Colorimetric determination of N-acetylhexosamine-terminating O-glycosidically linked saccharides in mucins and glycoproteins

Abstract

A sensitive colorimetric assay for detecting mucins and glycoproteins rich in O-glycosidically linked saccharides is reported. The method combines the susceptibility of N-acetylgalactosamine terminating O-glycosidically linked saccharides to β-elimination with the Morgan-Elson reaction for N-acetylhexosamines with free reducing ends. All mucin and mucin-type glycoproteins but none of the serum-type glycoproteins tested resulted in characteristic color production. All mucins tested gave linear responses in the range 5 to 200 μg and the assay was also adapted to the microscale involving the use of 96-well microtiter plates. The microassay in which the volumes of samples and reagents are scaled down 2.5-fold was particularly useful in monitoring of mucins, in the presence of other glycoconjugates, in large numbers of samples obtained during fractionation procedures. Cesium chloride, cesium bromide, potassium thiocyanate, and various detergents do not interfere with the colorimetric determination. Guanidine hydrochloride, cesium trifluoroacetate, and β-mercaptoethanol decreased color by 30 to 45%; however, the interference was not serious to prevent the use of the method for detection of mucins in their presence. The use of the method for the specific detection of mucin during fractionation by gel filtration and density gradient centrifugation of cystic fibrosis sputum samples is demonstrated.