Coordinators: Șerban Andrei Gâz Florea Bogdan Cormoș Authors: Diana Ciurca Anca-Teodor a Berdan University of Medicine and Farmacy of Tîrgu Mures, Faculty of Pharmacy Marisiensis, 2014

From ancient times - scientists - tried to unlock the medical potential of snake venom; Greek religion - snake - divine and used in healing rituals; Snake venom - complex mixture of enzymes and toxins - fast-acting and potent molecules. Naja haje haje, the Egyptian cobra is a species from the Elapidae family, which possesses a neurotoxic venom, a grossly underexplored resource in pharmacological prospecting. A proper method of identification and separation New possible therapeutic agents Vonk F, Jackson K, Dole R et al - Snake venom: From fieldwork to the clinic, Bioessays, 2011, 33: 269–279;

Proteins constitute 90-95% of venom’s dry weight and they are responsible for almost all of its biological effects. Fox J, Serrano S - Exploring snake venom proteomes: multifaceted analyses for complex toxin mixtures, Proteomics, 2008, 8: 909–920;

The most important proteins - toxins and enzymes Mackessy S - Handbook of Venoms and Toxins of Reptiles, CRC Press, New York, 2010,

Venom analysis 3 steps gel electrophoresis RP-HPLC snake venom extraction Snake venom can be extracted only by experienced snake handlers ! After a secure immobilization of its head, the snake must be guided to bite a special membrane, covering the venom’s recipient. We kept the venom at low temperature to prevent protein degradation. Mackessy S - Handbook of Venoms and Toxins of Reptiles, CRC Press, New York, 2010, Snake venom extraction Objective: to develop an optimal RP-HPLC and gel electrophoresis methods for separation and identification of Naja haje haje snake venom components.

Venom analysis 2. Reversed-phase High-performance liquid chromatography (RP-HPLC) Due the high resolving power of the technique, RP-HPLC is the only method of choice for the separation, purification, and analysis of complex protein mixtures. HPLC chromatographic parameters: quaternary pump 2 mobile phases: A (0,1% TFA in 5%ACN) and B (0,1% TFA in 95% ACN) elution gradient (5-45% mobile phase B) at a flow of 0.6 mL/min mobile phase A at pH=2,4 and pH=7,4 the samples were prepared by diluting pure venom with mobile phase A injection: 100 µL, at a temperature of 6°C C 18 RP-HPLC column (4,6 mm x 250 mm) UV detection at different wavelength values (210, 254, 350 nm). “Ultimate 1100” Dionex HPLC system

Venom analysis 2. RP-HPLC pH=2,4 Optimization of HPLC method 6.32 pH=7,4

Venom analysis 2. RP-HPLC Optimization of HPLC method λ=350 nm λ=254 nm

Venom analysis 2. RP-HPLC Our venom contains short, long and miscellaneous neurotoxins, cytotoxins, phospholipase A 2. Shafqat J, Beg O, Yin S et al - Primary structure and functional properties of cobra (Naja naja naja) venom Kunitz-type trypsin inhibitor, Eur. J. Biochem, 1990, 194: The best resolution was obtained at a pH value of 7,4 and at a wavelength value of 210 nm

Venom analysis 3. Gel electrophoresis – agarose gel Electrophoretic parameters: Voltage = 90 V; Intensity = 110 mA. AccuGENE Water + Molecular Water 9:1; Agarose gel 2% + ethidium bromide (fluorescent tag). sample: 10µL of pure venom; Molecular Weight Marker.

Venom analysis 3. Gel electrophoresis Sample – pure venom and diluted venom - no migration; The best approach is by using a vertical gel electrophoresis system and a polyacrylamide gel (SDS-PAGE). Concentrated venom Weight Marker

Venom – therapeutic potential Venom analysis Presence of Phospholipase A 2 Development of new antibacterial agents? 1. Demonstration of Naja haje haje snake venom’s antibacterial properties We prepared a 0,5 McFarland inoculum (1,5 * 10 8 UFC/mL) using two reference strains of bacteria: Staphylococcus aureus ATCC and Escherichia coli ATCC We inoculated Muller-Hinton (Agar) medium with bacterial suspension. We inserted sterile filter paper discs and we inoculated Naja haje haje snake venom on them. We inoculated both newly collected venom, and refrigerated venom. Microbiologic parameters: - - Time of incubation: 24 hours; - Temperature: 37°C.

Venom – therapeutic potential 1. Antibacterial properties Escherichia coli ATCC 35218: left – refrigerated venom – no effect; right – fresh venom – no effect. Staphylococcus aureus ATCC 29213: left, up – 5 µL of fresh venom; right, up – 20 µL of fresh venom; down – 5 µL of refrigerated venom. The venom doesn’t inhibit bacterial growth on Escherichia coli; The venom inhibits Staphylococcus aureus growth; Newly collected venom will induce a greater antibacterial effect than refrigerated venom; even by refrigeration we can’t completely prevent protein degradation.

Conclusions Recent discoveries reveal an unexpectedly vast degree of variation in venom composition, showing that this resource represents an enormous potential for pharmacological prospecting. A proper identification and separation of its compounds is an important step towards finding new therapeutic agents. The best HPLC separation is obtained at a mobile A pH value of 7,4 and at a wavelength value of 210 nm. The HPLC separation will be followed by SDS-PAGE vertical gel electrophoresis. With the recent emergence of methicillin (MRSA) and vancomycin (VRSA) resistance, Staphylococcus aureus infection is a serious clinical problem that will have a grave socio-economic impact in the near future. From our best knowledge this is the first demonstration of Naja haje haje snake venom antibacterial properties by inhibiting the growth of Staphylococcus aureus cultures. This research can represent the starting point of developing new antibacterial drugs.

Acknowledgment We would like to express our sincere thanks to everyone who contributed at this research: University of Medicine and Pharmacy’s Research Laboratory and Experimental Station, and especially to Conf. Dr. Augustin Curticapean; University of Medicine and Pharmacy’s Department of Microbiology, and especially to Dr. Adrian Man and Dr. Anca Delia Mare; University of Medicine and Pharmacy’s Department of Genetics, and especially to Dr. Claudia Bănescu. *The work is a product of the intellectual environment of the whole team and all members have contributed in equal degrees to the analytical methods used!

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