Isolation, Kinetics and in Vitro Anti-leukemic Activity of Capsicum Annum Lglutaminase

Abstract

This study is aimed at exploring the potential of developing naturally occurring L-glutaminase isolated from green chilies as anti-leukemic drug which selectively targets cancer cell metabolism unlike other nonselective chemotherapeutics. This was done by determination of its specific activity and optimum condition for L-glutaminase activity followed by partial purification and in vitro cytotoxicity assay.Activity of L-glutaminase was measured in the aqueous extract of fruits by Nesslerization method after homogenization in liquid nitrogen and extraction with 0.05 M sodium borate buffer (pH 8.5). Results showed that L-glutaminase activity per milligram of total protein was 18.7 U/mg.Optimum conditions for the catalytic activity of crude L-glutaminase extracted from chili fruits were studied. Results showed maximum activity of L-glutaminase was achieved when the enzyme was incubated with 250 mM of l-glutamine at 37°C for 30 minutes in the presence of phosphate buffered saline at pH 7.2. The maximum velocity (Vmax) and affinity constant (Km) of L-gutaminase were 14.1 mM and 90.2 mM respectively.Crude L-glutaminase was purified by salting out using seven different concentrations of ammonium sulfate ranging from 20% to 80% saturation. Specific activity of purified L-glutaminase was increased from 18.7 to 98.5 U/mg at when salted out in 50% ammonium sulfate solution. This result indicated the high efficiency of ammonium sulfate precipitation as purification technique.Assessment of anti-leukemic property was done by comparing the cytotixicity of L-glutaminase with doxorubicin and combination of them against THP-1 cell line by MTT assay. Results showed significant anti-cancer activity with inhibition percent of 72.75 % in case of combination of the enzyme with doxorubicin when compared to the enzyme alone (60.28%).These findings concluded that L-glutaminase of green chilies could be developed as anti-leukemic agent owing to its high content in chilies, ease of purification and signicant cytotoxicity against human monocytic leukemia cells.