Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Purification And Characterization Of A New Toxin From Lycosa erythrognatha With Antimicrobial And Insecticidal Activities. 1,2Santos, 1 Lab.Venenos 5Lab. D.M., 1,2Pimenta, A.M.C., 2,3Richardson, M., 2Bemquerer, M.P; 4Galuppo, C.D., 4Farias, L.M; 4Carvalho, M.A.R.; 5 De Maria, M.; 1,2De Lima, M.E. e Toxinas Animais; 2Núcleo de Biomoléculas - Depto. Bioquímica e Imunologia, 4Lab. de Microbiologia Oral e Anaeróbios – Depto. De Microbiologia; Aracnologia, Depto. Zoologia – ICB – Universidade Federal de Minas Gerais. 3Centro de Pesquisa e Desenvolvimento – Fundação Ezequiel Dias. Belo Horizonte, MG. Brasil. •Introduction ESI-Q-TOF mass espectrometry analyses Wolf spiders from Lycosa genus, are very common in urban areas in southeastern region of Brazil. Their venoms are poorly studied and knowing their components can be a step forward to prospect new molecules with biological activity. In this work, we initiate the biochemical characterization of the L. erythrognatha venom by using ion-exchange high performance liquid chromatography (RP-HPLC). We verify interspecifc variations in the composition of the venom related to the sex. Using ion-exchange and reverse phase high performance liquid chromatography we purify new peptide (LyeTx I, II, III and IV) with molecular mass of and 2830,90 and 2847.3 Da determined for ESI-Q-TOF mass spectrometry. LyeTx was sequenced by automatic Edman degradation and ESIQ-TOF and its predicted secondary structure display amphipathic a-helix, character typical of antimicrobial poreforming peptides. Antimicrobial assays showed this toxin potently inhibits the growth of pathogenic bacteria at micromolar concentrations. Yet, biological assays with the fraction 12C.4 have demonstrated a high lethality in house fly (Musca domestica) at nanomolar concentrations. Chemical synthesis of this molecule is in progress to obtain sufficient material to improve biological and structural studies. •Objectives 2831.10±0.02 2847.10±0.03 709.2862 A4 708.7852 B4 713.0342 713.2850 % 945.0499 945.3839 713.5367 A3 944.7153 950.3821 A5 567.4257 567.6243 950.7171 713.7865 A5 B5 567.2230 570.6227 951.0465 570.8254 571.0231 450 500 714.0401 A4 708.5255 571.2308 473.0190 544.8091 0 The main objective of this work was to initiate the characterization of Lycosa erythrognatha venom and the purification of bioatives compounds. A: B: A4 709.0356 100 952.3806 717.0351 721.0239 1417.0796 958.3829 m/z 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 Figure 3. Mass spectrometry (ESI-Q-TOF) of fraction 12C.4 (fig. 2). It is observed presence of two main masses. :A:2830,90 e B: 2847,30. Electrospray ionization source operated in positive ion mode. Samples were diluted in 50% acetonitrile/0.1% TFA in Milli-Q® water and applied with flow rates of 5-10 mL/min. •Material and methods: Simulation of the secondary structures of toxins obtained from the venom of L. erythrognatha. Animals and venom obtention. Spider were collected from both Belo Horizonte, MG, and Santa Bárbara, MG, regions, maintened in the aracnidarium at Laboratório de Venenos e Toxinas Animais, Departamento de Bioquímica e Imunologia (ICB– UFMG),. Spiders were classified as Lycosa erythrognatha and the venom was obtained by electrical stimulation of spider´s fangs. Cation exchange chromatography in HPLC system. Liophylized venoms were solubilized in Milli-Q water and loaded onto HPLC system using a Tosoh (TSK gel CMSW, 25cm x 4,6 mm) column. Reverse phase chromatography in HPLC system. Liophylized frations of venom were solubilized in 0.1% TFA and loaded onto HPLC system using a supelco C18 (25cm x 4.6mm) column. Gradient was carried out with 0,1% TFA in acetonitrile. ESI-Q-TOF mass espectrometry analyses. ESI-Q-TOF mass spectrometry analyses were carried out using a Q-TOF MicroTM (Micromass, UK) equipped with an electrospray ionization source operated in positive ion mode. Capillary voltage was 3-3.5 kV and sample cone voltages were 40-60V. Mass spectrometer calibrations were made by using sodium iodide with ceasium iodide in 2000 Da range. Samples were diluted in 50% acetonitrile/0.1% TFA in Milli-Q® water and introduced by using a syringe pump with flow rates of 5-10 mL/min in electrospray source. Collision induced dissociation (CID) was carried out using argon and collision energies in the range 30-45V. Data were analyzed by MassLynx® 3.5 software. LyeTx I and II LyeTx III LyeTx IV Figure 4. a-helical wheel plot of LyeTx I, II, III and IV showing amphipathic character. The toxins had been sequenced from the fraction 12C.4 for automatic degradation of Edman and spectrometry of mass (ESI-Q-TOF and MALDI-TOF-TOF), The obtained sequences presented great similarity with the Lycotoxin I purified from venom of Lycosa carolinensis (YAN & ADAMS, 1998). The peptides in the a-helical wheel plot present periodic variation in the hydrophobicity value of the residues along the peptide backbone with a 3.6 residues/cycle period characterizes an ahelix. The amino acid sequence of LyeTx begins with isoleucine (blue arrow) and proceeds in a clockwise direction. Hydrophobic aminoacids; Polars aminoacids; Charged aminoacids (+); Charged aminoácids (-). Tests of antimicrobian activity Antimicrobial tests. The bacterias were cultered on BHI (brain Heart Infusion) plus levedure extract (5%). Tha agar diffusion method was performade for screening the antibacterial activity. The fractions of venom were applied on the discs (0,5mm diameter) and added to petri plates previously inoculated with the bacterial strain. After 24 h at 37o C, the inhibition zone was evaluated. A B C •Results Cation exchange chromatography in HPLC system. A 200 100 80 Figure 5. Tests of inhibition of the growth of bacteria: (A) Staphylococcus epidermides; (B) Staphylococcus aureus; (C) Escherichia coli. Paper of filter containig 5 mg of the fraction 12C.4. Bacteria. Halo of inhibition of the growth. 150 60 Biological assay in house fly. 100 40 20 50 0 The effect of the intratoraxic injections of the fraction 12C.4 in flies had started immediately. The flies had presented a fast contraction of the members. The minimum dose for activity was of 0,75 ng/fly and the calculated DL50 was of 14 ng/fly 0 0 20 40 60 80 100 Time (min) B 200 100 80 •Conclusions 150 60 100 40 * The venom of L. erythrognatha presents intraspecific variations. 20 50 * * 0 Five peptides were sequenced from the venom of L. erythrognatha. 0 0 20 40 60 80 100 Time (min) The obtained sequences presented great similarity with the Lycotoxin I purified from venom of Lycosa carolinensis. Figure 1: Gender variations in Lycosa erythrognatha venoms. HPLC cation exchange profiles of venoms from adult male (A) and female (B) specimens. ( ) red marks show fractions present only in female specimens. * Green arrow show the fraction 12C. Tosoh (TSK gel CM-SW, 25cm x 4,6 mm) column.Gradient was carried out with NaCl (1M) in sodium acetate (10mM), pH 5,0. Flow rate was 1,0 mL/min The fraction 12C.4 presented antibacterial action in pathogenic bacterias. Reverse phase chromatography in HPLC system 500 100 400 80 300 60 200 40 100 20 Biological assays with the fraction 12C.4 have demonstrated a high lethality in house fly. •References YAM, L., ADAMS, M. E. Lycotoxins, antimicrobial peptides from venom of the wolf spider Lycosa carolinensis. J. Biol. Chem., 273, 2059-2066, 1998. 0 0 20 Time (min) 40 60 Figure 2. HPLC reverse phase profile of fraction 12 C. Red arrow show the fraction 12C.4. Supelco (C18 Supecosil) column, equilibrated with 0,1 % TFA and eluted by linear gradient of 0,1 % TFA in acetonitrile. Supported by CAPES, CAPES-PRODOC, FAPEMIG, MCT-INFRA and CNPq.