Dr. Murai Éva szerk.: Parasitologia Hungarica 14. (Budapest, 1982)

The time course of inhibition is demonstrated in Fig. 1. Both enzymes require about five minutes for complex formation with the inhibitor. (This is the reason for applying in the experiments a ten minute preincubation prior to the addition of substrate.) Fig. 2 shows the effect of increasing quantities of inhibitor on enzyme activity. There is a linear relationship with both enzymes between the amount of inhibitor and the decrease in enzyme activity up to about 90%. The resulting enzyme-inhibitor complex is stable, it was not dissociated even by 4 M KCl during a ten minute incubation. The molecular weight of the inhibitor was estimated by gel chromatography. The inhibitor proved to be homogenous and was eluted in a single symmetrical peak (Fig. 3). The estimated molecular weight is about 6 000. The sensitivity of the inhibitor was tested with different agents. Treatment with 2-mercaptoethanol (0.2 M final concentration) for 90 minutes resulted in the complete disappearance of both inhibitory activities, indicating the presence of disulfide bridge(s). In contrast, 8 M urea was quite ineffective in a 90 min treatment at 25°C, suggesting the lack of non-covalent bridges. The inhibitor is heat-stable: treatment for 30 min at 100°C resulted in 20% reduction of the inhibitory activity only. The inhibitor activity was not changed at pH 1.6 - pH 9.0 interval during 3 days. Number of fractions Fig. 3. Gel chromatography of purified P. daubneyi inhibitor on Sephadex G-75 column. Column: 2.6x40 cm; sample: 2 ml, trypsin inhibitor activity: 183 U, chymotrypsin inhibitor activity: 316 U; eluant: 0.1 M TRIS-HC1/0.2 M NaCl buffer, pH 8.0; fractions: 5 ml; • • : trypsin inhibitor activity, o o : chymotrypsin inhibitor activity DISCUSSION The data concerning the protease inhibitors of trematodes are partly incomplete, partly contradictory, (see: introduction). That was the reason for studying the problem with reliable, modern methods. The fluke of the rumen, P. daubneyi proved to be a suitable model because of its size and its easy accessibility and as it was earlier proved that the worm contained inhibitory activity (JUHÁSZ, 1980).