Interaction between carbohydrate residues of alpha(1)-acid glycoprotein (orosomucoid) and progesterone. A fluorescence study.
Interaction between progesterone and the carbohydrate residues of alpha(1)-acid glycoprotein was followed by fluorescence studies using calcofluor white. The fluorophore interacts with polysaccharides and is commonly used in clinical studies. Binding of progesterone to the protein induces a decrease in the fluorescence intensity of calcofluor white, accompanied by a shift to the short wavelengths of its emission maximum. The dissociation constant of the complex was found equal to 8.62 microM. Interaction between progesterone and free calcofluor in solution induces a low decrease in the fluorescence intensity of the fluorophore without any shift of the emission maximum. These results show that in alpha(1)-acid glycoprotein, the binding site of progesterone is very close to the carbohydrate residues. Fluorescence intensity quenching of free calcofluor in solution with cesium ion gives a bimolecular diffusion constant (k(q)) of 2.23 x 10(9) M(-1) s(-1). This value decreases to 0.19 x 10(9) M(-1) s(-1) when calcofluor white is bound to alpha(1)-acid glycoprotein. Binding of progesterone does not modify the value of k(q) of the cesium. Previous studies have shown that the terminal sialic acid residue is mobile, while the other glycannes are rigid [Albani, J. R.; Sillen, A.; Coddeville, B.; Plancke, Y. D.; Engelborghs, Y. Carbohydr. Res. 1999, 322, 87-94]. Red-edge excitation spectra and Perrin plot experiments performed on sialylated and asialylated alpha(1)-acid glycoprotein show that binding of progesterone to alpha(1)-acid glycoprotein does not modify the local dynamics of the carbohydrate residues of the protein.
Laboratoire de Biophysique Moléculaire, Université des Sciences et Technologies de Lille, Bâtiment C6, 59655 Villeneuve d'Ascq Cédex, France.
Melanie
M
De Ceukeleire
Jihad René
JR
Albani
D016880:Anisotropy; D001557:Benzenesulfonates; D002241:Carbohydrates / Q000737:chemistry*; D002586:Cesium; D005453:Fluorescence; D005456:Fluorescent Dyes; D009961:Orosomucoid / Q000737:chemistry*; D011374:Progesterone / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D017186:Titrimetry
D001557:Benzenesulfonates; D002241:Carbohydrates; D005456:Fluorescent Dyes; D009961:Orosomucoid; D002586:Cesium; D011374:Progesterone; C007061:C.I. Fluorescent Brightening Agent 28