Molecular spectroscopic studies on the interaction of glycosaminoglycans with brilliant cresol blue and its analytical application

Abstract

The interaction of brilliant cresol blue (BCB) with glycosaminoglycans (GAGs), such as heparin (Hep) and chondroitin 4-sulfate (CS), in aqueous solution has been studied by spectrophotometry and light scattering spectroscopy. Absorbance of BCB at 632 and 594 nm decreased on addition of Hep or CS with the appearance of a new blue-shifted absorption band at 550 nm, which indicated that new metachromatic complex formed. The linear decrease in absorbance of BCB at 632 nm was observed. In addition, Hep was more effective than CS (1.7 times) in decreasing absorbance of BCB. The stoichiometry of Hep or CS with BCB was determined by spectrophotometric titration and the MacIntosh extraction method. The result showed that the stoichiometry of BCB/Hep was 1.8 times that of BCB/CS. These results suggested that the interaction between GAGs and BCB was the result of electrostatic forces, and the differences between Hep and CS were attributed to the different negative charge numbers on repetitive disaccharides unit. Studies on the effects of alcohol and urea indicated that GAGs only interacted with the aggregates of BCB. Moreover, a strong light scattering signal was observed after mixing BCB with GAGs. Furthermore, the light scattering intensity at light scattering bands was proportional to the concentration of Hep or CS added when the concentration of BCB was constant.