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

For gel chromatography Sephadex G-75 gel (Pharmacia) was used in a column of 2.6 x 40 cm, with 0.1 M TRIS-HC1/0.2 M NaCl buffer (pH 8.0) as eluant, collecting 5-ml fractions at room temperature. The column was calibrated with proteins of known molecular weight: bovine serum albumin (Calbiochem, MW 67 000), soybean trypsin inhibitor (BDH, MW 21 500), cytochrom C (Koch-Light, MW 12 400), Cysticercus pisiformis protease inhibitor (own preparation: NÉMETH et al. 1979, MW 7 100). The molecular weight was calculated from the following regression equation: log MW = 5.8084 - 1.5902K (r

2 = 0.9989) RESULTS Trypsin activity of the crude worm extracts Was 84 U/ml, chymotrypsin activity 148 U/ml, that is, 420 U/g worm trypsin activity and 740 U/g worm chymotrypsin activity. The crude extracts were applied on a Sepharose-chymotrypsin column. The results are summarized in Table 1. Both tx-ypsin and chymotrypsin inhibitory activities were bound onto the column, no inhibitory activity could be eluted by borate buffer. The desorbtion of the inhibitory activities by HCl resulted an about 709[ recovery. This procedure supplied the inhibitor ina highly purified form and, besides, suggested that both trypsin and chymotrypsin inhibitory activities are the properties of the same molecule. The Sepharose-chymotrypsin can adsorb only the trypsin inhibitor, and as in our case the trypsin inhibitor was adsorbed as well, this can be explained by the fact that the molecule inhibiting the trypsin is the same as the chymotrypsin inhibiting molecule. Fig. 2. Effect of increasing amounts of P. daubneyi inhibitor on trypsin and chymotrypsin activities In order to know the more precise structure of the inhibitor, the purified inhibitor solution was incubated with chymotrypsin. The activity of the enzyme was 50 fold over that of the inhibitor. After the treatment the complex could not inhibit the activity of trypsin. Similarly, tosylphenylalanine chloromethyl ketone-treated trypsin (without chymotrypsin impurities) given to the inhibitor solution in a 20 fold activity made it quite incapable of inhibiting the activity of chymotrypsin.These two experiments suggest that the inhibition of both enzymes depends on the same or closely adjacent active sites of the inhibitor molecule, that is, the inhibitor is not "double headed". Hi)