Boundary refinement of the lysozyme antigenic site around the disulphide bond 6-127 (site 1) by 'surface-simulation'...

1. We have previously shown that an antigenic site (site 1) in native lysozyme resides around the disulphide bond 6-127 and, by classical synthesis of nine disulphide peptides, the antigenic site was accurately narrowed down to the structure Cys((6))-Arg((14))-[Cys((6))-Cys((127))] -Gly((126))-Arg((128)). Only a few residues on this disulphide peptide were proposed to be involved in the reactivity with antibody. However, this lacked direct verification and the role of Arg-128 remained uncertain. 2. In the present work, several peptides were designed and synthesized by the surface-simulation concept devised in our laboratory. These enabled the precise definition of the site as well as the investigation of its conformational and directional requirements. 3. The results showed that the antigenic site (site 1) is made up of the spatially contiguous surface residues: Arg-125, Arg-5, Glu-7, Arg-14, Lys-13. The surface-simulation synthetic peptide Arg-Gly-Gly-Arg-Gly-Glu-Gly-Gly-Arg-Lys (which does not exist in native lysozyme, but copies a surface region of it) accounted entirely for the maximum expected reactivity of the site (i.e. about one-third of the total antigenic reactivity of lysozyme). An immunoadsorbent of the peptide also removed about one-third of the total lysozyme antibodies. 4. The antigenic site exhibited restricted conformational freedom. The achievement of the full reactivity of the site by surface-simulation synthesis requires the appropriate choice of spacer separation between its reactive residues. The surface-simulation synthetic site exhibits the same mono-directional preference (Arg-125 to Lys-13) for the rabbit and goat antisera so far tested. The site describes a line which encircles a part (3.01 nm in C((alpha))-to-C((alpha)) distance from Arg-125 to Lys-13) of the surface of the molecule.