SEPARATION AND CHARACTERISTICS OF GLYCOPROTEINS IN TEARS WHICH INHIBIT COATING AND PRECIPITATION OF PROTEIN

Using a modified turbidimetric assay to determine the protein concentr ation in human tears by precipitation with trichloroacetic acid (TCA) we found lower protein concentrations if compared with other methods f or protein determination. This implies that a factor in human tears is able to inhibit the precipitation of protein by TCA. Earlier a coatin g inhibitory factor in human tears was described which is able to prev ent coating of a polyacrylate surface by proteins using a ELISA method ology. Because of the similarity in its behaviour towards protein we i nvestigated whether the same factor could be responsible for both inhi bitory effects. A pool of human tears was separated into various fract ions using HPLC whereafter inhibitory activity in the turbidimetric an d the coating assay could be found in the same fractions. Characteriza tion of the inhibitory factor was performed by minigel-electrophoresis (SDS-PAGE), after which blotting and staining with a lectin (Jacalin) revealed two subunits of a glycoprotein with a molecular weight of 30 and 70 kD. The inhibitory factor also could be isolated if human tear s were incubated for 30 min at 100-degrees-C whereafter precipitated p rotein was removed by centrifugation. Inhibitory activity could be det ected in the supernatant and an identical glycoprotein profile could b e produced after staining with lectin (Jacalin). The results of this s tudy suggest that a soluble glycoprotein serves as a coating and preci pitation inhibitor in tears and may play an important role in the prot ein to protein interaction on the surface of the eye.