Amino acid sequence in lysozyme. I. Displacement chromatography of peptides from a partial hydrolysate on ion-exchange resins.

It has been shown (Fraenkel-Conrat, 1950) that the enzymic activity of lysozyme depends upon the presence of some of its amino, carboxyl and hydroxyl groups. A study of the structure of the lysozyme molecule as awhole has been undertaken in an approach to the problem of defining that portion of the molecule which is concerned in its activity. The lysozyme molecule probably consists of a single polypeptide chain (Fraenkel-Conrat, Mohammad, Ducay & Mecham, 1951) internally crosslinked by disulphide bridges. The N-terminal residue has been shown to be lysine (Green & Schroeder, 1951; Thompson, 1951a) by the fluorodinitrobenzene method (Sanger, 1945), and the C-terminal residue leucine with the enzyme carboxypeptidase (Thompson, 1952a). A sequence of four residues at the N-terminal end of the chain (Lys. Val . Phe . Gly) has been established by Schroeder (1952) by a study of the 2:4-dinitrophenyl-(DNP-)peptides liberated on hydrolysis of DNP-lysozyme. This sequence has been confirmed by Landmann, Drake & Dillaha (1953), who reported that the fifth residue is serine, and by R. Acher & J. Thaureaux, whose results indicate, however, that arginine is the fifth residue (private communication from Professor C. Fromageot, 1953). A tripeptide sequence Arg. His. Lys and two dipeptides containing tyrosine have also been identified in partial hydrolysates by Acher, Thaureaux, Crocker & Fromageot (1952). The successful investigation of the sequence of amino acids in fractions A and B of oxidized insulin (Sanger & Tuppy, 1951 a, b; Sanger & Thompson, 1953a, b) suggested that a considerable portion of the sequence in lysozyme could be elucidated by similar methods. The most generally useful method for degrading proteins to smaller peptides is partial hydrolysis with concentrated acid at low temperatures (Synge, 1943). Since the lysozyme molecule contains approximately 130 amino acid residues, the mixture of peptides produced on partial hydrolysis would be expected to be much more complicated than that from either of the insulin chains. Obviously a method for the preliminary separation of the peptides into fractions as well defined as possible was necessary. A preliminary experiment in which the peptides were separated into groups by ionophoresis on filter paper had indicated the complexity of the fractions so obtained. The successful separation ofamino acids by means of displacement chromatography on ion-exchange resins (Partridge & Westall, 1949; Partridge, 1949; Partridge, Brimley & Pepper, 1950; Partridge & Brimley, 1951a, b, 1952) suggested the use of such columns for fractionation of the complex mixture of peptides in a protein hydrolysate. The high capacity of these columns is an obvious advantage. The behaviour of peptides on ion-exchange columns had not previously been studied. But on the assumptions that the order of displacement of peptides is governed by the pK value of the ionizing group as is the case with amino acids (Partridge & Brimley, 1951 b), and that other factors are not much involved, it was envisaged that effluent fractions from the column would be considerably simpler than the original peptide mixture. The two experiments described in this paper involving displacement chromatography on ionexchange resins are exploratory and do not necessarily give the best conditions for obtaining final fractions for subsequent identification of the peptides. Nevertheless, a large number of peptides was isolated from these experiments. It was possible to determine the structure of a number of them and finally deduce certain of the amino acid sequences in lysozyme. These experiments showed, however, that a method of much higher resolving power is required for preliminary fractionation of the peptides in partial hydrolysates of proteins. Further experiments have shown that elution chromatography on ion-exchange resins (cf. Moore & Stein, 1951; Dowmont & Fruton, 1952) is very successful for this purpose and will be described in