Chemical and Physical Variables Affecting the Properties of Fluorescein Isothiocyanate and Its Protein Conjugates

Abstract

Summary The effect of pH, time, temperature, and buffer systems on solutions of fluorescein isothiocyanate and its protein conjugates was examined fluorometrically. The fluorescence of the isothiocyanate and of the protein conjugate was maximal at about pH 8.7 and pH 10.7, respectively. The stability of their fluorescence above pH 7 was affected adversely by increases in pH or temperature. The protein conjugate, however, showed maximum stability at pH 10.5 and above. The type of buffer (carbonate, phosphate, borate, Tris and barbiturate) did not affect the fluorescence of the free dye significantly. On the other hand, increasing the molarity of the buffer caused a decrease in stability of fluorescence of the free dye but did not seriously affect the fluorescence of the conjugate. The absorbance of fluorescein isothiocyanate and its conjugates increased with increasing pH. In 1 N NaOH, the effect of pH variation on absorbance was minimized. By increasing the pH or temperature of the reaction mixture during conjugation, fluorescein isothiocyanate reacted more readily with the protein. Conditions may be selected to obtain the desired degree of label with short conjugation periods. Conjugation of a bovine anti-Brucella abortus globulin sample for 30 min at pH 9.45 and room temperature was as effective as conjugating at pH 8.75 for 18 hr at 5°C. No apparent loss of biologic activity as the result of conjugation was observed. The binding of fluorescein isothiocyanate to protein resulted in a loss of fluorescence efficiency, the extent of loss being dependent upon the pH of measurement. Existing procedures in fluorescent antibody studies should be reevaluated on the basis of the variables discussed.