Nanosecond fluorescence spectroscopy of pyrenebutyrate-anti-pyrene antibody complexes

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Abstract
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[Ig]G. The time dependent anisotropy decay data points showed very little scatter in the time interval 0-350 ns, which is more than 3 times the comparable time interval observed with .epsilon.-1-dimethylamino-5-naphthalenesulfonyllysine (DNS-lysine) bound in the combining sites of anti-DNS antibodies. 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, the presence of Fab segmental flexibility was confirmed in IgG molecules specific for a hapten different from DNS-lysine. A value of about 135 ns was obtained for the longer rotational correlation time which probably represents global rotation of the entire molecule. The combining sites of antipyrenebutyrate antibodies are relatively nonpolar.