Amino Acid Sequence in Lysozyme

The previous paper in this series (Thompson, 1955) reported some amino acid sequences in lysozyme which were deduced from the structures of a number of small peptides isolated from partial acid hydrolysates by displacement chromatography on ion-exchange resins and further fractionation on paper chromatograms. Despite the success achieved by Partridge and co-workers (see Partridge & Brimley, 1952) in the separation of amino acids by displacement chromatography and the high capacity of such columns, it was apparent in these experiments that the relatively non-specific van der Waals interactions between the larger peptide molecules and the resin outweighed the effect of differences in the pK values of their ionizing groups, with the result that many peptides were similarly absorbed and thus not differentiated. Nevertheless, a large number of peptides were isolated and identified in these experiments. Experiments using elution chromatography on ion-exchange resins for the preliminary separation of peptides from an acid hydrolysate of lysozyme are described in this paper. The high resolving power of this method for amino acids was elegantly demonstrated by Moore & Stein (1951). Dowmont & Fruton (1952) have shown that a number of simple peptides may be satisfactorily chromatographed in this way. Schroeder, Kay, Le Gette, Honnen & Green (1954) have chromatographed a partial acid hydrolysate of gelatin in Dowex-50 x 8 and examined the material in the eluate. After further resolution of the peptides as N-2:4-dini-trophenyl (DNP) derivatives on silicic acid-Celite columns a number of diand tri-peptides were identified in gelatin hydrolysates. A similar examination was made of silk-fibroin hydrolysates by Kay & Schroeder (1954). This procedure has the advantage that no desalting step is necessary to remove buffer salts from the eluant fractions. The present experiments have borne out the expectation that the elution procedure would possess the highest general resolving power of any method yet developed for the separation of