Interaction Between Carbohydrate Residues Of Alpha(1)-Acid Glycoprotein (Orosomucoid) And Saturating Concentrations Of Calcofluor White. A Fluorescence Study

Calcofluor White is a fluorescent probe that interacts with polysaccharides and is commonly used in clinical studies. Interaction between Calcofluor White and carbohydrate residues of alpha(1)-acid glycoprotein (orosomucoid) was previously followed by fluorescence titration of the Trp residues of the protein. A stoichiometry of one Calcofluor for one protein has been found [J.R. Albani and Y.D. Plancke, Caubohydr. Res., 318 (1999) 193-200]. alpha(1)-Acid glycoprotein contains 40% carbohydrate by weight and has up to 16 sialic acid residues. Since binding of Calcofluor to alpha(1)-acid glycoprotein occurs mainly on the carbohydrate residues, we studied in the present work the interaction between Calcofluor and the protein by following the fluorescence change of the fluorophore. In order to establish the role of the sialic acid residues in the interaction, the experiments were performed with the sialylated and asialylated protein. Interaction of Calcofluor with sialylated alpha(1)-acid glycoprotein induces a red shift of the emission maximum of the fluorophore from 438 to 450 nm at saturation tone Calcofluor for one sialic acid) and an increase in the fluorescence intensity. At saturation the fluorescence intensity increase levels off. Binding of Calcofluor to asialylated acid glycoprotein does not change the position of the emission maximum of the fluorophore and induces a decrease in its fluorescence intensity. Saturation occurs when 10 molecules of Calcofluor are bound to 1 mol of alpha(1)-acid glycoprotein. Since the protein contains five heteropolysaccharide groups, we have 2 mol of Calcofluor for each group. Addition of free sialic acid to Calcofluor induces a continuous decrease in the fluorescence intensity of the fluorophore but does not change the position of the emission maximum. Our results confirm the presence of a defined spatial conformation of the sialic acid residues, a conformation that disappears when they are free in solution. Dynamics studies on Calcofluor White and the carbohydrate residues of alpha(1)-acid glycoprotein are also performed at saturating concentrations of Calcofluor using the red-edge excitation spectra and steady-state anisotropy studies. The red-edge excitation spectra experiments show an important shift (13 nm) of the fluorescence emission maximum of the probe. This reveals that emission of Calcofluor occurs before relaxation of the surrounding carbohydrate residues occurs. Emission from a non-relaxed state means that the microenvironment of bound Calcofluor is rigid, inducing in this way the rigidity of the fluorophore itself a result confirmed by anisotropy studies. (C) 2000 Elsevier Science Ltd. All rights reserved.