BIOCHEMICAL AND HISTOCHEMICAL-STUDIES OF PLASMINOGEN-ACTIVATOR OF UROKINASE-TYPE (U-PA) ACTIVITY .1. A SIMPLE RAPID SEMIQUANTITATIVE FLUORESCENT METHOD FOR ITS DETECTION IN THE TEAR FLUID

A simple rapid fluorescent method for the detection of plasminogen act ivator activity of urokinase type (u-PA) in the tear fluid is describe d. Small filter paper punches were soaked in the substrate solution (Z -Gly-Gly-Arg-trifluoromethylcoumarinyl-7-amide, 1 mg/1 ml) and aprotin in 100 mu g/1 ml) dissolved in 0.1 M Tris-HCl buffer, pH 7.2 and dried . The dried punches were soaked with tears (by direct contact of the p unch with the site where the activity should be assessed or by droppin g of 3-5 mu l of tears collected by a glass micropipette). The punches were incubated in a thermostat (37 degrees C) together with punches c ontaining a known u-PA activity (callibrated punches) in preheated (37 degrees C) Petri dishes. In 1 min intervals (during the first 15 min) and in 5 min intervals thereafter the probes were exposed to UV light , and the time of the first appearance of a bright yellow fluorescence was recorded. In punches containing 5 IU u-PA activity fluorescence a ppeared after 2 min incubation; 2.5 IU were detected after 5 min, 1.25 IU after 15 min, 0.625 IU after 30 min, 0.313 IU after 60 min, 0.156 IU after 90 min, and 0.078 IU after 120 min incubation. This simple me thod is recommended for use particularly in clinical laboratories. It enables e. g. to obtain a rather quick information about the urokinase activity in the tear fluid and to start the treatment with an appropr iate inhibitor, if necessary.