FLUORESCENCE LINE-NARROWING STUDIES OF ANTIBODY-BENZO[a]PYRENE TETROL COMPLEXES

Benzo a ]pyrene tetrol (BPT) was used as a fluorescent probe to investigate the nature of antigen binding by two different monoclonal antibodies (MAb) that recognize a variety of derivatives of anti -7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo a ]pyrenes (BPDE). Fluorescence line-narrowed spectra of the physical complexes of BPT formed with antibodies 8E11 and 3C3 were recorded at 4 K by employing vibronic excitation into the S, electronic state. The frequencies of the vibrational modes of the S₁ state were only marginally affected, though changes in relative intensities of some bands were observed. Fluorescence spectra recorded at 77 K by excitation into the S₂ state showed that the (0,0) fluorescence emission of BPT was shifted to red on complex formation. Intensity ratios of the (0,0) band and the main vibrational band at 1300 cm^-1 were used to assess the degree of interior binding of the chromophore. Quenching studies with acrylamide were employed to designate the complexes as type I, solvent inaccessible, or type II, solvent accessible. These studies also indicated that antibody 3C3 complexes tend to be more heterogeneous compared to the 8E11 complex. Deuterated BPT-d-12 also formed complexes with both antibodies, however, with different quenching behavior.