Studies on the isolation and composition of human ocular mucin

Abstract

A method for the isolation and purification of human ocular mucin from the brief saline extract of human ocular mucus is reported. Initial purification of ocular mucin was achieved by sequential chromatography of the saline-soluble mucus extract from an individual donor's mucus pool on columns of Sephadex G-50 and Sepharose CL-4B. A portion of such mucin isolate was subjected to quantitative analysis of the O-seryl (threonyl)-N-acetylgalactosaminyl linkage, characteristic of mucins, by alkaline beta-elimination and tritiated borohydride reduction. Following Bio-Gel P-2 filtration, the mucin isolate whose cleaved oligosaccharides contained tritiated galactosaminitol greater than 0.5 microCi mg-1, a value that represents at least 64% of that observed for bovine and ovine submaxillary reference mucins, was considered to be mucin-rich. These isolates were subjected to further purification on Sephacryl S-500 and DEAE-Trisacryl M column chromatographies. The purified mucin had a minimum molecular weight of 120 kDa. It consisted of 25-30% protein and 54-55% carbohydrate. Its amino acid and carbohydrate compositions are characteristic of a mucin structure. The purity of the mucin was verified by SDS-gradient PAGE. Upon isoelectric focusing, polydispersity/microheterogeneity were exhibited in the pI range 5.0-6.6.