Determination of the effect of EDTA and PCMB on purified bromelain activity from pineapple core and in vitro antiplatelet activity

Abstract

Bromelain is an enzyme belongs to the cysteine protease. In this study, bromelain isolated from pineapple core (Ananascomosus [L.] Merr) was purified by fractionation using ammonium sulfate followed by dialysis and then ion exchange chromatography. The fraction of bromelain obtained from each purification step showed an increase in specific activity. The highest specific activity of protease was found in 20-50% ammonium sulfate fraction of 104.018 U/mg with a purity level 3.2-fold compared to crude extract. Further purification by ion exchange chromatography using DEAE-Cellulose, the fraction of bromelain showed an increase in specific activity to 278.333 U/mg with a purity level 8.8-fold compared to crude extract. The determination of kinetics parameter of purified bromelain using Lineweaver-Burk plot gives Km value of 0.15 % (w/v) and Vmax of 0.056 U/min. This bromelain can be strongly inhibited by EDTA and PCMB. The addition of EDTA and PCMB at a concentration of 0.5 mM can decrease the activity of the enzyme up to 88.50% by showing the competitive and mix-inhibition types of inhibition, respectively. The antiplatelet activity of the bromelain fraction was tested in-vitro based on the Born method, by using plasma (PRP), acetosal as a positive control and ADP as an aggregator. The purified bromelain showed the ability of an antiplatelet agent with percentage of aggregation 29.51% and percentage of inhibition 68.91%.