Abstract
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.
Highlights
To avoid oxidation of the cysteine residue during cyanogen bromide cleavage, the albumin preparations used in this work were converted to the mixed disulfide form by reaction with L-cystine or to the S-carboxamidomethylated derivative with iodoacetamide
Isolation of Cyanogen Bromide Fragments of Bovine Plasma Albumin-Five peptides should result on cyanogen bromide cleavage of BPA, since albumin contains 4 methioninyl residues
There was significantly more of the materials eluted in front of Fragment C when the cleavage was done in HCl rather than in formic acid
Summary
Amino acid analyses were made on a Beckman-Spinco model 120-B analyzer after hydrolysis of the samples in 6 N HCl at 110” for 22 and 72 hours [7]. Amino acid analyses were made on a Beckman-. For good separation of the homoserine lactone and ammonia peaks, a short column (15 cm) was used; for resolution of homoserine and glutamic acid peaks, analyses on the long column (55 cm length) were started at 48”, and the temperature was changed to the normal operating value of 55” at the position of threonine peak. For. 22-hour hydrolysates, the following decomposition factors were used for carboxymethylcysteine, threonine, cystine, and serine: 3%, 5%,, 7%, and lo%, respectively [7]. The dansyl amino acids were identified by chromatography on polyamide sheets [12], with the exception of dansyl-arginine which was identified on paper electrophoresis [10] and verified by Sakaguchi reaction
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