the S30 portion was obtained by ultracentrifugation. The gene sequences encoding the DC/HF3-Mut Acid and DC/HF3-Mut Alkaline were optimized, synthesized and inserted in the vector pIVEX2.3d. For expression of DC/ HF3-Mut Acid, DC/HF3-Mut Alkaline and wild-type proteins, a small-scale expression was used to optimize the reaction conditions, such as magne- sium, potassium concentration and redox conditions. Results and Discussion: To evaluate the protein composition of the S30 lysate, the extract was analyzed by mass spectrometry, and many proteins for translation and expression were identified. The mutant proteins DC/ HF3-Mut Alkaline and the wild-type protein DC/HF3 were expressed in the soluble form using different proportions of GSH and GSSG. The proteins were purified, and their identity was confirmed by WB using an anti 6X- His Tag antibody. Financial support: FAPESP: 2017/09648-4 and 2013/ 07467-1. 008 GLYCOPROTEOMIC COMPLEXITY OF BOTHROPS SNAKE VENOMS AND SIALIC ACID CONTRIBUTION IN TOXIN FUNCTION Carolina Br as Costa, D ebora Andrade Silva, Daniela Cajado Carvalho, Solange Maria de Toledo Serrano. Laborat orio Especial de Toxinologia Aplicada, Cetics, Instituto Butantan, S~ ao Paulo, SP, Brazil Introduction and Objectives: Protein glycosylation is one of the major post-translational modifications (PTMs) in viperid venoms, and contrib- utes to diversification of proteomes. We have shown that Bothrops venoms are markedly defined by their content of glycoproteins. We also reported the N-glycan structures present in eight Bothrops venoms and demon- strated that most of them contain sialic acid. To further investigate pro- teome venom variation and the mechanisms involved in the generation of different venoms by related snakes, in this investigation we are analyzing the subproteomes of glycoproteins selected by lectins with different saccharide specificities and the role of sialic acid in the activity of venom proteinases. Material and Methods: Venom from B. cotiara, B. insularis, B. jararaca, B. moojeni, B. neuwiedi, B. jararacussu, B. erythromelas, B. atrox and B. fonsecai was subjected to affinity chromatography using the lectin from Sambucus nigra (SNA-agarose), which binds to sialic acid attached to terminal galactose in a-2,6 linkage, and the Phaseolus vulgaris eritroaglutinin (PHA- E-agarose), which recognizes bisecting N-acetylglucosamine structure. Protein profiles were compared by SDS-PAGE and identification was car- ried out by in-solution trypsin digestion and LC-MS/MS. Sialic acid was removed from venom toxins using neuraminidase, and confirmation of removal was evaluated by SDS-PAGE and Western blot using Mackia amurensis lectin, which recognizes terminal sialic acid. Venom hydrolytic activities were determined using gelatin, fibrinogen and Bz-Arg-pNA. Results and Discussion: The identification of PHA-E-binding proteins in Bothrops venoms revealed around 80 unique toxins in each venom, and most of identified proteins were proteinases. The number of SNA-binding proteins, contrarily, was lower than that observed with PHA-E, suggesting that the presence of sialic acid attached to galactose in a-2,6 linkage in Bothrops venoms may be limited to specific toxins. Removal of sialic acid changed the pattern of gelatinolysis of most Bothrops venoms, and decreased the hydrolytic activity of proteinases on fibrinogen. In contrast, the amidolytic profile of Bothrops venoms did not change after the treat- ment with neuraminidase. This study reveals a hitherto little explored form of Bothrops venom proteome variability. Supported: CNPq and FAPESP (2017/09929-3). 009 ANTIPARASITIC, ANTIPROLIFERATIVE AND TOXICITY ACTIVITIES OF TWO ANALOGS PEPTIDES FROM STIGMURIN OF THE SCORPION TITYUS STIGMURUS Adriana Marina E Silva Parente 1 , Bruno Amorim-Carmo 1 , Alessandra Daniele Da Silva 1 , Allanny Alves Furtado 1 , Claudia Jassica Moreno 2 , Johny Wysllas De Freitas Oliveira 2 , Marcelo De Sousa Da Silva 1 , Matheus De Freitas Fernandes Pedrosa 1 . 