how protein denaturation affects the time-resolved fluorescence intensity and anisotropy decays. Specifically, we report results for the denaturation of BSA by urea and guanidine hydrochloride (Gd.HCl). The results demonstrate that the fluorescence intensity decay of dansylated BSA, native and denatured, is best described by a double exponential decay law. The short lifetime component reflects dansyl at the BSA exterior and the long component arises from dansyl located in a more hydrophobic environment. Anisotropy decay results are best described by a model with three discrete rotational reorientation times. At low denaturant concentrations, the slower motion represents the reorientational dynamics for the entire BSA molecule. Upon addition of high concentrations of denaturant, the slow motion begins to reflect some segmental mobility within the protein. The two faster rotational reorientation times are attributed to the local motion of dansyl. Without denaturant, these two motions are a manifestation of dansyl located at the BSA interface or buried within an internal hydrophobic site. As the denaturant concentration increases, we see that the rotational reorientation time for the interfacial dansyl remains unchanged. In contrast, the local motion of the internal dansyl increases substantially. Eventually, as BSA unfolds, these two motions coalesce; they cannot be distinguished from one another. The semiangle for the cone through which the dansyl group rotates increases for the internal dansyl as the BSA denatures (i.e., unfolds). The corresponding semiangle for dansyl at the exterior of BSA is not affected by the denaturant. Our experimental results also argue that Gd.HCl is a stronger denaturant compared to urea.