Nanosecond fluorescence spectroscopy of pyrenebutyrate anti-pyrene antibody complexes

The utility of the long-lived fluorophore, pyrene, as a probe in nanosecond fluorescence depolarization measurements was investigated using pyrenebutyrate bound in the combining sites of rabbit antipyrenebutyrate immunoglobulin G. The time dependent anisotropy decay data points showed very little scatter in the time interval 0-350 ns, which is more than three times the comparable time interval observed with epsilon-1-dimethylamino-5-naphthalenesulfonyllysine (DNS-lysine) bound in the combining sites of anti-DNS antibodies [Holowka, D.A., and Cathou, R.E. (1976), Biochemistry 15, 3379]. Thus, the use of pyrene can significantly extend the range of macromolecular rotational correlation times that can be measured by the single photon technique. In the present investigation, we confirmed the presence of Fab segmental fexibility in immunoglobulin G molecules specific for a hapten different from DNS-lysine. We obtained a value of about 135 ns for the longer rotational correlation time which probably represents global rotation of the entire molecule. In the course of these experiments, we have also found that the combining sites of antipyrenebutyrate antibodies are, as expected, relatively nonpolar.