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CORE Metadata, citation and similar papers at core.ac.uk Provided by MUCC (Crossref) Hindawi Publishing Corporation BioMed Research International Volume 2013, Article ID 612649, 12 pages http://dx.doi.org/10.1155/2013/612649 Research Article Biochemical, Pharmacological, and Structural Characterization of New Basic PLA2 Bbil-TX from Bothriopsis bilineata Snake Venom Victor Corasolla Carregari,1 Rafael Stuani Floriano,2 Lea Rodrigues-Simioni,2 Flavia V. Winck,3 Paulo Aparecido Baldasso,1 Luis Alberto Ponce-Soto,1 and Sergio Marangoni1 1 Department of Biochemistry, Institute of Biology (IB), Faculty of Medical Sciences, State University of Campinas (UNICAMP), Campinas, SP, Brazil 2 Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Campinas, SP, Brazil 3 Max Planck Institute of Molecular Plant Physiology and University of Potsdam, Potsdam, Germany Correspondence should be addressed to Luis Alberto Ponce-Soto; [email protected] Received 9 May 2012; Revised 17 August 2012; Accepted 1 September 2012 Academic Editor: Laura Leiva Copyright © 2013 Victor Corasolla Carregari et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Bbil-TX, a PLA , was puri�ed from Bothriopsis bilineata snake venom aer only one chromatographic step using RP-HPLC on -Bondapak C-18 column. A molecular mass of 14243.8 Da was con�rmed by �-Tof �ltima API ESI/MS �T�� MS mode� mass spectrometry. e2 partial protein sequence obtained was then submitted to BLASTp, with the search restricted to PLA from snakes and shows high identity values when compared to other PLA s. PLA activity was presented in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 25–37 C. Maximum PLA activity2 required 2 2 Ca and in the presence of Cd , Zn , Mn ,andMg it was reduced in the presence or∘ absence of Ca . Crotapotin from 2 Crotalus2+ durissus cascavella rattlesnake2+ 2+ venom2+ and antihemorrhagic2+ factor DA2-II from Didelphis albiventris opossum2+ sera under optimal conditions signi�cantly inhibit the enzymatic activity. Bbil-TX induces myonecrosis in mice. e fraction does not show a signi�cant cytotoxic activity in myotubes and myoblasts �C2C12�. e in�ammatory events induced in the serum of mice by Bbil-TX isolated from Bothriopsis bilineata snake venom were investigated. An increase in vascular permeability and in the levels of T��-a, IL-6, and IL-1 was was induced. Since Bbil-TX exerts a stronger proin�ammatory e�ect, the phospholipid hydrolysis may be relevant for these phenomena. 1. Introduction in a number of patients as a consequence of excessive or poorly regulated immune response to the injured organism Viperidae snakes are represented in South America by Cro- [3]. is imbalanced reaction may harm the host through a talus, Bothrops, Bothriopsis and Lachesis. Bothriopsis bilineata maladaptitive release of endogenous mediators that include is the endemic and rare bothropic snake species [1]. cytokines and nitric oxide. e envenomation is characterized by a generalized PLA s are abundant in snake venoms and have been in�ammatory state. e normal reaction to envenomation widely employed as pharmacological tools to investigate their involves a series of complex immunologic cascades that role in2 diverse pathophysiological processes. Viperid and ensures a prompt protective response to venom in humans crotalid venoms contain PLA swiththeabilitytocause [2]. Although activation of the immune system during rapid necrosis of skeletal muscle �bers, thus being referred envenomation is generally protective, septic shock develops toasmyotoxicPLA s[4]. Local2 in�ammation is a prominent 2 2 BioMed Research International characteristic of snakebite envenomations by viperid and by preincubating the protein (Bbil-TX) and each