Enzymic and immunochemical properties of lysozyme: XIX. Definition of the direction and conformational restrictions of the antigenic site around the disulfide bonds 64–80 and 76–94 (site 2) by ‘surface-simulation’ synthesis

Abstract

We have previously shown that an antigenic reactive site is situated in native lysozyme around the disulfide bonds 64–80 and 76–94 and we also established that the site comprised the spatially contiguous surface residues: Trp-62, Lys-97, Lys-96, Asn-93, Thr-89 and Asp-87. The identity of the site was confirmed by the ‘surface-simulation’ synthetic approach that we recently devised in which the aforementioned six residues were linked directly via peptide bonds into a single peptide (Phe-Gly-Lys-Lys-Asn-Thr-Asp) with an intervening spacer where appropriate and substituting tryptophan by phenylalanine. The peptide does not exist in native lysozyme but simulates a surface region of the protein. In the present work, several surface-simulation synthetic peptides were synthesized in order to investigate the conformational restrictions of the site and to determine if the spatially constructed antigenic site has a preferred ‘direction’. The results showed that the above peptide possessed the maximum inhibitory activity towards the lysozyme immune reaction and, remarkably, accounted quantitatively for the maximum expected reactivity of the site (i.e. about a third of the total antigenic reactivity of lysozyme). An immunoadsorbent of the peptide also removed about a third of the total lysozyme antibodies. The antigenic site exhibited restricted conformational freedom. Deletion of the glycine spacer between phenylalanine and lysine had a vastly detrimental effect on the immunochemical reactivity of the peptide. Furthermore, when the above sequence was reversed the immunochemical reactivity of the resultant peptide was drastically decreased with goat antisera but was unchanged with rabbit antisera. The antigenic site therefore exhibits a preferred ‘direction’ towards the goat antisera and none towards the rabbit antisera tested. The site describes a line which encircles part (27.3 Å in extended C a -to-C a distance from Trp-62 to Asp-87) of the surface of the native molecule.