Fluorescence quantum yields, excitation and emission spectra, decay times, and polarization values have been measured for conjugates of gamma globulin containing various dyes. The labeling agents examined were fluorescein isothiocyanate, dansyl chloride, rhodamine B isothiocyanate, anthracene-2-isocyanate, Lissamine rhodamine B sulfonyl chloride, and 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid. The quantum yield data do not support the idea that conjugation of a dye to a protein generally causes a decrease in fluorescence efficiency. Conjugates containing fluorescein, rhodamine B, and anthracene moieties show decreasing quantum yields and polarizations as labeling increases, but conjugates containing large numbers of dansyl groups have higher quantum yields and blue-shifted emission spectra compared with more lightly labeled conjugates. In these dansyl conjugates, the sites most accessible to labeling are in polar environments in contrast to the case of serum albumin which is labeled first in hydrophobic areas. Fluorescence depolarization experiments on the fluorescein- and anthracene-containing conjugates yielded different apparent rotational relaxation times which were shorter than that expected for gamma globulin; dye rotation independent of the rest of the macromolecule is thus indicated. In spite of its wide use in immunochemistry, Lissamine rhodamine B sulfonyl chloride yields conjugates with quantum efficiencies an order of magnitude lower than those from rhodamine B isothiocyanate. Re-evaluation of the latter dye for immunologic staining procedures appears warranted. The stilbene dye exhibited marked photosensitivity, presumably due to a trans-to-cis isomerization. © 1969.
