FRACTIONATION & CHARACTERIZATION OF OCULAR MUCUS

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The object of the proposed research is to acquire information leading to a clearer understanding of the roles of various ocular mucus and tear components in mucus gel formation and tear film stability in health and disease. This will involve isolation and characterization of the responsible components in mucus from both normal and diseased eyes. Special attention will be paid to the isolation of ocular mucin. Other mucus/tear components to be studied include serum and tear specific proteins, lipids, glycosaminoglycans, and proteoglycans. For the purification of ocular mucin, specimens will be separated according to blood type of individual ocular mucus donors. Methods of isolation and purification will include selective solvent extraction, affinity and ion exchange chromatographies, gradient ultracentrifugation, and immunological methods. Chemical compositional analyses of isolates will include carbohydrates by GLC, amino acids and amino sugars by amino acid analysis, and lipid components by TLC and GLC. Structural analyses of mucin will involve amino acid sequencing of the peptide core, and GLC, MS, and NMR spectroscopy in conjunction with sugar analysis and immunological assays of the oligosaccharide chains. (Monoclonal) antibodies to mucin, tear prealbumin, and possibly de-glycosylated mucin isolates will be prepared. Physical property studies of individual mucus components, particularly mucin, will include measurements/determinations of viscosity, solubility, molecular weight, surface activity, and mode of binding among structural elements. Recent advances in microanalyses and our experience with the application of these techniques will provide an excellent opportunity for achieving these objectives.