Characterization of fragments of human albumin purified by Cibacron blue F3GA affinity chromatography

Abstract

Controlled limited proteolysis of human plasma albumin (0.3 mM; 37 degrees C; 15 min; pH 3.7) with pepsin [pepsin/albumin, 1:1000 (w/w)] in the presence of octanoic acid (4.2 mM) yields at least 14 fragments in the range of 5000--56000 Da. By utilizing a combination of conventional and affinity-chromatographic procedures, two fragments with mol. wts. 25000 and 27000 were purified to more than 99% homogeneity. The larger fragment consists of a continuous polypeptide chain and has been shown to contain the primary bilirubin-binding site. The small fragment contains an internal cleavage site. On the basis of amino acid compositions, N-terminal sequences, C-terminal sequences, molecular weights and other internal markers the locations of these fragments within the known sequence of human albumin were determined to be residues 49--308 for the 27000 Da peptide and 309--585 for the 25000 Da peptide. Peptide 309--585 contains an internal cleavage site and appears to be missing residues 408--423. These non-overlapping fragments should be useful for investigations of individual ligand-binding sites and for the determination of antigenic sites.