[Chromatography and rechromatography in high-performance liquid chromatography of peptide mixtures: the complete primary structure of an immunoglobulin L-chain of kappa-type, subgroup I (Bence-Jones Protein Den) (author's transl)].

In continuation of our work on the separation of enzymatic hydrolysates by high-pressure liquid chromatography with reverse-phases we present two new buffer systems: firstly 0.005M potassium phosphate, pH 6.0 and secondly 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 Bence-Jones protein Den (Mr = 23000). Like ammoniumacetate these buffers are well suited for the first separation of peptide mixtures but can be used rather effectively in rechromatographies. If peaks are not or not fully resolved during the first chromatography they can be separated by a rechromatography in one 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 one rechromatography. The amino acid analysis yielded also integer numbers and by a modified Edman degradation the primary structure could be determined. As with protein Wes (Kratzin, H., Yang, C.Y., Krusche, T.U. & Hilschmann, N. (1980) Hoppe-Seyler's Z. Physiol. Chem. 361, 1591--1598) all the peptides were recovered after high-pressure liquid chromatography, none was missing. From these data and from homology reasons the complete amino acid sequence of protein Den could be established. It contains 214 residues and belongs to subgroup I of the kappa-chains. The valine residue in position 191 indicates that it belongs to allotype Inv b+.