Hapten-antibody interactions as determined by fluorescence polarization

Abstract

A new method of data analysis has been developed to analyze hapten-antibody binding reactions using the results of intensity and polarization of hapten fluorescence measurements. The method was applied to the study of functional differences between water-soluble (PG) and water-insoluble (EG) globulin fractions of rabbit anti-phenylarsonate antisera. The new method of data analysis provides: (1) a correction for hapten binding nonspecifically to non-antibody globulin; (2) a more accurate determination of equilibrium constants than has previously been possible by using this correction in calculation of the fraction of antibody sites occupied as a function of hapten concentration; and (3) computation of the change of hapten fluorescence as a function of the fraction of antibody sites occupied when hapten moves from the free to the bound state. The latter computation provides a new parameter to describe heterogeneity of antibodies. Antibody was prepared against phenylarsonate. To carry out the binding measurements, a new, highly fluorescent hapten, 4-(4′-arsonophenylazo)-2,7-dichlorofluorescein (F-F10, was synthesized. It was found that although EG comprised only one-third of the total globulin, it contained almost two-thirds of the total anti-phenylarsonate antibody. This finding contrasts with observations for several anti-protein systems in which two-thirds of the antibody was found in the pseudoglobulin fraction. It was found that PG and EG antibodies have similarly valued mean association constants ( K o s = 2.1 × 10 7 M −1 for PG vs 1.5 × 10 7 M −1 for EG) but differ with respect to heterogeneity of association contants; antibodies in PG are more heterogeneous than in EG. The two antibody fractions also differ with respect to a parameter which we call average specific quenchence ( Q post ): Q post = 0.45 for EG vs Q post = 0·38 for PG . Nonspecific binding by pre-immunization PG was greater than by EG (3.5 × 10 −7 m M/mg protein vs 0.8 × 10 −8 m M/mg protein). The higher value of Q post for antibodies in EG is interpreted to imply that EG antibody combining sites are more hydrophobic than those of PG.