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

John H. Rockey, Weiye Li and John F. Eccleston
Experimental eye research, v 37(5), pp 455-466
1983
DOI: https://doi.org/10.1016/0014-4835(83)90021-0
PMID: 6671474
Featured in Collection :   UN Sustainable Development Goals @ Drexel

Abstract

association rate constants carboxyfluorescein equilibrium association constants equilibrium dialysis fluorescein human serum proteins stopped-flow kinetic spectroscopy
The binding of fluorescein and 5- and 6-carboxyfluorescein by human serum proteins was measured at 37 and 4°C by equilibrium dialysis. The equilibrium association constants ( K A) for fluorescein were 3·7 × 10 3 and 7·1 × 10 3 m −1, and for carboxyfluorescein were 3·5 × 10 3 and 7·5 × 10 3 m −1 at 37 and 4°C, respectively. The molar concentration of binding sites in human serum, determined from the 37°C equilibrium dialysis data, was 3·9 × 10 −3 m for fluorescein and 3·3 × 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°C. Experimental binding data obtained after prolonged equilibrium dialysis (four days) at low total fluorochrome concentrations (1·5 × 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°C by undiluted human serum proteins. Stopped-flow kinetic spectrophotometric studies of the changes in absorptivity at 487–488 and 510 nm that occured 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°C of 3·0 × 10 3 and 1·5 × 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.

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Ophthalmology
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