1 Universidade Federal Do Rio Grande Do Norte, Natal, RN, Brazil; 2 Universidade Federal Do Rio Grande Norte, Natal, RN, Brazil Introduction and Objectives: Stigmurin (FFSLIPSLVGGLISAFK) is an anti- microbial peptide identified by transcriptomic analysis of the venom gland of the scorpion Tityus stigmurus that present activity against Gram-positive bacteria with antiproliferative activity and low hemolytic activity. From this molecule, StigA6 (FFSLIPKLVKGLISAFK) and StigA16 (FFSLIPKLVKGLI- SAFK) analog peptides with higher net charge and hydrophobic moment were designed aiming an enhanced antimicrobial activity. The analog peptides showed lower minimum inhibitory concentration and broader activity spectrum against Gram-positive and Gram-negative bacteria. Thus, the aim of this study was to evaluate the antiparasitic and anti- proliferative activity as well as the toxicity of the analog peptides comparing with the native peptide. Material and Methods: For the antiparasitic activity, epimastigote and trypomastigote forms of Trypanosoma cruzi were incubated with the peptides in serial dilution and T. cruzi inhibition was evaluated by the MTT reduction assay, and epimastigote forms of Leishmania amazonensis, also incubated with serial dilution of the peptides, and the inhibition were evaluated using resazurin reduction assay. The peptides were also tested against normal and cancerous cell lines in order to assess their toxicity and antiproliferative activitie, and cell inhibition was evaluated using the MTT reduction assay. Results and Discussion: StigA6 and StigA16 were able to inhibit 100% of both forms of T. cruzi at 5 mM, while Stigmurin was able to inhibit at 25 mM. For the Leishmania assay, the analog peptides showed 100% inhibition at lower concentrations than Stigmurin, indicating a higher antiparasitic activity. For the antiproliferative assay we calculated the IC50 for the cell lines tested, the lowest IC50 observed for the cancerous cells was 4.60mM to Panc-1 ATCC cell line. Regarding the normal cell line, 3T3 ATCC, the peptides showed higher IC50 (14 and 13 mM) indicating that the analogs are less toxic for normal cell lines. Therefore, the analog peptides showed higher antiparasitic activity against Trypanosoma and Leishmania than Stigmurin and presented lower toxicity to normal cell while showing antiproliferative activity against cancerous cell lines. 010 PONTENT BIOATIVE AND BROAD-SPECTRUM ANALOGS OF THE SCORPION PEPTIDE STIGMURIN Bruno Amorim-Carmo 1 , Adriana Marina E Silva Parente 1 , Alessandra Daniele-Silva 1 , Allanny Alves Furtado 1 , Johny Wysllas De Freitas Oliveira 1 , Eneas Carvalho 2 , Marcelo De Sousa Da Silva 1 ,S ergio Ruschi Bergamachi Silva 1 , Matheus De Freitas Fernandes Pedrosa 1 . 1 Universidade Federal Do Rio Grande Do Norte, Natal, RN, Brazil; 2 Instituto Butantan, S~ ao Paulo, SP, Brazil Introduction and Objectives: Scorpion venom constitutes a rich source of biologically active compounds with high potential for therapeutic and biotechnological applications that can be used as prototypes for the design of new drugs. The aim of this study was to characterize the structural conformation, evaluate the antimicrobial activity, and gain insight into the possible action mechanism underlying it, for two new analog peptides of the scorpion peptide Stigmurin, named StigA25 and StigA31, with higher net charge and hydrophobic moment. Material and Methods: We used circular dichroism techniques and mo- lecular modeling to characterize the peptides structure, as well as their stability at different pHs and temperatures. The antimicrobial activity was evaluated by broth microdilution technique. Molecular dynamics and scanning electron microscopy (SEM) were used to evaluate a possible mechanism of action. In addition, we evaluated the antiparasitic activity in Y strains of Trypanosoma cruzi. Cytotoxicity was determined by the rate of hemolysis in human erythrocytes. Results and Discussion: Stigmurin, StigA25, and StigA31 showed the ca- pacity to modify their structural conformation in different solvents, with stability to high pH and temperature range. The analog peptides demon- strated broad-spectrum antimicrobial activity, and a high parasite inhibi- tion rate, showing an effect superior to the native peptide, not being hemolytic at biologically active concentrations. At the molecular dynamics StigA31 presented the highest propensity to interact with the microbial membranes, corroborating the data obtained in vitro. We observed through SEM that the peptides act on the cell wall of Staphylococcus aureus without cell lysis. Therefore, this study demonstrates the therapeutic Abstracts / Toxicon 168 (2019) S1eS42 S11