Dr. Murai Éva szerk.: Parasitologia Hungarica 16. (Budapest, 1983)

RESULTS After 6 h and 16 h incubation, proteolytic enzymes (trypsin, chymotrypsin) were not demonstrable in the maintenance medium. The values of trypsin and chymotrypsin inhibitory activities are shown in Table 1. Activity of trypsin and chymotrypsin inhibitor secreted by P. daubneyi (U inh. /g worm) Time of No incubation Trypsin Chymotrypsin inhibitor 6 h 5 16 h 5 36 (18-47) 46 (25-70) 76 (50-91) 90 (58-130) A 50 ml volume of medium, containing the released inhibitors of 5 g worms was applied on a Sepharose-chymotrypsin column (Table 2,) Recovery of trypsin and chymotrypsin inhibitor activities by affinity chromatography Trypsin | Chymotrypsin inhibitor act. (U) Maintenance medium (50 ml) 260 480 Fractions eluted by borate buffer 0 0 Fractions eluted by 0. 05 M HCl 205 395 Recovery % 78. 9 82. 3 Absence of activity in the borate buffer fractions indicated the binding of both inhibitory substances to the column. Desorption by 0. 05 M HCl resulted in about 80% recovery of both inhibitory activities. At the same time, it was proved that the same molecule had both trypsin and chymotrypsin inhibitory activités. After treatment with excess of chymotrypsin the inhibitor could not inhibit the activity of trypsin. In a similar way, the treatment with trypsin removed chymotrypsin inhibitory activity. These two experiments suggest that the sites of the inhibitor molecule active against trypsin and chymotrypsin are identical or closely adjacent. The reaction of trypsin and chymotrypsin with the inhibitor is not instaneous, it requires about 5 min. The enzyme-inhibitor complex is stable, it does not dissociate in 4 M KCl during 10 min. incubation. The inhibitory activity against both enzymes disappeared during a 90 min treatment with 2mercaptoethanol, while 8 M urea had no effect (during 90 min. as well). The inhibitor is heat-stable, over 90% of its activity was maintained after 15 min incubation at 100°C. During storage at 4°C for two weeks, all inhibitor activity remained unchanged at both pH 1. 6 and pH 9. 0.