Separation and characteristics of glycoproteins in tears which inhibit coating and precipitation of protein.
Using a modified turbidimetric assay to determine the protein concentration in human tears by precipitation with trichloroacetic acid (TCA) we found lower protein concentrations if compared with other methods for protein determination. This implies that a factor in human tears is able to inhibit the precipitation of protein by TCA. Earlier a coating inhibitory factor in human tears was described which is able to prevent coating of a polyacrylate surface by proteins using a ELISA methodology. Because of the similarity in its behaviour towards protein we investigated whether the same factor could be responsible for both inhibitory effects. A pool of human tears was separated into various fractions using HPLC whereafter inhibitory activity in the turbidimetric and the coating assay could be found in the same fractions. Characterization 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 tears were incubated for 30 min at 100 degrees C whereafter precipitated protein was removed by centrifugation. Inhibitory activity could be detected in the supernatant and an identical glycoprotein profile could be produced after staining with lectin (Jacalin). The results of this study suggest that a soluble glycoprotein serves as a coating and precipitation inhibitor in tears and may play an important role in the protein to protein interaction on the surface of the eye.
Meijer
F
F
Biochemical Laboratory, The Netherlands Ophthalmic Research Institute, Amsterdam.
van Haeringen
N J
NJ
D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D006023:Glycoproteins / Q000302:isolation & purification* / Q000494:pharmacology; D006358:Hot Temperature; D006801:Humans; D019158:N-Acetylneuraminic Acid; D009391:Nephelometry and Turbidimetry; D012794:Sialic Acids / Q000032:analysis; D013666:Tears / Q000378:metabolism
D005136:Eye Proteins; D006023:Glycoproteins; D012794:Sialic Acids; C042434:tear proteins; D019158:N-Acetylneuraminic Acid