057 IDENTIFICATION AND PURIFICATION OF PHOSPHOLIPASE A2 INHIBITORS (PLIS) FROM SNAKE BLOOD MAINTAINED IN THE LABORATORY OF HERPETOLOGY OF BUTANTAN INSTITUTE Victor Koiti Kavazoi 1 , Daniela Miki Hatakeyama 2 , Caroline Serino- Silva 2 , Caroline Fabri Bittencourt Rodrigues 2 , Kathleen Fernandes Grego 2 , Karen Morais-Zani 2 , Anita Mitico Tanaka- Azevedo 2 . 1 Universidade Presbiteriana Mackenzie, S~ ao Paulo, SP, Brazil; 2 Instituto Butantan, S~ ao Paulo, SP, Brazil Introduction and Objectives: The complex mixture of the venom com- ponents of venomous snakes is mainly composed of proteins, most of them responsible for the venom toxicity. Venomous and non-venomous snakes can have phospholipase A 2 (PLA 2 ) inhibitory proteins (PLIs) in their serum. The PLIs are acid oligomeric and globular glycoproteins that form soluble complex with PLA 2 s, thereby inhibiting their enzymatic activity. These inhibitors are classified into a, b and g, according to their structural fea- tures. The aim of this study is to identify PLIs in the serum of several snake species maintained in the Laboratory of Herpetology of Butantan Institute. Material and Methods: Initially, nineteen snake specimens were selected, and their blood samples were collected via puncture of the caudal vein. The collected blood was kept in the refrigerator for blood coagulation prior to centrifugation, at 1200 g. Then, affinity chromatography was performed coupling 1mg of crotoxin, isolated from Crotalus durissus terrificus, to 0,1g of CNBr-activated Sepharose, as described by the manufacturer (GE Healthcare). SDS-PAGE (12%) was performed to verify the PLIs present in different snake serum. Western blotting was made to confirm the inter- action of PLIs and PLA 2 . Results and Discussion: Through affinity chromatography we purified the PLIs from different snake species serum and we identified them by SDS- PAGE. The molecular mass obtained by SDS-PAGE was around 25kDa, which corresponds to PLI described by other studies. The Western blotting was performed to confirm the interaction between PLI and PLA 2 , thus most snakes’ serum have PLIs. It is interesting to observe that some of them have already been identified, like Bothrops jararaca, B. moojeni, B. alternatus, B. atrox, and other PLIs were observed for the first time in this project: B. marajoensis, B. matogrossensis, B. leucurus and B. fonsecai. Acknowledgment: FAPESP (2017/01890-0; 2017/16908-2; 2017/17165-3). 058 EVALUATION OF CROTALUS NEUTRALIZING FACTOR (CNF) ON CROTOXIN PARALYZING ACTIVITY IN MICE NEUROMUSCULAR PREPARATIONS ^ Emylle Karoline Ramos Pinto 1 , Nat alia Muradas Val erio Souza 1 , Paula Ladeira Ortolani 2 , Consuelo Latorre Fortes-Dias 2 , Walter Luís Garrido Cavalcante 1 . 1 Department of Pharmacology, Institute of Biological Science, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil; 2 Research and Development Center, Fundaç~ ao Ezequiel Dias (Funed), Belo Horizonte, MG, Brazil Introduction and Objectives: Crotalus Neutralizing Factor (CNF) is a phospholipase A2 inhibitor present in the blood plasma of Crotalus dur- issus terrificus (C.d.terrificus) snake. This substance is capable to neutralize the in vivo toxic effects of Crotoxin (CTX), a potent neurotoxin found in the C.d.terrificus venom. However, there are no in vitro studies in neuromus- cular preparations showing the inhibitory action of CNF. Thus, the objec- tive of this study was to evaluate the ability of CNF to inhibit the neuromuscular blockade induced by CTX. Material and Methods: Paralyzing activity was evaluated through the recording of isolated contractions (0.5 Hz) evoked indirectly in phrenic nerve-diaphragm muscle preparations during 120 minutes. The prepara- tions were exposed to CTX (5 mg/mL) or CNF (50 mg/mL) or the mixture of preincubation of CTX (5 mg/mL) with CNF (5 or 20 or 50 mg/mL) for 15 minutes at 35  C. The experimental procedures were submitted and approved tothe Ethics Committee, Protocol CEUA: 17/2017. Results and Discussion: The Control and CNF groups did not induce al- terations in the amplitude of indirectly evoked twitches over the time. CTX 5 mg/mL alone induced the total blocked of muscle contractions in 60 minutes. This toxic effect was not neutralized when CTX 5 mg/mL was preincubated with CNF 5 mg/mL. However, a partial neutralization was noted when CTX 5 mg/mL was preincubated with CNF 20 mg/mL. The total inhibition was observed in 120 minutes when CTX 5 mg/mL was pre- incubated with CNF 50 mg/mL. Therefore, the present study showed for the first time that CNF is able to inhibit the paralyzing activity of CTX in neuromuscular preparations in vitro. Acknowledgments: Coordenaç~ ao de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundaç~ ao Ezequiel Dias (FUNED) and Pr o-Reitoria de Pesquisa da UFMG (PRPQ). 