Investigation of the antigen-antibody rection by fluorescence polarization: Measurement of the effect of the fluorescent label upon the bovine serum albumin (BSA) anti-BSA equilibrium

Abstract

The concept of utilizing labeled molecules as tracers to elicidate chemical mechanisms has played a predominant role in the development of modern chemistry and biology. In the area of macromolecules, fluorescence labeling has been particularly valuable as, for example, in the measurement of absolute values of rotational relaxation times, in the detection of conformational changes as reflected in local brownian motion, in the characterization of macromolecular equilibria and kinetics and in the fluorescent antibody technique.In all of the above instances, as a result of the labeling (i.e. the environment during the labeling procedure and the mere presence of the label itself), it is the behavior and properties of the labeled and hence altered molecule that are being studied. The actual importance of any such alterations may be assessed by comparing experimetally the properties of the native molecule with those of the labeled one.The present paper deals with this problem as it relates to macromolecular reactions. A theoretical and experimental approach has been devised for measuring the extent of the effect of the label on macromolecular equilibria, and for computing the value of the equilibrium constant when the label is not present. Experimental results for the system dansyl-labeled BSA and rabbit anti-BSA have been found to be of the form predicted by the theory. For this system, the presence of 2.7 dansyl residues per BSA molecule lower the association constant from an average value of 3.7 × 108M−1 (for the unlabeled, native molecule) to 2.1 × 108M−1.