Studies on a human 19-S immunoglobulin M The arrangement of inter-chain disulphide bridges and carbohydrate sites

Abstract

1. 1. Amino acid analysis of carboxymethylated and aminoethylated polypeptide chains from a human 19-S immunoglobulin M has shown that 1 cysteine residue per light chain and 4 cysteine residues per heavy chain are most probably involved in the inter-chain disulphide bridges. This has been substantiated by the isolation and amino acid analysis of a single-labelled peptide from the [ 14C] carboxymethylated light chain and 4 labelled peptides from the [ 14C]carboxymethylated heavy chain. Each of these peptides contains 1 residue of carboxymethylcysteine. Analogous peptides have also been isolated from oxidized IgM and contain 1 residue of cysteic acid in each case. 2. 2. The results show that 1 disulphide bridge links a light chain to a heavy chain and that each heavy chain is most probably linked to other heavy chains by 3 disulphide bridges. 3. 3. The peptides arising from the 3 inter-heavy chain bridges are also glycopeptides, and a fourth glycopeptide has been isolated. This indicates that the heavy chain has at least 4 major carbohydrate sites. 4. 4. Treatment of the human 19-S immunoglobulin M and its reduced and alkylated subunits, with trypsin, produces 3.6-S Fab u, 6.3-S F(ab′) 2u and 4.5-S Fab′ u fragments. Immunochemical and peptide analysis has shown that the Fab u fragment consists of a light (κ) polypeptide chain linked by a single disulphide bridge to a piece (Fd u) of the heavy (μ) polypeptide chain. The F(ab′) 2u fragment consists of 2 Fab′ u fragments linked by a single inter-heavy chain disulphide bridge. Each Fab′ u fragment contains a κ-chain linked by a single disulphide bridge to a piece (Fd′ u) of the μ-chain that is slightly larger than the Fd u. 5. 5. Treatment of reduced and alkylated subunits with papain in the presence of cysteine produces Fab u and Fc u fragments. Immunochemical and peptide analysis of the Fc u fragment has shown that it consists of a part of the μ-chain which is distinct from that in the Fab′ fragment. Two inter-heavy chain disulphide bridges have been found in the Fc u, one of which forms the linkage between subunits. 6. 6. The Fd u part of the Fab u fragment contains a major site of carbohydrate and the Fd′ u part of the F(ab′) 2u fragment contains an additional carbohydrate site situated near the inter-heavy chain disulphide bridge. The Fc u contains 2 further carbohydrate sites also situated near the inter-heavy chain disulphide bridges.