Chromatographie und Rechromatographie in der Hochdruckflüssigkeitschromatographie von Peptidgemischen. Die vollständige Primärstruktur einer Immunglobulin L-Kette vom κ-Typ, Subgruppe I (Bence-Jones-Protein Den)

Abstract

In the separation of enzymatic hydrolysates by high-pressure liquid chromatography with reverse-phases 2 new buffer systems are presented: 0.005 M potassium phosphate, pH 6.0 and aqueous trifluoroacetic acid, pH 2.15. As organic solvent, acetonitrile is always used. The excellent properties are demonstrated by the separation of the tryptic peptides of human Bence-Jones protein Den (MW = 23,000). Like ammonium acetate these buffers are well suited for the 1st separation of peptide mixtures but can be used rather effectively in rechromatographies. If peaks are not or not fully resolved during the 1st chromatography they can be separated by a rechromatography in 1 of the other systems. This was most successful since in both cases the same high resolving technique is employed. From the primary separation 14 peptides could be isolated in analytically pure form. The remaining fragments were completely purified after 1 rechromatography. The amino acid analysis yielded integer numbers and by a modified Edman degradation the primary structure could be determined. As with protein Wes, all the peptides were recovered after high-pressure liquid chromatography. The complete amino acid sequence of protein Den was established. It contains 214 residues and belongs to subgroup I of the k-chains. The valine residue in position 191 indicates that it belongs to allotype Inv b+.