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ISOLATION, KINETICS AND IN VITRO ANTI-LEUKEMIC ACTIVITY OF CAPSICUM ANNUM LGLUTAMINASE

Mohannad Idress, Mohammed Salim, Asia El-Hassan

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 Lglutaminase 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 Lglutaminase 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.