Purification and characterization of camel liver L-asparaginase

L-asparaginase from camel liver was isolated and purified by heat denaturation followed by QAE-Sephadex A-50 column chromatography and SP-Sepharose column chromatography. The purified camel liver L-asparaginase had a molecular weight of 180 kDa [consistent with a homotetramer] and a pI value of 8.6. The enzyme was thermostable with relative structure rigidity and an optimum temperature at 65°C. It had a pH optimum at 9.6 and was stable for storage at 4°C in the refrigerator for 7 days

Expression of recombinant escherichia coli L-asparaginase II, purification and characterization

L-Asparaginase [ASNase] is an anti-cancer [[antineoplastic] or [cytotoxic]] chemotherapy drug that is used for the treatment of acute lymphoblastic leukemia [ALL]. An efficient and economical scheme was developed for over expression and rapid purification of the Escherichia coli enzyme. The gene encoding for the Escherichia coli L-asparaginase was PCR-amplified and cloned in pGEX-4Tl expression vector. The recombinant L-asparaginase was purified to homogeneity by affinity chromatography on glutathione Sepharose column. The recombinant enzyme had an apparent MW of 152 kDa and a K[m] value of 12.5 microM for the main physiological substrate L-asparagine. The pI value was 5.6 while the turnover number [catalytic constant] was 1 x 10[2] s[-1] and the K[cat]/K[m] value [specificity constant] was 0.8 x 10[7] M[-1]s[-1]

Influence of ionizing radiation on Echis pyramidium snake venom: Biochemical and immunological aspects

The effect of a single LD[50] dose of native Echis pyramidum venom [27.69 micro g/mouse] on the activities of certain serum enzymes levels: aspartate aminotransferase [AST], alanine aminotransferase [ALT], alkaline phosphatase [ALP], urea, creatinine, lactate dehydrogenase [LDH], creatine phosphokinase [CPK], creatine kinase isoenzyme [CK-MB] were studied. Samples from the serum were collected 4hr following LD50 venom dose intraperitonealy injected in male Swiss albino mice. The activities of these enzymes showed significant elevation compared to the non-envenomated group. In contrast, an equivalent dose of 1.5 kGy gamma irradiated Echis pyramidum venom [27.69 micro g/mouse] did not cause any significant increase compared to non-envenomated group. The effect of a dose that is equivalent to ½ LD[50][13.8 micro g/50 micro l] of native Echis pyramidum venom on plasma creatine phosphokinase [CPK] induced a significant increase of creatine phosphokinase [CPK] level compared to normal control [P<0.01]. In contrast, an equivalent dose of 1.5 kGy gamma irradiated Echis pyramidum venom showed non significant difference in creatine phosphokinase activity when compared to the normal control. Light microscopic examinations of gastrocenemius muscles of mice injected with native Echis pyramidum venom [½ LD[50]; 13.8 micro g/50 micro l] showed fragmentation, disorganization, loss of myofibrils in some of the muscle fibers, hemorrhage in-between the muscle fibers and mononuclear cellular infiltration. While light microscopic examinations of gastrocenemius muscles of mice injected with 1.5 kGy gamma irradiated Echis pyramidum venom [13.8 micro g/50 micro l; a dose identical to that used from native venom] showed that most muscle fibers were of normal appearance except for small area of fragmentation and disorganized myofibrils and oedema of the intercellular connective tissue. Double immunodiffusion test revealed a similar reactivity for native, 1 kGy, 1.5 kGy and 3 kGy gamma irradiated Echis pyramidum venoms against a commercial polyvalent Egyptian antivenin. The visible lines obtained in the immunodiffusion reactions were identical and joined smoothly at the corners, indicating that there was no change in their antigenic reactivity. These results demonstrate that the ability of the venom antigens to react with its corresponding antibodies was maintained in spite of being exposed to radiation doses of 1 kGy, 1.5 kGy and 3 kGy. Both antivenins raised against native or 1.5 kGy gamma irradiated venoms recognized Echis pyramidum venom when submitted to protein blotting, but the anti 1.5 kGy gamma irradiated venom show a higher intensity bands than the antivenin raised against native Echis pyramidum venom, in spite of having less neutralizing activity [native neutralize 50 LD[50], 1.5 kGy gamma irradiated neutralize 40 LD[50]], this indicates that antibodies were formed not only for toxic fraction but also for non toxic fraction. Irradiation of the whole Echis Pyramidium Venom with 1.5KGy reduced its lethality 12.5 times though keeping its immunogenicity. The 1.5KGy dose was shown to be the best radiation dose to promote detoxification without significantly affecting its immunogenicity. Thus results of this study confirm the conclusion that gamma radiation is a suitable way to detoxify Echis Pyramidium Venom without affecting its immunogenicity provided that proper dose is used