Enzymic and Immunochemical Properties of Lysozyme. Accurate Definition of the Antigenic Site Around the Disulphide Bridge 30-115 (site 3) by 'surface-simulation' Synthesis

1. Previous reports from this laboratory have shown that both Lys-33 and Lys-116 are parts of an antigenic site in native lysozyme. Similar studies of tyrosine derivatives indicated that one or both of Tyr-20 and Tyr-23 are located in or very close to an antigenic site in lysozyme. The site, which was located around the disulphide bridge 30-115, was recently shown unequivocally to include the residues Tyr-20, Arg-21, Lys-116, Asn-113, Arg-114, Phe-34 and Lys-33. This was confirmed by the ;surface-simulation' synthetic approach that we have recently developed, in which the foregoing eight surface residues were directly linked via peptide bonds, with intervening spacers where appropriate, into a single peptide. The peptide does not exist in native lysozyme, but simulates a surface region of it. 2. In the present work several surface-simulation peptides were synthesized representing various parts of the region, to determine the minimum structural feature that retains full antigenic reactivity and to investigate if the spatially constructed antigenic site has a preferred direction. 3. The peptide Lys-Asn-Arg-Gly-Phe-Lys exhibited a remarkable inhibitory activity towards the immune reaction of lysozyme and accounted entirely for the maximum expected reactivity of the site in the native protein (i.e. about one-third of the total lysozyme reactivity). An immunoadsorbent of the peptide bound about one-third of the total antibody to lysozyme. 4. The residues Tyr-20 and Arg-21 are not part of the site. The previously reported immunochemical effect observed on nitration of Tyr-20 was due to a deleterious ionic effect exerted by the modified tyrosine residue on the adjacent Lys-96, which is in an entirely different antigenic site of lysozyme. Thus the modification of Tyr-20 impairs the reactivity of an adjacent antigenic site, even though the residue itself is not part of a site. The conformational and immunochemical implications of this finding are discussed. 5. The antigenic site therefore comprises the five spatially adjacent residues Lys-116, Asn-113, Arg-114, Phe-34, Lys-33. The antigenic site exhibited a preferred direction (Lys-116 to Lys-33), since the reverse surface-simulation synthetic sequence was immunochemically inefficient. The site describes a line which circumscribes part [2.1nm in C((alpha))-C((alpha)) distance from Lys-116 to Lys-33] of the surface of the molecule.