Interaction between calcofluor white and carbohydrates of α1-acid glycoprotein

Abstract Interactions between the fluorescent probe, calcofluor white, and human serum albumin (HSA) and α 1 -acid glycoprotein (orosomucoid) are compared. The two proteins have comparable isoelectric points, but α 1 -acid glycoprotein is highly glycosylated (40% of glycans by weight), while the serum albumin is not. Binding of calcofluor to the proteins induces an increase in both the fluorescence anisotropy and the fluorescence intensity of the fluorophore. Also, we found that the calcofluor exhibits a fluorescence emission with a maximum located at 432, 415 or 445 nm, respectively, in the absence of proteins, in the presence of HSA, and in the presence of α 1 -acid glycoprotein. The stoichiometries of the calcofluor–serum albumin and calcofluor–α 1 -acid glycoprotein complexes are 2:1 and 1:1, respectively. The association constants are 0.04 and 0.15 μM −1 , respectively. The calcofluor does not interact with Lens culinaris agglutinin (LCA), although the protein has a hydrophobic site. Nevertheless, one cannot exclude that the binding of the fluorophore to the HSA is nonspecific. Our results, when compared with those obtained with calcofluor dissolved in the hydrophobic solvent isobutanol, and with the fluorescent probe, potassium 6-( p -toluidino)-2-naphthalenesulfonate (TNS), bound to α 1 -acid glycoprotein, indicate that the emission of calcofluor bound to HSA occurs from a hydrophobic state, while that of calcofluor bound to α 1 -acid glycoprotein occurs from a hydrophilic state. The fluorescence intensity of calcofluor decreases in the presence of carbohydrates isolated from α 1 -acid glycoprotein, while it increases in the presence of α 1 -cellulose. Thus, calcofluor interacts mainly with the glycan moiety of α 1 -acid glycoprotein, and its fluorescence is sensitive to the secondary structure of the glycans.