A colorimetric assay for mucous glycoproteins using Alcian Blue [proceedings

Abstract

The present paper describes a simple, sensitive technique for measurement of respiratory mucous glycoproteins that involves their precipitation with the cationic dye Alcian Blue. The assay was adapted from that described by Whiteman (1973) for measurement of urinary glycosaminoglycans. This method is useful for measurement of mucins secreted in response to stimulating agents in isolated airway preparations of animals, and of human airway secretions obtained by bronchoswpy. The procedure is also useful for measurement of mucin content of fractions obtained after column chromatography. The Alcian Blue assay was developed as an alternative to the usual methods of glycoprotein determination, which measure total hexose wntent (phenol/sulphuric acid, anthrone, orcinol), which are of limited sensitivity (Mantle & Allen, 1978) and are hazardous to operate, requiring the use of large amounts of sulphuric acid. Mucous glycoproteins are polyanionic, having both sialic acid and sulphate residues attached to their oligosaccharide structures. It is these groups that interact with Alcian Blue to form insoluble complexes. A 0.1% (w/v) solution of Alcian Blue in 0.1 M-sodium acetate/acetic acid buffer, pH 5.8, containing 25 mM-MgC1, was clarified by sedimentation at 1870ga, for 30min at 2OoC in an MSE Mistral 2L centrifuge. To 3.0ml portions of tracheal mucus samples in plastic test tubes was added 1 ml of the purified Alcian Blue solution. After at least 2 h equilibration at room temperature, mucus-Alcian Blue complexes were sedimented at 1870ga, for 30min at 2OOC. Supernatant solutions were discarded, and each pellet was washed twice by successive resuspension in 40% (v/v) ethanoV0.1 Msodium acetate buffer, pH 5.8, containing 25 mM-MgCI,, and sedimentation at 1870ga, for IOmin at 2OOC. A 40% (w/v) Manoxol 1 B (sodium dibutylsulphosuccinate) solution was clarified by filtration. Mucus-dye complexes were dissociated by addition of 1 ml of the Manoxol 1B solution and ultrasonication at SOW for 10s with a Dawes soniprobe. Samples were sedimented for 1 min as described above to eliminate the foam generated during sonication. Absorbance at 620nm was measured in a Cecil CE272 spectrophotometer. A calibration curve for Alcian Blue precipitation was determined for a cat tracheal mucus preparation (Fig. 1) obtained as described by Gallagher et al. (1975). The protein content of this preparation was determined by alkaline hydrolysis followed by ninhydrin reaction, and the carbohydrate content was determined by g.1.c. (Hall, 1978). The carbohydrates present were those typical of cat tracheal mucins (Gallagher et al., 1975) and contained no deoxyribose, ribose or uronic acids. This mucus preparation was not contaminated by nucleic acids or proteoglycans. The resultant absorbance at 620nm of the Alcian Blue 0.6 r