The precipitation of mucin by aluminium
Introduction
Mucins are integral components of the epithelial secretions of the alimentary, respiratory and genital tracts of a wide diversity of biota including man. The mucin glycopeptide is composed of a flexible, linear polypeptide chain supporting a heavily branched network of O-glycosidically linked carbohydrate chains of variable length [1]. The mucin macromolecule has a molecular weight in the range 105–107 Daltons and upon its hydration is responsible for the viscoelastic properties of epithelial mucus. Changes in the rheological properties of mucus are associated with a number of abnormal physiologies, for example, the disease cystic fibrosis in man [2]. A significant body of research has investigated how mucus rheology is altered in vivo [3]and in vitro [4]. Perhaps surprisingly, considering their omnipresence, very little work has investigated how metal ions influence the rheology of mucus. Calcium, perhaps as a consequence of its putative role in cystic fibrosis, has been studied and along with magnesium and copper was described as a “mucus thickening agent” [5]. The “thickening” being attributed to a change in the hydration of mucus brought about by metal-induced conformational changes in the mucin macromolecule. Research has also documented associations of both iron [6]and aluminium [7]with mucus in the gastrointestinal tract of man. Such interactions are likely to be involved in both the transport and excretion of these metals. However, the bioinorganic chemistry underlying these processes is unresolved.
The recent proposition of a contributory role for a mucus–Al interaction in acute aluminium toxicity in fish [8]prompted the current investigation into the solution chemistry of the reactions of mucin with Al. The aim of the research was to establish whether there were any reactions between mucin and Al and to offer insight into how any interaction might influence mucin viscosity. Crucial to both the experimental design and the physiological significance of the results was the understanding that mucins undergo post-exocytotic swelling [9](they are secreted by cells as a concentrate) and hydrate according to a Donnan equilibrium [4]. Thus the composition of hydrated mucins are significantly influenced by the hydration medium. Experiments were thus designed to mimic a number of possible hydration environments. Al was found to influence the viscosity of mucin and to have concomitant effects upon its solubility, solution structure and precipitation.
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Experimental system
Lyophilized bovine salivary mucin was obtained from Sigma (Poole, UK) and in each experiment was used at a concentration of 0.5 mg ml−1 (approximately 0.5 μM assuming an average MW of 106 for mucin). Higher concentrations of mucin showed a tendency to form gels which reduced the accuracy with which measurements of mucin viscosity could be made. All solutions were made up using 10 mM NH4HCO3 as the background electrolyte. This was chosen to mimic the solution conditions immediately adjacent to
Mucin hydrated at pH 3 (Protocol I)
The viscosity of the pure electrolyte was 1.0 mPa s. Hydration of mucin at pH 3 produced a clear solution of viscosity ca. 1.3 mPa s. The titration of this solution to pH 8 neither altered the visible appearance of the solution nor influenced its viscosity (see Fig. 1 solid circles). The addition of Al to hydrated mucin at pH 3 to give a concentration of 0.62 mM had no effect upon the appearance or viscosity of the mucin solution. The same was true upon its subsequent titration to pH 4.
Discussion
The research has demonstrated the precipitation of mucin by Al and the concomitant abolition of the viscosity of its hydrated macromolecular network. Clearly, the precipitation was influenced by the different hydration environments and, in particular, by the Al chemistry. In the absence of any other strong ligands in the precipitation assays the predominant Al chemistry will be with hydroxide and the components of mucin.
The mucin included a number of constituents which either bound or
Acknowledgements
CE thanks Dr. A.J. Milling for helpful research discussions and for carrying out the PCS. CE is a Royal Society University Research Fellow.
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