The precipitation of mucin by aluminium

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Abstract

The interactions of Al with a mucin glycopeptide have been studied. A number of specific reactions were identified the nature of which were dependent upon the Al chemistry in the hydration environment. In particular, Al was observed to precipitate mucin and it is suggested that this proceeded via the intercalation of the hydroxide within the hydrated macroreticular network of the mucin biopolymer. This precipitation of mucin was visible by eye and abolished the viscosity of native mucin. Viscometry indicated that Al was bound by mucin at low pH. At pH > 3 Al formed a low molecular weight complex with mucin which was hydrolytically stable and was not precipitated at pH up to 8. In an additional and competitive reaction Al was bound by mucin and the resultant mucin–Al complex was suggested to be the precursor to self-assembled mucin–Al spheres identified in solution, by photon correlation spectroscopy, and in precipitate using selective histochemistry. The majority of these spherical structures were of sub-micron diameter and, through their interaction with each other, were probably responsible for the observed pH-dependent peaks of mucin solution viscosity. The larger spheres, between 20 and 80 μm in diameter, were only identified in isolated mucin/Al precipitates and, being comparatively rare, were unlikely to have influenced solution viscosities. These large spheres were observed to act as possible nucleation sites for the flocculation of mucin/Al precipitate. Al at concentrations as low as 0.015 mM induced changes in the rheological properties of mucin. Considering the ubiquitous nature of mucin and the degree to which it is conserved within biota the interactions of Al with mucin may have wide ranging implications for biological systems.

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.

Section snippets

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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