Investigation of antigen-antibody kinetics by fluorescence polarization.

Abstract Fluorescence labeling techniques afford a direct, general approach to the study of biologically important macromolecular reactions. In particular, fluorescence polarization methods have previously provided direct equilibrium measurements for antigen-antibody systems. Kinetic studies utilizing fluorescence polarization have now been initiated and have led to detailed information concerning the elementary steps of combination for fluorescein-labeled ovalbumin (AG) and antiovalbumin (AB). Fluorescence polarization changes were measured directly as functions of time for reaction mixtures at 1·5 ± 0·5° in neutral Tris buffer containing 0·15 M NaCl with the use of a specially designed fluorescence polarometer. Initial rate and integrated rate studies indicated that the empirical rate law in the initial stages of combination is Rate= k (AB) (AG) This law was obeyed over wide ranges of initial antibody concentrations (5·5 × 10 −9 to 1·8 × 10 −7 M ) and initial antigen concentrations (8·0 × 10 −10 to 8·0 × 10 −8 M ). The mechanism proposed involves the reversible bimolecular combination fo AG and AB. The rate constants for association and dissociation were determined to be 2 × 10 5 M −1 sec −1 and approximately 1 × 10 −3 sec −1 , respectively. Deviations from the rate law during latter stages of reaction can be attributed to heterogeneity of combining sites.