The Effects of pH and Temperature on Cysteine Protease (Cathepsin B) Activity in Miracidia and Eggs of Fasciola hepatica

Abstract

Abstract Background: Fascioliasis is a worldwide zoonotic disease caused by the trematodes Fasciola hepatica in humans and animals. Proteases are essential for the survival of parasites and have important activities such as penetration, tissue migration, and egg hatching. This study was conducted to analyze cysteine protease of the miracidia and eggs of F. hepatica, and to assess the effects of pH and temperature on the proteases activity and stability. Methods: Adults F. hepatica were isolated from infected livers and were morphologically identified in 2018. Eggs collected from the adults and incubated in distilled water at 28 °C for 16 d to produce miracidia. The extract was collected from miracidia and eggs. A substrate for cathepsin B (Z-Arg-Arg-Pna) was used to assess the enzyme activity at different (2-12) pH levels. After homogenization, protein level was measured with Bradford method. Estimation of optimum temperature and pH was performed in the temperature range of 10-90 ° C and pH values from 2-12. Results: The highest activity of the miracidia and eggs enzyme extracts for Z-ArgArg-pNA was at pH 4. The miracidia extract was most stable at neutral pH and the eggs extract was most stable in acidic pH. The optimum temperature activity for both stages was 40 °C. These proteases were stable up to 40 °C. Conclusion: Upon the importance of pH and temperature in the life cycle of F. hepatica, the current findings can be used for induction of some modifications in pH and preventing the activity of the enzymes for decrement of the efficacy of miracidia penetration into the intermediate snails and egg hatching of this zoonotic parasite.