Purification and characterization of rabbit ocular mucin

Ocular mucin, the major product of conjunctival goblet cells, constitutes the innermost layer of preocular tear film. Ocular mucin is known for its limited amount and inaccessibility. Using impression cytology, mucus strands collected from the inferior fornix of either rabbit or human eyes were found to contain inflammatory cellular debris. In order to circumvent these difficulties and to isolate native mucin molecule(s), we bathed rabbit eyes in fluid containing isotonic PBS and 5.5 X 10(-4) M acetylcholine for 4 or 12 hr. Bathing fluids containing rabbit ocular mucin (ROM), 1 ml per eye, were pooled and combined with 1M guanidine HCl and protease inhibitors containing EDTA, PMSF, and sodium azide to avoid any possible enzymatic degradation, and then separated under the same conditions by Sepharose CL-4B. In parallel, commercial porcine stomach mucin (PSM) was purified and used to compare with ROM. We also developed nitrocellulose-based dot semi-quantitative assays for nucleic acid, protein, and glycoprotein. PAS-positive fractions monitored by such a dot assay were collected at CL-4B void volume and then separated from nucleic acid contaminants by CsCl-gradient ultracentrifugation. A protein fraction, 65K, poorly-glycosylated, with high contents of Asx, Glx, and Gly was found strongly associated with both ROM and PSM, and was only separable by ultracentrifugation in 4M guanidine HCl and CsCl. Purification of the ROM was verified by SDS-polyacrylamide gel electrophoresis, amino acid analysis, and carbohydrate analysis. These results will allow future exploration of the molecular mechanism by which tear film is achieved.