The amino-acid compositions of CNBr fragment C1 from antihapten antibodies. Use of guanidine for reproducible isolation of the C1 fragment.

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Most rabbit antibodies contain a methionine residue about 240 residues from the NH2‐terminus of the heavy chain sequence. Cleavage of heavy chains by CNBr at this methionine leads to the formation of a fragment, C1, which contains the variable portion of the H‐chain but is slightly longer than the Fd fragment. Published amino acid compositions of fractions from different antibodies, all taken to represent fragment C1, showed marked similarity. In this work it is shown that the primary reason for this similarity is that previous methods of isolating C1, by gel filtration using 6 M urea or 1 M acetic acid as solvent, led to the isolation of aggregates contaminated with an extensive section of the constant region. Six M guanidine · HCl prevented the formation of these aggregates and gel filtration in this solvent yielded a C1 fragment with an amino acid composition quite different from that of the contaminated aggregate. The amino acid compositions of the uncontaminated C1 fragments from antihapten antibodies from individual rabbits showed large differences. The differences were apparent because of the restricted heterogeneity of the antibodies being compared. Amino acid compositions of C1 fragments from γ‐globulins from individual uninjected rabbits showed appreciably less variation because, unlike the antibodies, the γ‐globulins were complex mixtures.