ANTIBODIES FOR FLUORESCENT MOLECULAR ROTORS

We have prepared monoclonal antibodies for the fluorescent molecular r otors 9-(2-carboxy-2-cyanovinyl)julolidine (CCVJ) and 9-(dicyanovinyl) julolidine (DCVJ). Mouse monoclonal antibody (IgG2b) prepared against CCVJ-conjugated bovine serum albumin strongly bound CCVJ and DCVJ. The CCVJ (or DCVJ) binding to IgG and Fab was accompanied by a drastic in crease in fluorescence quantum yield, suggesting the restriction of in tramolecular rotational relaxation about the donor-acceptor bond of th e fluorophores. Nonspecific IgG never changed the quantum yield of the fluorophores. From the Scatchard plots, the association constants of CCVJ to IgG and Fab were 6.8 x 10(7) and 5.4 x 10(7) M-1, respectively , and the numbers of moles of CCVJ bound per mole of IgG and Fab were calculated to be 2.0 (+/-0.1) and 1.0 (+/-0.05), respectively. The flu orescence spectra of the IgG-bound CCVJ were quite similar to those of Fab-bound CCVJ. The fluorescence lifetimes of the IgG-bound and Fab-b ound CCVJ were 388 and 383 ps at 25-degrees-C, respectively. They were 6.3 times as long as the fluorescence lifetime of CCVJ free in soluti on (62 ps). These results indicated that the drastic increases in quan tum yields were due to the decreases of the nonradiative rate constant s of the antibody-bound CCVJ, as well as due to the changes of the int rinsic radiative rate constant, and that the nonradiative internal rot ations about the donor-acceptor bond of CCVJ were not dependent on the size of the bound antibody molecules. From the nonradiative rate cons tants, it was suggested that the donor-acceptor bond of the fluorophor e may possibly rotate inside the antigen combining site at a rate of 2 .4 ns-1 at 25-degrees-C. This result is interesting because previous r otational anisotropy studies have examined the molecular motion of the entire protein, not the motion of the probe within a binding site as described here.