The binding of fluorescein and 6-carboxyfluorescein by normal, diabetic and in vitro glycosylated human serum proteins was analyzed by absorption and fluorescence spectroscopy, gel filtration and equilibrium dialysis. The absorption spectra of bound fluorochromes showed red shifts and hypochromic changes, and fluorochrome fluorescence was reduced by serum proteins. Bio-gel P-6 and Sephadex G–200 gel filtration in solvents containing free fluorochrome demonstrated that fluorescein and carboxyfluorescein were bound by serum proteins of varying molecular weights and/or diffusion rates. Equilibrium dialysis at 37°C showed that human sera contained 3.5 ± (SD) 0.4 × 10––3 Mof fluorescein and carboxyfluorescein binding sites with an average association constant of 3.9 ± 0.2 ×103 M––1. Undiluted serum proteins bound 93% of the fluorochrome at a total concentration of 2×10––4 M or less. Glycosylation of serum proteins in vivo or in vitro did not change the concentration-dependent or pH-dependent binding of fluorescein or carboxyfluorescein.

Keywords:
Fluorescein, Carboxyfluorescein, Human serum proteins, Nonenzymatic glycosylation, Gel filtration, Equilibrium dialysis, Absorbance red-shift
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© 1982 S. Karger AG, Basel
1982
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