Enzymic and immunochemical properties of lysozyme XVI. A novel synthetic approach to an antigenic reactive site by direct linkage of the relevant conformationally adjacent residues constituting the site

Abstract

Previous studies from this laboratory on the immunochemistry of specific chemical derivatives of native lysozyme and of the two disulfide peptide 62–68 (Cys 64–Cys 80) 74–97 (Cys 76–Cys 94) (i.e.(SS) 2-peptide), have established an antigenic reactive site to comprise the spatially contiguous surface residues: Trp 62, Lys 97, Lys 96, Asn 93, Thr 89 and Asp 87. In the present work, the identity of the site was verified by an entirely different and novel approach. The aforementioned amino acids were linked directly into a single linear peptide with an intervening spacer where appropriate and substituting phenylalanine for tryptophan (i.e. Phe-Gly-Lys-Lys-Asn-Thr-Asp). This peptide (which does not exist in native lysozyme but simulates a surface region of the protein) possessed a remarkable inhibitory activity towards the reaction of lysozyme with its antisera. The immunochemical reactivity of the peptide was equal to the maximum expected reactivity of the site (i.e. a third of the total antigenic reactivity of lysozyme). These findings define quite conclusively and accurately the reactive site which is clearly composed of spatially adjacent residues that are distant in sequence reacting as if in direct linear linkage. The unequivocal establishment of this concept indicates that antigenic sites need not always be composed of residues in direct peptide linkage in the sequence. The nature of the site may depend on the protein. This unorthodox attack at the problem provides a novel and powerful approach for final delineation of the antigenic reactive sites (and perhaps other types of but we subsequently have not found it to exist in myoglobin [27], at least in early course antisera. Studies are now being carried out to determine if the present site has a “direction”. Also, the remaining two antigenic sites of lysozyme are being synthesized in a similar fashion. These results will be reported elsewhere in detail.