Secondary structural changes of N-bromosuccinimide-cleaved bovine serum albumin in solutions of sodium dodecyl sulfate, urea, and guanidine hydrochloride

Abstract

Two tryptophyl peptide bonds of bovine serum albumin (BSA), Trp 134-Gly 135 and Trp 212-Ser 213, were cleaved with N-bromosuccinimide (cleaved BSA). The relative extents of secondary structures in cleaved BSA were determined by curve-fitting of the circular dichroism spectrum. Cleavage at these two sites caused an appreciable disruption in the helicity of the protein. The cleaved BSA contained 55% α-helical structure as against 66% for the intact BSA. The cleavage also made the helical structures unstable in solutions of urea and guanidine hydrochloride. The extent of α-helical structure of cleaved BSA decreased to 50% in sodium dodecyl sulfate (SDS) solutions. However, some moieties, the helical structures of which had been disrupted by N-bromosuccinimide cleavage, appeared to re-form the helical structure in SDS, although the cleavage effect remained through the urea and guanidine denaturations and also through the thermal denaturation. On the other hand, the three resultant polypeptides, Asp 1-Trp 134, Gly 135-Trp 212, and Ser 213-Ala 582, were separated by reduction of the disulfide bridges joining them. These three separated polypeptide fragments showed only 20% helix content. However, the extent of the helical structure in the three polypeptide fragments increased to 35–40% in SDS. Secondary structural changes in the three fragments were also examined in urea and guanidine hydrochloride solutions.