The specific cleavage of immunoglobulin polypeptide chains at cysteinyl residues.

A method has been developed for fragmenting immunoglobulin light and heavy polypeptide chains at the cysteinyl residues that participate in intrachain disulfide bridges. The method involves the quantitative blockage of positive charges associated with lysyl and arginyl residues, the reduction and subsequent aminoethylation of disulfide bridges, and the treatment of aminoethylated, blocked chains with trypsin to yield the desired blocked peptide fragments. Lysine and arginine are modified by succinic anhydride and cyclohexanedione respectively, to yield stable “trypsin-insensitive” residues. The blocked peptides of heavy and light chains are separated on the basis of size by gel filtration chromatography in either 8 M urea or 0.01 M ammonium bicarbonate. The blocked peptides from a K-type Bence-Jones protein can be resolved into six chromatographic peaks. The peaks, in order of their elution, have been assigned loci within the known over-all structure of a Bence-Jones protein: E and D have not been characterized and probably represent partial digestion products; C and C’ probably correspond to regions between the two intramolecular disulfide bridges; B contains the peptide corresponding to the “switch region” as judged by amino-terminal analysis and amino acid composition; A contains the carboxyl-terminal cysteinyl residue and corresponds to peptide fragments from the amino and carboxylterminal regions of the starting Bence-Jones protein. The peptide patterns obtained from whole normal mouse and human light chain are closely similar to the BenceJones profile and strongly suggest that the disulfide bridge pattern found for Bence-Jones proteins applies to the majority of K and X chains in the whole normal light chain population. A comparison of the gel filtration patterns of a y2a mouse myeloma heavy chain and whole normal mouse heavy chains also indicates that a single heavy disulfide bridge pattern is