Tseng S C, Zhang S H
Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami School of Medicine, FL 33101, USA.
Cornea. 1995 Jul;14(4):427-35. doi: 10.1097/00003226-199507000-00013.
Bindings of rose bengal to several proteins were determined by Sephadex G-75 chromatography. Their respective blocking effect against dye uptake was demonstrated in an assay using a rabbit corneal epithelial cell layer. The total binding capacity of nonmucin proteins was measured using fluorometry and Scatchard analysis. The results showed that albumin, lactoferrin, transferrin, and lysozyme could--but serum prealbumin, IgA, carboxymethyl cellulose (CMC), and Sepharose 4B-purified porcine stomach mucin (PSM) could not--bind rose bengal. Lysozyme formed precipitates with rose bengal. Sufficient concentrations of albumin, lactoferrin, transferrin, or lysozyme premixed with rose bengal could block dye uptake by cells, but IgA and serum prealbumin could not. Premixed PSM was not as effective as precoated PSM in blocking dye uptake. The dissociation constant (Kd) was 1.2 x 10(-7) M, 3.6 x 10(-7) M, 3.9 x 10(-7) M, and 1.6 x 10(-6) M for albumin, transferrin, lactoferrin, and lysozyme, respectively. Based on these values, the total maximal binding capacity of nonmucin proteins in normal 7-microliters tears was extrapolated to be 0.249 micrograms rose bengal, which is too small to explain the negative staining of rose bengal on the normal ocular surface. Rose bengal, but not fluorescein, could interact with carbohydrate-containing Sephadex, CMC, and PSM to slow down its elution via Sephadex column chromatography. Therefore, the normal negative staining to rose bengal might be caused by the blocking effect of preocular mucus tear layer, which serves as a diffusion barrier. Rose bengal remains a unique dye for detecting the protective function of the preocular mucus tear.
通过葡聚糖G - 75色谱法测定孟加拉玫瑰红与几种蛋白质的结合情况。在使用兔角膜上皮细胞层的测定中证明了它们各自对染料摄取的阻断作用。使用荧光法和Scatchard分析测量非粘蛋白的总结合能力。结果表明，白蛋白、乳铁蛋白、转铁蛋白和溶菌酶能够结合孟加拉玫瑰红，但血清前白蛋白、IgA、羧甲基纤维素（CMC）和琼脂糖4B纯化的猪胃粘蛋白（PSM）则不能。溶菌酶与孟加拉玫瑰红形成沉淀。与孟加拉玫瑰红预混合的足够浓度的白蛋白、乳铁蛋白、转铁蛋白或溶菌酶可以阻断细胞对染料的摄取，但IgA和血清前白蛋白则不能。预混合的PSM在阻断染料摄取方面不如预包被的PSM有效。白蛋白、转铁蛋白、乳铁蛋白和溶菌酶的解离常数（Kd）分别为1.2×10⁻⁷ M、3.6×10⁻⁷ M、3.9×10⁻⁷ M和1.6×10⁻⁶ M。基于这些值，正常7微升泪液中非粘蛋白的总最大结合能力推断为0.249微克孟加拉玫瑰红，这太小以至于无法解释正常眼表上孟加拉玫瑰红的阴性染色。孟加拉玫瑰红而非荧光素能够与含碳水化合物的葡聚糖、CMC和PSM相互作用，通过葡聚糖柱色谱法减缓其洗脱。因此，正常情况下对孟加拉玫瑰红的阴性染色可能是由眼前粘液泪液层的阻断作用引起的，该层起到扩散屏障的作用。孟加拉玫瑰红仍然是检测眼前粘液泪液保护功能的独特染料。
