ACTIVITY OF SERINE PROTEASES FROM FASCIOLA HEPATICA EGGS IN RELATION TO PH AND TEMPERATURE

摘要

This study was conducted to analyse the serine protease of Fasciola hepatica eggs by specific substrates and inhibitors, and investigation of the effects of pH and temperature on proteases’ activity and stability. Adult worms were isolated from infected livers. After homogenisation, their protein levels were measured with the Bradford method. Total proteolytic activity of the Fasciola hepatica extract was evaluated with azocasein substrate at pH values from 2 to 12. N-benzoyl–arginine–pnitroanilide (BApNA) trypsin and N-succinyl-alanine-alanine-prolin-phenylalanine-p-nitroanilide (SAAPFpNA) chymotrypsin substrates were used to measure specific protease activities. The effect of protease inhibitors phenylmethane sulfonyl fluoride (PMSF), pepsin, and ethylenediaminetetraacetic acid (EDTA) on these enzymes was evaluated. Estimation of optimum temperature and pH was performed in the temperature range of 10–90 °C and pH values from 2–12. The optimum pH activities for trypsin and chymotrypsin were at alkaline pH and for total proteolytic activity at acidic pH. The results using protease inhibitors showed that the eggs had serine protease activity. The optimum temperature activity of trypsin and chymotrypsin was 50 °C. These proteases were stable up to 40 °C. Due to the importance of pH and temperature in life cycle of Fasciola hepatica, these findings can be used for induction of some modifications in pH and preventing the activity of the enzyme for decrement of the efficacy of embryonic development and egg hatching of this zoonotic parasite.