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Chemical modifications by mass spectrometry from the PLA2 Bbil-TX isolated from the Bothriopsis bilineata venom: 'de novo' sequencing using the merge of different kinds of fragmentation and an overlaping of the peptides from a new PLA2 from B. taeniata

Grant number: 14/09869-2
Support type:Regular Research Grants
Duration: August 01, 2014 - July 31, 2015
Field of knowledge:Biological Sciences - Biochemistry - Chemistry of Macromolecules
Principal Investigator:Sergio Marangoni
Grantee:Sergio Marangoni
Home Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil

Abstract

The snake venoms are a complex functional molecules and manly a protein mixture, which along of the evolution trough of mutations and the natural selection for the species maintenance inside of the micro-evolution it was created a several toxins with a high functional complexity and high structural similarity. These toxins has different pharmacological effects such as neurotoxicity and myotoxic that generate a physiological issues paralyzing and initiating the digestion of the snake prey. The search for a new medicines trough comparing and chemical modeling with the site specificity for theirs targets, the pharmacy industry could take more than 11 years to development a new drug and spent a lot of money with the researches. The snake venoms toxins has a high specificity with theirs targets, they are used each time more as a biological models and has contributed to get a better understanding how each aminoacids of the polypeptide chain participate on the biological expression of the molecule. These toxins are widely used as a pharmacological tools and natural prototypic for the medical development, for example the Captopril (Power Hypotensive) drawn from peptide present on the Bothropsjararacasnake venom (BPF-bradiking-potentiating factor) improved by the natural selectionin millions of years. What supports this industrial strategy is that the toxins are biological model, isolated and purified by chromatography methods, having a high molecular homogeneity, physical and chemical characterized and pharmacologically analyzed. All results arising from this molecule could be attributed to the structural characteristics (Obtained from the protein sequencing) that contribute for the spatial organization of the molecule making a functional protein (Landres, P., 2004).This project pretends realize the chemical modifications of specific regions from PLA2, using a mass spectrometry to analyze this modifications and to determinate with a high precision which aminoacids was modified and the efficiency that this modifications occurs, we could infer the rule of this specifics regions on the pharmacological activities. We also propose to optimize the purification method of a new PLA2 isolated from the Bothriopsistaeniata venom, and with this allowed to do the "de novo" sequencing of this protein through an automatic "de novo" sequencing by Peaks Studio and using the high resolution mass spectrometer (VelosOrbitrap) to merge a different kinds of fragmentation HCD/CID/ETD/ to obtain a longer coverage and reliability of the data from the sequencing of this PLA2. Snakes commonly knew as a forest pit vipers are exceptionally lengthy and could vary the size (Campbell and Lamar., 2004). Bothriopsistaeniata are a rare viper from Brazil (Santos, 2003) and almost never could be found. This happen due the similarities with their body colors that fake the liver and lichens on the forest floor (Melgarejo, 2003). Has been reported one authentic snake bite with this snake also popularly knew as jararaca star and jararacatigrina (Torrez, et al, 2009; Miranda-Silva 2009). (AU)