Immunochemistry of serum albumin—II: Isolation and characterization of a fragment from the first third bovine serum albumin carrying almost all the antigenic reactivity of the protein

Abstract

Previously we had introduced a novel approach for obtaining fragments from tight (i.e. disulfide-containing, proteolytically inaccessible) proteins. Reversible masking of the amino groups by citraconic anhydride induces in these proteins conformational changes which renders them readily accessible to tryptic cleavage at the arginine peptide bonds. After unmasking of the amino groups, cleavage at the lysyl residues can be achieved, if desired, thus yieling the tryptic peptides with intact disulfide bonds. In the present work, bovine serum albumin (BSA) was subjected by this procedure to cleavage at the arginine residues (giving the Arg-peptides) or at the arginine as well as the lysine residues (i.e. giving the (Arg, Lys)-peptides). The Arg-peptides inhibited 83% the reaction of BSA with its antisera and 97% its reaction with the IgG fraction of the antisera. The (Arg-Lys)-peptides had no (0%) inhibitory activity. A homogeneous fragment was isolated from the Arg-peptides by gel filtration on Sephadex G-100. Its purity was established by disc electrophoresis and its mol. wt was 25,000 by gel filtration on a calibrated Sephadex column and 23,000 by SDS-electrophoresis. From its amino acid composition and N-terminal sequence determination of the first 14 residues, it was possible to assign the location of this fragment to sequence 11–193 (less Arg 144) of BSA. Its mol. wt, calculated from its sequence, was 20,947. This fragment had an inhibitory activity of 80% towards the precipitin reaction of BSA with its antiseraa and 88% towards the reaction of BSA with the IgG fraction of the antisera. An immunoabsorbent of the peptide held 84–89% of the total anti-BSA antibody. By fluorescence conjugation and co-elution with antibody in gel filtration, two moles of antibody were bound per mole of peptide. Intactness of the disulfide bonds was essential for maintenance of the inhibitory activity of the peptide. Its activity was completely destroyed on rupture of the disulfide bonds by performic acid oxidation or by reduction followed by carboxymethylation. Also, cleavage at the lysyl peptide bonds, without rupture of the disulfides, destroyed the inhibitory activity of the peptide, due to scission of the antigenic reactive sites. Since the peptide comprised less than a third of the BSA molecule but accounted for almost all the BSA antigenic reactivity, it was concluded that native BSA carried repeating identical antigenic reactive sites.