A synthetic peptide consisting of the aminoacid sequence of residues 64-82 of lysozyme, with alanine replacing cysteine as residue 76, was prepared by the solid-phase technique. Mild reduction followed by reoxidation in air of the deprotected peptide led to the formation of a closed loop containing an intrachain disulfide bond. A conjugate consisting of this "loop" attached to multi-poly(DL-alanyl)-poly(L-lysine) elicited, in rabbits and goats, the formation of antibodies capable of reacting with lysozyme and with the loop peptide prepared from it. These immunological interactions can be inhibited by either lysozyme or the loop peptide, but not by the performic acid-oxidized open-chain peptide. Thus, the antibodies elicited by the completely synthetic antigen show specificity toward the "loop" structure (residues 64-80) of native lysozyme.
The "loop') peptide region of hen egg-white lysozyme was labeled with the dansyl group. The binding of the labeled peptide to the anti-"loop" antibodies led to a specific excitation energy transfer from the antibody to the dansyl group on the hapten. This sensitized fluorescence was used for quantitative evaluation of the binding and homogeneity parameters of the "loop"-anti-"loop" interaction. The association constant observed was 3.2 x lo6 M-l and the homogeneity index interpreted as a result of excitation energy transfer to these haptens, and was used for quantitative studies of the affinity and heterogeneity of the antibodies.More recently, antibodies to the dansyl group have also been prepared [13], and the fluorescence behaviour of the antibody-bound hapten yielded information about the nature of the environment of the binding site [14]. I n these studies the antibody specificity was directed to determinants in which the dansyl group played an immunodominant role.Unusual Abbreviations. Dansyl, l-dimethylaminonaphthalene-5-sulfonyl; "loop", amino acid sequence 60 to 83 of lysozyme, containing a disulfide bridge between half-cystine residues 64 and 80.Enzyme. Lysozyme or mucopeptide N-acetylneuraminylhydrolase (EC 3.2.1.17).
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