Isolation, Partial Purification and Characterization of L-Asparaginase from Hedgehog Serum

Abstract

The need to further explore other sources of therapy for leukemias prompted this current work. It’s already known that L-asparaginase possess a lot of potentials in the treatment of cancers of the white blood cell but the sources of these enzyme and its properties determines the efficacy of this drug. Generally, for an asparaginase to be ideally suited for use in anti-tumour therapy, it had to satisfy a variety of criteria. The organism that is selected should produce the asparaginase in high quantity or yield, and it should be capable of being isolated from simple mammalian sources. The procedures developed for purification of the enzyme should be as rapid and simplified as possible. The purified enzyme should have long term stability on storage, maximal activity at a physiological pH, and a Km for substrate below the concentration of the substrate in the blood. Therefore the aim of this present research was to partially purify and characterize this enzyme from Hedgehog serum in order to compare its anti-leukemic potentials with those from documented literature for possible future medical application. L-asparaginase was isolated and partially purified from Hedgehog serum which is also known to be a reservoir of many other useful proteins including anti venoms using a four step profile of ammonium sulphate fractionation, dialysis, and ion exchange and gel filtration chromatography. The enzyme gave an overall yield of 77.58%, optimum pH and temperature of 7.8 and 39oC respectively, and a Km of 0.0125 mM. Gel filtration gave an approximate molecular weight of 139,000 Da, while SDS PAGE indicated a subunit molecular weight of 36,000 and 34,600 Da respectively. The enzyme also catalyses the hydrolysis of glutamine though slightly. Mg2+ and Zn2+ serve as activators for Hedgehog serum L-asparaginase, while inhibition from heavy ions like Hg2+ was also observed. This work shows that mammalian sources of this enzyme could be more preferable for treatment of asparagine dependent tumours in time to come.