[Three-dimensional structure determination of antibodies. Primary structure of crystallized monoclonal immunoglobulin IgG1 KOL, I].

The crystallizable myeloma immunoglobulin IgG1 KOL [allotype Gm(-1,4), (gamma 1, gamma)2] which is well characterized in its three-dimensional structure by X-ray diffraction analysis of high resolution has been proved to be homogenous by polyacrylamide gel electrophoresis. The H- and L-chains were separated by gel filtration after complete reduction and carboxymethylation and were characterized by amino acid analysis, end group determination and polyacrylamide gel electrophoresis, respectively. The intact IgG1 KOL was cleaved by cyanogen bromide and all CNBr-fragments were isolated and characterized. The reduced and carboxymethylated H-chain was digested by trypsin and the tryptic hydrolysate was separated by ion-exchange chromatography. Using different procedures of rechromatography 35 out of 37 tryptic H-chain peptides could be isolated in sufficient amounts, the missing 2 peptides were produced by tryptic digestion of 2 CNBr-fragments. The amino acid sequences of all tryptic peptides were determined using a modified Edman degradation method after separation of the enzymatic cleavage products by high-performance liquid chromatography (HPLC). The complete primary structure of the VH-part of the H-chain was established by isolation and partial sequence determination of overlapping peptides obtained from cleavage of the intact H-chain by Staphylococcus aureus proteinase. The gamma 1-H-chain KOL comprises 455 amino acid residues and belongs to subgroup III. The switch from the variable to the constant part occurs at position 126/127, thus making VH-KOL one of the longest variable parts among the yet known immunoglobulin H-chains. This is due to the hypervariable region Hhv4 which is made up by 17 amino acid residues (4-9 residues more compared with other VH-parts). Within this region a so far not described additional intrapeptidal disulfide bridge could be localized (Cys 105-Cys 110) that creates a short loop with antiparallel running peptide strains in beta-pleated sheet conformation. Its role in the three-dimensional structure of the antigen-binding site of the IgG1 KOL molecule is discussed using the data obtained from X-ray diffraction analysis.