FLUORESCENCE LINE-NARROWING STUDIES OF ANTIBODY-BENZO[A]PYRENE TETROLCOMPLEXES

Benzo[a]pyrene tetrol (BPT) was used as a fluorescent probe to investi gate the nature of antigen binding by two different monoclonal antibod ies (MAb) that recognize a variety of derivatives of roxy-9,10-epoxy-7 ,8,9,10-tetrahydrobenzo[a]pyrenes (BPDE). Fluorescence line-narrowed s pectra of the physical complexes of BPT formed with antibodies 8E11 an d 3C3 were recorded at 4 K by employing vibronic excitation into the S -1 electronic state. The frequencies of the vibrational modes of the S -1 state were only marginally affected, though changes in relative int ensities of some bands were observed. Fluorescence spectra recorded at 77 K by excitation into the S-2 state showed that the (0,0) fluoresce nce 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 com plexes 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 a lso formed complexes with both antibodies, however, with different que nching behavior.