الفهرس 
Snake venomOnly 14 pages are availabe for public view
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Abstract
In this study, a cytolytic compound (Fraction VIb) was isolated from the venom of Egyptian Cobra Snake, Naja nigricollis nigricollis. First, the crude venom was fractionated by gel filtration chromatography into four fractions (Ia, IIa, IIIa, and IVa). The cytotoxicity of each fraction was determined by measuring the percentage of red blood cells’ hemolysis.
Although Fraction IVa showed the highest hemolytic effect on red blood cells, Fraction IIIa was chosen for further fractionation since it had the highest protein content. Using ion exchange chromatography Fraction IIIa was separated into six fractions: Ib, IIb, IIIb, IVb, Vb, and VIb. The six fractions were tested on red blood cells for their hemolytic effect. Fraction VIb was the one with highest cytolytic effect on red cells.
The cytolytic activity on red cells was also re-estimated after Fraction VIb was subjected to different temperatures and incubated at different pH values. Our protein of interest was found to be stable at different temperatures (up to 100ºC) and at different pH ranges (5-9).
The isolated protein was found to be partially pure as indicated by disc polyacrylamide gel electrophoresis (SDS-PAGE), with molecular weight approximately 10 kDa. Fraction VIb was also tested for protease and phospholipase activity and the results showed that, it is devoid of any protease or phospholipase activity.
On testing the carbohydrate content of fraction VIb, it was found that Fraction VIb is a glycoprotein.
Spectroscopy of fraction Fraction VIb by U.V. absorbance was done and showed that the protein had a maximum absorbance at 280nm. This means that Fraction VIb contains tyrosine and/or tryptophan amino acid.
Antigenicity of the cytotoxic fraction was assessed by Ouchterlony technique, and it was found to be non antigenic (most probably due to small molecular size, around 10 kDa). It did not show any precipitin bands with the polyvalent antivenin from VACSERA.
The lethality of Fraction VIb was determined by injecting mice with different doses and LD50 was found to be 1.83 mg/ kg body weight.
Histopathological changes in mice liver, kidney and bone marrow were tested after injection with Fraction VIb. The liver tissue showed areas of vacuolation and swelling due to cloudy and hyDROPic degeneration.
Kidney biopsy showed areas of pallor due to cloudy swelling, infiltration with sheets of inflammatory cells and accumulation of hyaline casts inside tubular lumens due to hyDROPic degeneration.
Bone marrow biopsy revealed hypercellular bone marrow with dilated congested blood vessels and increased granulocytic series.
Electron microscope examination for mouse liver after injection with Fraction VIb showed accumulation of fat DROPlets, dilated endoplasmic reticulum, swollen mitochondria and ruptured mitochondrial cristae.
To study the mechanism of action of cytotoxin Fraction VIb, DNA extraction was done from mouse liver tissue after invitro incubation with Fraction VIb. This step was followed by agarose gel electrophoresis which revealed that the extracted DNA was separated as an intact single band, indicating that apoptosis is not involved in the cytotoxic mechanism.
DNA extraction was also done from hepatocellular carcinoma (HEPG2) cell line after incubation with Fraction VIb. The DNA was separated as an intact single band with no fragmentation.
Fraction VIb probably causes cell lysis by a series of events including, damage to cell membrane, degeneration in the cytoplasm, presented by vacuolization as a result of cloudy and hyDROPic degeneration, organelle damage, presented by mitochondrial swelling and disruption of mitochondrial cristae.
The effect of Fraction VIb was assessed on malignant cells by incubating the fraction with different types of malignant cells in tissue culture plates. It was found that small doses caused cytotoxicity in breast carcinoma cell line, colon carcinoma cell line and hepatocellular carcinoma cell line
Conclusion
Fraction VIb isolated from crude Naja nigricollis nigricollis venom is a cytolytic protein, approximately 10 kDa. It has neither phospholipase nor protease activity, stable at different pH values and different temperatures up to 100ºC. It is lethal to mice, with LD50 1.83 mg/kg. The cytotoxic protein is non antigenic and is glycoprotein in nature. Testing the cytotoxic activity of Fraction VIb revealed that small doses induced cytolytic effect on hepatocellular carcinoma, colon cancer and breast cancer cells while larger doses caused hemolysis of red blood cells. Thus, this protein can be considered a target compound for further researches as it can be a potential biotherapeutic agent for cancer treatment, or a model for new drug design.