Proteinase activity in normal human tears: Male-female dimorphism

Recent studies using radial caseinolysis suggested that preoperative plasmin and plasminogen-activators were predictive indicators of surgical outcomes. In this study, zymography was used to determine if other caseinolytic proteases were present in normal human tears. Tear samples were collected and proteins extracted with a buffer of 2% sodium dodecyl sulfate (SDS), 100 mM Tris/HCl, pH 6.8. Tears were run on casein zymograms (4 mg/ml) which were incubated in 50 mM Tris/HCl, 5 mM CaCl2, 0.15 M NaCl and NaN3, (pH 7.5) at 37 degrees C for 48 h. After staining and destaining, the caseinolytic activity was seen as cleared areas. Plasminogen activator activity was analyzed with zymograms of copolymerized casein and plasminogen incubated in phosphate buffer saline (pH 8.0) overnight at room temperature. Results showed that besides a 50 kDa plasminogen activator, human tears contained 3 other caseinolytic activities of molecular weights 90 kDa, 70 kDa and 30 kDa. These were serine proteases with optimal activities at pH 7-8 and did not have plasminogen-activator properties. The 70 kDa activity required calcium but the others did not. The 30 kDa caseinolytic activity was gender-related as it was readily detected in males and was absent or weakly expressed in females.