FLUORESCENCE PROPERTIES OF FREE AND PROTEIN BOUND FLUORESCEIN DYES

Received for publication May 31, 1977, and in revised form December 6, 1977 (MS 77- 161) Excitation and emission properties of fluorescein derivatives were studied macrofiuorometrically. Measurements were performed with solutions of various concentrations (0.07-100 �g/ml) of free sodium fluorescein prepared from fluorescein diacetate (FDA), fluorescein isothiocyanate (FITC) and FITC bound to rabbit y-globulin. Both excitation and emission spectra as well as fluorescence intensities at constant excitation/emission wavelengths (496/515 nm) were recorded. The findings indicate that (1) FDA gives about twice the fluorescence intensity compared to equal concentrations of FJTC. (2) The fluorescence properties of FITC upon excitation with blue light (A = 496 nm) are only slightly altered by the conjugation to rabbit y-globulin. (3) Considerable quenching due to conjugation could, however, be shown to occur upon UV excitation (A = 340 nm). (4) Fluorescence emission excited by visible blue light (496 nm) increases linearly to dye concentration in a range of 0.07-2.5 �g/ml. Beginning at 5 �g/ml (10� Mu) all three compounds show a sharp decrease of fluorescence intensity with further increasing concentration. Practical aspects of these data for the immunofluorescence method are discussed.