Some chemical and physical properties of a fluorescent protein, human y-globulin conjugated with fluorescein isothiocyanate (FITC), were described in this journal earlier by one of us (1). The relative quenching of the fluorescence of the conjugate and its apparent dissociation constant, were calculated and used as criteria for the purity and stability of the conjugate. In these calculations the molar ratio of FITC to protein (F/P) was obtained from optical absorption measurements, tacitly assuming that the molar extinction coefficient (E) of the bound FITC was the same as that of the free FITC. This assumption is generally made when calculating the F/P ratio (2).
The validity of this assumption was questioned by Dandliker and others, who found 1.12 times more FITC in a fluorescent ovalbumin by SO1 analysis than by direct absorption measurement, indicating a decrease in absorption upon conjugation (3). In recent studies on the equilibrium and kinetics of the immunoreaction between conalbumin and anticonalbumin we used fluorescence quenching for following the reaction (4, 5). In these and similar quantitative fluorescence studies it is desirable lo know the exact F/P ratio in order t'o calculate the fluorescence quenching properly and also to estimate correctly the effect of 6he presence of the fluorescent label on the immunoreaction. For this reason we reinvestigated the absorption and fluorescent properties of fluorescent conalbumin preparations used in immunochemical studies.
MATERIALS A11SD METHODS
The protein in the experiments was a 5X crystallized conalbumin (CA) of about 95% purity by chromatography (Nutritional Biochemical Co.).