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

Inhibitory activities were determined by measuring the residual enzyme activity in an assay medium of known enzyme activity after addition of test material. One unit of enzyme activity: The amount of enzyme hydrolysing 1 jimol substrate per min. at 25°C. One unit of inhibitor activity: the amount of inhibitor which inactivates one unit of enzyme. Table 1 Recovery of trypsin and chymotrypsin inhibitors from Paramphistomum daubneyi extracts by affinity chromatography Inhibitor activity (U) Trypsin Chymotrypsin Crude worm extracts (6 ml) 504 888 Fractions eluted by borate buffer 0 0 Fractions eluted by 0.05 M HCl 370 632 Recovery (fc) 73.4 71 .2 For affinity chromatography, according to the method of FEINSTEIN (1971), chymotrypsin A (bovine pancreatic, 4 x crystallized; BDH) was coupled to CNBr-activated Sepharose 4B (Pharmacia). The conjugate was packed into a column of 0.9 x 10 cm and was equilibrated with 0.1 M borate buffer (pH 9. 0). The worm extracts had been applied onto the column and the unretarded substances were eluted by borate buffer. The bound inhibitor was desorbed by 0.05 M HCl (pH 1.6), collecting 2-ml fractions at room temperature. Fig. 1. Time course of enzyme inhibition by P. daubneyi inhibitor 50 jig trypsin (13.2 U) was incubated with 100 Jul purified inhibitor solution (9.3 U trypsin inhibitor activity), 0.30 ug chymotrypsin (15.2 U) incubated with 50 p.1 inhibitor (7.9 U chymotrypsin inhibitor activity), at 25°C 6 S