Biochemical characterization of proteases isolated from the latex of Tabernaemontana divaricata L. and Carissa carandas L.: Their role in hemostasis

Abstract

In the present investigation, comparative profile of proteases isolated from the latex of Tabernaemontana divaricata L. and Carissa carandas L. of Apocynaceae and their role in hemostasis was studied. The crude enzyme was prepared from the latex of Tabernaemontana divaricata L. and Carissa carandas L. by repeated freeze-thaw followed by centrifugation. The protein yield was 30mg/ml for Tabernaemontana divaricata L. and 66mg/ml for Carissa carandas L. Sephadex G-75 fractionation yielded major peak TDP4 (19kDa) from Tabernaemontana divaricata L. and CCP1 (22kDa) from Carissa carandas L. with significant proteolytic activity. The enzymes showed dose-dependent caseinolytic, fibrinogenolytic, blood and plasma clot activities. TDP4 and CCP1 exhibited optimum pH at 6.8 and 7.8 & temperature at 400C ±20C and 250C ±20C respectively. TDP4 hydrolyzed blood clot, while CCP1 hydrolyzed plasma clot. Caseinolytic activity of TDP4 and CCP1 increased by 4-folds upon I²-mercaptoethanol treatment and inhibited by iodine suggesting that the enzymes are serine proteases.