Dr. Murai Éva szerk.: Parasitologia Hungarica 12. (Budapest, 1979)
preincubation, in a final volume of 3 ml. One unit of protease activity has been defined as the amount of enzyme causing on increase of absorbance at 253 and 256 nm of 1.000 per minute upon hydrolysis of BAEE or BTEE as substrate, respectively. Inhibitor activity was assessed by measuring the residual enzyme activity of trypsin and of chymotrypsin in an assay medium of known enzyme activity after addition of test material. The inhibitor unit was defined amount of inhibitor required to depress the activity of trypsin or chymotrypsin so as to cause a reduction of BAEE and BTEE hydrolysis by 1.000 O. D. per minute. Table 5 Effect of protease inhibitors on the BTEE-splitting activity of the liver fluke extracts (0. 15M Tris-HCl buffer, containing 8. 3mM CaCl • pH 7.8) Inhibitors BTEE- splitti extract rig activity U/ g extract+inhibitor Inhibition % soybean trypsin inhibitor 0. 001% 3. 25 1. 85 43 phenylmethyl sulphonylfluoride ImM 8. 00 2. 50 68 tosylphenylalanil- chloromethane ImM 8. 00 1. 50 81 Acid protease activity was also determined on 2. 5% haemoglobin substrate. The proteolytic unit was expressed as the increase of optical density over the substrate-free control at 280 nm (A E 280) (MYCEK, 1970). The variation coefficient was assessed as 20% for 20 routine measurements in each test system. In the inhibitor-activity assays, the test system, of 3 ml final volume, contained 2. 3 ml 0. 15 M TRIS-HC1 buffer (pH: 7. 8), containing 8. 3 mM CaCl 2 , 0. 1 ml incubation medium, 0. 1 ml trypsin or chymotrypsin solution of known activity, and 0.5 ml substrate. The activities of trypsin and chymotrypsin were measured after 5-min and 15-min preincubation of enzyme plus sample, respectively. Koultg I. Protease activity of fluke extracts a) BAEE-splitting activity The BAEE-splitting activity of extracts was 0. 138 U/g in 0. 15 M TRIS-HC1 buffer (pH 7.8), containing 8.3 mM CaC^ . The pH optima for BAEE-hydrolyzing enzyme activity are shown in Table 1. b) BTEE-splitting activity This proved to be 1. 53 U/g under the above conditions. Tables 2 and 3 show the pHoptimum of BTEE-splitting activity in different buffer systems.
