Isolation of domain-sized fragments of bovine serum albumin by limited peptic digestion and their secondary structural changes in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate

Abstract Three fragments of bovine serum albumin (BSA) by limited peptic hydrolysis were isolated and their positions in the parent molecule were identified on the basis of the analyses of plural amino acid residues at N- and C-terminals of each fragment. The sequences of the three fragments correspond to residue Nos. 1–306, 193–368, and 383–511. The molecular weights of the three fragments were determined to be 34,900, 20,100, and 14,700 from their sequences. As is well known, BSA consists of three domains, each comprising three subdomains (disulfide double loops) and containing about 190 amino acid residues with a molecular weight of approximately 22,000. The present three fragments approximately corresponded to regions representing 1.5, 1, and 0.5 domains of BSA based on their positions in the parent molecule and their molecular weights. The relative proportions of a-helix, β-structure, and disordered structure in conformations of the fragments were determined by the curve-fitting method of circular dichroism (CD) spectra. The a-helical portions occupied 50–60% in these fragments. Most of the helices in the intact BSA molecule appeared to remain after digestion into the fragments. The helical proportions of the fragments decreased stepwise in SDS solutions in the same way as intact BSA. This suggests that the decrease in helical content of BSA might represent the sum of the reductions in helical content of the three domains by SDS binding. The helices in the fragments resisted structural changes by dilute urea and guanidine in a manner similar to that of BSA.