Colorimetric Determination of N-Acetylhexosamine-Terminating O-Glycosidically Linked Saccharides in Mucins and Glycoproteins

"A sensitive colorimetric assay for detecting mucins and glycoproteins rich in 0-glycosidically linked saccharides is reported. The method combines the susceptibility of N-acetylgalactosamine terminating 0-glyco idically linked saccharides to P-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 ""g 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 triftuoroacetate, and B-mercap- . toethanol 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. Mucins are an important class of biomacromolecules whose structure-function relationship is poorly understood (1,2). The investigations of mucins are hampered by difficulties in purifying these very large size molecules which appear to avidly bind other components of the mucus (3,4). One of the problems during purification is the specific detection of mucins. For example, during the various purification steps, the fractions containing mucins are typically detected by assaying for neutral sugars (hexoses) (5,6), for sialic acids (7), or for periodate oxidizable saccharide units (8). However, these methods are not entirely satisfactory because neutral sugars, sialic acids, and periodate-oxidizable saccharides are also components of other glycoconjugates. In addition, the neutral sugar and sialic acid measurements are also subject to interference from high levels of proteins and nucleic acids."