CHEMICAL AND PHYSICAL VARIABLES AFFECTING FLUORESCEIN ISOTHIOCYANATE AND ITS PROTEIN CONJUGATES

Abstract

The effect of pH, time, temperature, and buffer system on the fluorescence, absorbance, and stability of fluorescein isothiocyanate and its protein conjugates was studied. The effect of these parameters on dye-binding procedures was also studied. 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. This was confirmed by paper electrophoresis. The type of buffer (carbonate, phosphate, borate, tris(hydroxymethyl)aminomethane, and barbiturate) had little effect on fluorescence, but increased molarity of buffer reduced the fluorescence stability of the isothiocyanate. 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 and temperature in dye-binding procedures, the desired degree of labeling can be obtained with short conjugation periods.