FRACTIONATION AND CHARACTERIZATION OF OCULAR MUCUS

拟议研究的目的是获取信息 从而更清楚地了解各种眼的作用 粘液凝胶形成和泪膜中的粘液和泪液成分 健康和疾病的稳定性。 这将涉及隔离， 粘液中负责成分的表征 正常和患病的眼睛。 将特别注意 眼粘蛋白的分离。 其他粘液/泪液成分， 被研究的包括血清和泪液特异性蛋白质，脂质， 糖胺聚糖和蛋白聚糖。 用于纯化 眼粘蛋白，标本将根据血型分离 单个眼粘液捐献者的情况 分离方法和 纯化将包括选择性溶剂萃取、亲和和 离子交换色谱法、梯度超离心法和 免疫学方法。 化学成分分析 分离物将包括通过GLC测定的碳水化合物、氨基酸和 通过氨基酸分析测定氨基糖，通过TLC测定脂质组分 和GLC。 粘蛋白的结构分析将涉及氨基酸 肽核心测序，GLC、MS和NMR 光谱学结合糖分析和免疫学 寡糖链的测定。 （单克隆）抗体 粘蛋白、泪液前白蛋白和可能的去糖基化粘蛋白 将制备分离物。 个人的物理性质研究 粘液组分，特别是粘蛋白，将包括 测量/测定粘度、溶解度、分子量 重量、表面活性和结构之间的结合模式 元素 微量分析的最新进展和我们的经验 这些技术的应用将提供一个很好的 实现这些目标的机会。

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.