Fractionation of the Venom of Iranian Cobra Snake by Gel Filtration Chromatography and Reverse-Phase HPLC and Evaluation of Antibacterial Activity of Isolated Fractions on Standard Bacterial Strains

For many years, antibiotic resistant infections including septicemia, burn infection, and peritonitis have been reported from many hospitals and still no antibiotic guarantees comprehensive treatment. Antimicrobial peptides are one of suggested solutions to overcome the problem. The goal of this study was tracing for an antimicrobial peptide in the venom of Iranian cobra snake, Naja naja oxiana and evaluation of its toxicity on human Red Blood Cells as well.
Snake venom was firstly fractionated using Gel filtration chromatography. The resulted fractions controlled for antibacterial activity against E. coli and Staphylococcus aureus. The fractions responsible for antibacterial activity were fractionated by Reverse Phase- HPLC. All major peaks were collected and controlled for activity against E. coli. Positive fractions selected to examination of activity against Pseudomonas aeruginosa (ATCC27853), Acinetobacter baumannii (ATCCBAA747), and Staphylococcus aureus (ATCC29213). Toxicity of candidate antibacterial fraction was evaluated by in vitro hemolysis assay.
The weight of proteins and peptides approximately ranged from 7KDa to 170 KDa. According to results, 8 fractions were obtained gel filtration chromatography. Among 8 fractions only 3 fractions were peptide and had the most area percent. Three fractions, F6, F7, and F8, selected for determination of MIC against E. coli and S. aureus. Two fractions had antibacterial activity at 50µg for both of examined bacteria. As the amount of fraction F7 was greater than F6, it was candidated for further fractionation by HPLC. 12 peaks were eluted and collected from column. Among the fractions, F4, F9, F10, and F12 were more effective against E. coli. Fraction 12 had the most activity as compared to the others. The weight of F12 approximately estimated as an 8KDa peptide. The MIC for F12 against E. coli, Staphylococcus aureus, and Pseudomonas aeruginosa were determined at 6.25, 25, and 50µg respectively.
We could find a bactericidal peptide. It is the first report for antibacterial activity of Iranian cobra snake venom. This peptide did not show any direct or indirect hemolytic activity. Natural compounds like antimicrobial peptides derived from venomous animals could be an effective solution to deal with multidrug resistant pathogen.