Beitrag zur dreidimensionalen Strukturaufklärung der Antikörper. Die Primärstruktur des kristallisierbaren monoklonalen Immunglobulins IgG1 KOL, I

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Abstract
The crystallizable [human] myeloma IgG1 KOL [allotype Gm(-1,4),(.gamma.1,.lambda.)2] which is well characterized in its 3-dimensional structure by X-ray diffraction analysis of high resolution was 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 CNBr 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 of 37 tryptic H-chain peptides were 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, making VH-KOL one of the longest variable parts among the yet known Ig 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 was localized (Cys 105-Cys 110) that creates a short loop with antiparallel running peptide strains in .beta.-pleated sheet conformation. Its role in the 3-dimensional structure of the antigen-binding site of the IgG1 KOL molecule is discussed using data obtained from X-ray diffraction analysis.

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