Characterization of fluorescent 2-dimethylaminonaphthalene-5-sulfonyl-immunoglobulin G conjugates for application in fluorescence polarization studies.

Preparations of normal rabbit IgG antibody (NgG) and purified rabbit IgG antibody of anti-2,4-dinitrophenyl (Anti-Dnp) specificity were covalently labeled with the fluorescent probe 2-dimethylaminonaphthalene-5-sulfonyl chloride (2,5-Dns-Cl). 2,5-Dns-immunoglobulin G preparations were shown to have average fluorescence lifetimes of 29--32 ns by cross-correlated phase and modulation fluorescence lifetime measurements. Excitation maxima were observed at 275 and 368 nm, while the emission maximum occurred at 465 nm. With 390 nm excitation, an intrinsic fluorescence polarization of 0.43 was observed. Molar extinction coefficients of 36 000 M-1 . cm-1 (at 258 nm) and 2090 M-1 . cm-1 (at 352 nm) were determined by synthesis of 2,5-Dns-L-[4,5-3H]leucine, a tritiated sulfonamide of 2,5-Dns. Low labelling (1 mol 2,5-Dns/mol of antibody) of rabbit Anti-Dnp IgG antibody with 2,5-Dns-Cl did not significantly alter the antibody association constant with Dnp. Fluorescence polarization measurements were accurately measured (0.3% error) at antibody concentrations greater than 10(-7) M, using photon counting instrumentation. Rotational relaxation times of 128 and 131 ns were determined for 2,5-Dns-NgG and 2,5-Dns-Anti-Dnp, respectively.