Structural Studies and Organic Ligand-binding Properties of Bovine Plasma Albumin

Abstract Cyanogen bromide cleavage of bovine plasma albumin in 75% formic acid gave two fragments, N and C, in yields of 80%. After reduction of the cross-linking disulfide bonds of Fragment N, two peptides containing 88 and 98 amino acid residues were isolated. The single sulfhydryl group of albumin was found to be located in the 88 amino acid residue peptide which occupies the amino-terminal position of the molecule. After reduction of Fragment C, three peptides containing 211, 148, and 34 amino acid residues were isolated. These five peptides together account for the albumin molecule. Limited tryptic hydrolysis of defatted bovine plasma albumin at pH 8.8 and 0° gave a fragment of molecular weight of about 40,000 in a yield of 21%. The fragment is derived from the carboxyl-terminal two-thirds of the albumin molecule as shown by a comparison of its three cyanogen bromide peptides with those from albumin. These data were used for the alignment of the five cyanogen bromide peptides of albumin. Binding studies showed that the tryptic fragment and defatted albumin both had one primary site for octanoate and l-tryptophan. d-Tryptophan competitively displaced either of these two ligands from the fragment and from albumin, thus suggesting that all three ligands bind at the same site. The binding constants of the fragment for the three ligands were all about one-third of those for albumin. The ratio of the binding constants of l-and d-tryptophan for the fragment was nearly the same as that for albumin, and this was also the case for octanoate and l-tryptophan. These similarities strongly suggest that the binding site in the fragment is the one present in albumin.