[32] Structural analysis of carbohydrate moiety of bovine rhodopsin

This chapter discusses the structural analysis of carbohydrate moiety of bovine rhodopsin. Rhodopsin is a glycoprotein whose carbohydrate composition and biosynthesis have been studied by several laboratories. In the case of the opsin glycoprotein, any residual detergent interferes with the gas chromatography. The glycoprotein in detergent solution is therefore dried and the detergent is removed with chloroform/methanol and the insoluble glycoprotein is analyzed. Glycopeptides are usually heterogenous and microheterogenity in the sugar moiety can be studied by cleavage of the oligosaccharide from the glycopeptide by endo-β-N-acetylglucosaminidase. The anomeric configuration of each sugar can be determined by the treatment with specific exoglycosidases; α-mannosidase, β-mannosidase, and β-N-acetylglucosaminidase. To determine the position of the sugar linkage, the major oligosaccharide fractions of a Sephadex G-50 gel filtration column are pooled and subjected to methylation analysis. The partially methylated mannose and N-acetylglucosamine are analyzed after acid hydrolysis by a gas chromatograph-mass spectrometer. Total mass spectrometry analysis has been applied to the various glycoconjugates.