Binding of fluorescein and carboxyfluorescein by human serum proteins: significance of kinetic and equilibrium parameters of association in ocular fluorometric studies.

The binding of fluorescein and 5- and 6-carboxyfluorescein by human serum proteins was measured at 37 and 4 degrees C by equilibrium dialysis. The equilibrium association constants (KA) for fluorescein were 3.7 X 10(3) and 7.1 X 10(3)M-1, and for carboxyfluorescein were 3.5 X 10(3) and 7.5 X 10(3)M-1 at 37 and 4 degrees C equilibrium dialysis data, was 3.9 X 10(3) binding sites in human serum, determined from the 37 degrees C equilibrium dialysis data, was 3.9 X 10(-3)M for fluorescein and 3.3 X 10(-3)M for carboxyfluorescein. Utilizing these binding parameters it was calculated that a maximum of 93.5% of the total fluorescein and 92.0% of the carboxyfluorescein would be bound by undiluted human serum proteins at 37 degrees C. Experimental binding data obtained after prolonged equilibrium dialysis (four days) at low total fluorochrome concentrations (1.5 X 10(-4)M or less) indicated that 93.1 +/- 1.0 (S.D.)% of the fluorescein and 90.1 +/- 0.7% of the carboxyfluorescein were bound at 37 degrees C by undiluted human serum proteins. Stopped-flow kinetic spectrophotometric studies of the changes in absorptivity at 487-488 and 510 nm that occurred when the fluorochromes were bound by human serum proteins, indicated that the fluorescein and carboxyfluorescein binding reactions were 99% complete within 0.65 and 1.72 sec. These had second-order association rate constants at 25 degrees C of 3.0 X 10(3) and 1.5 X 10(3)M-1 sec-1, respectively. These findings offer a basis for calculation of bound and free fluorescein and carboxyfluorescein in vivo in human subjects.