059 OLEANOLIC AND URSOLIC ACIDS AS ANTIOPHIDIAN AGAINST BOTHROPS JARARACUSSU VENON IN ANIMAL MODEL Jocimar De Souza 1 , Bruna Stramandinoli Deamatis 1 , Fernanda Mayumi Ishii 1 , Ingrid Francine Araújo De Oliveira 2 , Gustavo Rodrigues Toledo Piza 3 , Edson Hideaki Yoshida 1 , Jos e Carlos Cogo 4 , Angela Faustino Jozala 2 , Denise Grotto 3 , Rauldenis Almeida Fonseca Santos 5 , Marcos Antonio De Queiroz Júnior 1 , Caroline Malavasi Barros 1 , Yoko Oshima- Franco 1 . 1 Laboratory of Research in Neuropharmacology and Multidisciplinary (Lapenm), Sorocaba, SP, Brazil; 2 Laboratory of Industrial Microbiology and Fermentation Process (LAMINFE), Sorocaba, SP, Brazil; 3 Laboratory of Toxicological Research (LAPETOX), Sorocaba, SP, Brazil; 4 Bioengineering and Biomedical Engineering Programs, Technological and Scientific Institute, Sorocaba, SP, Brazil; 5 Federal Institute of Education, Science and Technology of Rondonia, Sorocaba, SP, Brazil Introduction and Objectives: Plants are source of many bioactive mole- cules which are much attractive due to their medical properties, including complementary alternatives to antivenom therapy. Oleanolic (OA) and ursolic acids (UA) are phytochemical easily found in many plants and have several biological activities such as antibacterial, antimicrobial, anticancer/ antitumour, antidiabetic, antihypertensive, anti-inflammatory, antioxi- dant, antiparasitic, and hepatoprotective. To evaluate the potential of OA and UA as antiophidian, not yet described in the literature. Material and Methods: The experiment was approved by Animal Research Ethical Committee, CEUA-UNISO protocol number 93/2016. Paralysis and myotoxicity induced by Bothrops jararacussu venom were assayed using mouse phrenic nerve-diaphragm preparation. OA and UA 300 mg/mL were tested against 60 mg/mL of venom. Samples of nutrient solution were collected at time zero and after 2h. Oxidative stress biomarkers as reduced glutathione (GSH), Thiobarbituric acid reactive substance (TBARS), and Minimal Inhibitory Concentration (MIC) against Staphylococcus aureus were evaluated. Results and Discussion: Only OA protected against venom blockade (71.6%±6.4 at 120 min); while the venom injured 76.8±0.2, OA minimized the venom to 54.3±0.2. GSH has increased in all groups, and can be attributed to its release from the preparation, rather than to the antioxi- dant action of the acids. Only the venom increased TBARS, possibly due to membrane lysis and the release of fatty acids. OA and UA presented MICs of 31.3 mg/mL and 62.5 mg/mL, respectively, 10 and 5 times lower than the initial solution (300 mg/mL). OA, but not AU, minimized venous blockade and also myotoxicity. Antioxidant activity of OA and AU were observed only in TBARS, since levels increased in the group that received only the poison and decreased in the groups that received the acids + venom. In addition, OA and UA showed antimicrobial activity. 060 INTEGRATIVE STRUCTURAL ANALYSIS OF THE ANTIHEMORRHAGIC PROTEIN DM43 AND ITS TARGET TOXINS BAP1 AND JARARHAGIN Barbara Da Silva Soares 1 , Surza Lucia Gonçalves Da Rocha 2 , Diogo Borges Lima 3 , Mariana Fioramonte 4 , F abio Cesar Gozzo 4 , Teresa Escalante 5 , Alexandra Rucavado 5 , Jonas Perales 2 , Richard Hemmi Valente 2 , Francisco Gomes-Neto 2 , Ana Gisele Da Costa Neves- Ferreira 2 . 1 IOC/FIOCRUZ (Laboratory of Toxinology), Incttox, Rio De Janeiro, RJ, Brazil; 2 IOC/FIOCRUZ (Laboratory of Toxinology), Incttox, Rio De Janeiro, RJ, Brazil; 3 Pasteur Institute (Structural Mass Spectrometry and Proteomics Unit), Paris, France; 4 UNICAMP (Dalton Mass Spectrometry Laboratory), Campinas, SP, Brazil; 5 Instituto Clodomiro Picado (Facultad De Microbiología, Universidad De Costa Rica), San Jos e, Costa Rica Abstracts / Toxicon 168 (2019) S1eS42 S26