EFFECT OF PHOSPHATE ION ON OVALBUMIN-ANTIOVALBUMIN KINETICS AS MEASURED BY FLUORESCENCE POLARIZATION AND QUENCHING TECHNIQUES

The initial steps of the antigen-antibody reaction involve combinations between macromolecular ions, and therefore should be affected by changes in the surrounding ionic medium. Accordingly, for the reaction between ovalbumin (AG) and antiovalbumin (AB), at neutral pH and 1·5±0·5°, initial rate and half-time methods utilizing fluorescence labeling techniques have shown the rat law to be different in buffers containing phosphate, compared to those containing tris. In the presence of phosphate, the empirical rate law is - d(AG) dt= k″(AB)0·6(AG) whereas in tris, the rate law is - d(AG) dt= k′(AB)(AG). Immunospecific purification of the antibody of the use of a dansyl label instead of fluorescein (on the antigen) did not alter the fractional rate law. One mechanism compatible with the observed fractional order involves formation of a macromolecular encounter pair between the antibody and antigen, which rearranges prior to formation of the primary bonds of the antigen-antibody complex. A further necessary assumption for this mechanism is that the sites are non-uniform, and that a distribution of binding free energies is involved in the encounter pair formation. An alternative mechanism is proposed which involves at least a two-step process in the combination of antigen and antibody. Prior to a slow rate-determining step between the antigen and the active monomeric antibody, there would be a rapid, reversible dimerization of antibody. © 1969.