EXCITATION ENERGY TRANSFER AND THE QUANTITATIVE STUDY OF THE ANTIBODY HAPTEN REACTION

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Abstract
The tryptophane flourescence of proteins is quenched in complexes or conjugates of the proteins with molecules that have absorption in the 300 to 400 mu region. This phenomenon, applied previously to the study of enzyme coenzyme complexes is also applicable to antibody complexes with appropriate antigens or haptens. The fluorescence quantum yield of the antibody directed against the 2,4-dinitrophenyl group (DNP) is diminished about 70% by the binding of [epsilon]-N-DNP-lysine and serves as a sensitive indicator of complex formation. The binding stoichiometry is 2 moles of hapten per mole of purified antibody and the dissociation constant at 7[degree] is 2.4 10-9 [image]. Dissociation constants of this magnitude are not approachable by other methods. Hapten binding has been studied over the pH range 1.5 to 11 and over the temperature range 7[degree] to 60[degree]. [DELTA]H[degree]and [DELTA]F[degree]for complex formation are -11.3 and -8.6 kcal mole-1 and [DELTA]s[degree]is 9 eu. Weaker binding of some homologous univalent haptens and the formation of linear aggregates with a divalent hapten are described. It is established by direct titration that the soluble fragments I and II produced by papain proteolysis of the purified antibody, are univalent antibody pieces and that the crystalline fragment III, prepared by Porter''s method, carries no hapten binding site. The univalent antibody fragments may be titrated quantitatively, by the fluorometric method, with intact multivalent DNP antigen since they form soluble complexes in which the flourescence both of the antibody and antigen is quenched by the DNP ligands on the antigen. The binding sites of the purified anti-DNP antibody exhibit a high degree of homogeneity in their interactions with haptens but the statistical inhomogeneities, in reversible electrophoretic boundary spreading are of the same magnitude as those of nonspecific [gamma]-globulin. The fluorescence quenching method is also applied to the study of hapten interactions of an antibody directed against derivatives of p-azophenylarsonate. In the quenching interaction within the hapten complex each molecule of bound DNP captures the excitation energy of 8 to 9 tryptophane residues. These groups of tryptophane residues of the antibody lie within separate domains which do not overlap and the boundaries of which coincide roughly with the points of proteolytic cleavage by papain.