inhibitor crotalid species [5]. for 30 min at 37 C prior to assaying the residual enzyme Furthermore, PLA myotoxins are relevant tools for the activity under optimal∘ conditions. All assays were done in study of key general in�ammatory mechanisms. High levels triplicate and the absorbances at 425 nm were measured with of secretory PLA (sPLA2 ) are detected in a number of a VersaMax 190 multiwell plate reader (Molecular Devices, in�ammatory disorders in humans, such as bronchial asthma Sunnyvale, CA, USA). [6], allergic rhinitis2 [7], septic2 shock [8], acute pancreatitis [9], extensive burning [10], and autoimmune diseases [11]. 2.4. Electrophoresis. Tricine SDS-PAGE in a discontinuous In addition, increased expression and release of sPLA have gel and buffer system was used to estimate the molecular mass been found in rheumatoid arthritis, in�ammatory bowel of the proteins, under reducing and nonreducing conditions diseases, and atherosclerosis [12, 13]. Mechanisms involved2 [15]. in the proin�ammatory action of sPLA are being actively investigated, and most of this knowledge is based on studies 2.5. Amino Acid Analysis. Amino acid analysis was using puri�ed venom PLA s. 2 performed on a Pico-Tag Analyzer (Waters Systems) as is paper describes the isolation and biochemical and described by [16]. e puri�ed Bbil-TX sample (30 g) was pharmacological characterization2 of new PLA s from Both- hydrolyzed at 105 C for 24 h in 6 M HCl (Pierce sequencing riopsis bilineata venom, Bbil-TX, and also the study of its grade) containing 1 phenol (w/v). e hydrolyzates various toxic activities, including myotoxicity,2 cytotoxicity, ∘ were reacted with 20 L of derivatization solution (eth- and in�ammation. anol : triethylamine : water% : phenylisothiocyanate, 7 : 1 : 1 : 1, v/v) for 1 h at room temperature, aer which the PTC-amino 2. Materials and Methods acids were identi�ed and quanti�ed by HPLC, by comparing their retention times and peak areas with those from a 2.1. Venom and Reagents. Bothriopsis bilineata venom was standard amino acid mixture. donated by Dr. Corina Vera Gonzáles. All chemicals and reagents used in this work were of analytical or sequencing 2.�. Determination of the Molecular Mass of the Puri�ed grade and purchased from Sigma-Aldrich Co. (St. Louis, MO, Protein by Mass Spectrometry. An aliquot (4.5 L) of the USA). protein was inject by C18 (100 m 100 mm) RP-UPLC (nanoAcquity UPLC, Waters) coupled with nanoelectro- 2.2. Reversed-Phase HPLC (RP-HPLC). Five milligrams of spray tandem mass spectrometry on× a Q-T of Ultima API the whole venom from Bothriopsis bilineata was dissolved in mass spectrometer (MicroMass/Waters) at a �ow rate of 200 L ammonium bicarbonate 0.2 M pH 8.0. e resulting 600 nl/min. e gradient was 0–50 acetonitrile in 0.1 solution was clari�ed by centrifugation and the supernatant formic acid over 45 min. e instrument was operated in was applied to a -Bondapak C18 column (0.78 30 cm; MS continuum mode and the data acquisition% was from % Waters 991—PDA system). Fractions were eluted using a 100–3,000 at a scan rate of 1 s and an interscan delay of 0.1 s. linear gradient (0–100 , v/v) of acetonitrile (solvent× B) at e spectra were accumulated over about 300 scans and푚 the a constant �ow rate of 1.0 mL/min over 40 min. e elution multiple charged data produced by the mass spectrometer pro�le was monitored at% 280 nm, and the collected fractions on the scale were converted to the mass (molecular were lyophilized and conserved at 20 C. weight) scale using maximum-entropy-based soware (1) ∘ supplied푚 with Masslynx 4.1 soware package. e processing 2.3. PLA Activity. PLA activity− was measured using the parameters were: output mass range 6,000–20,000 Da at a assay described by Holzer and Mackessy, [14] modi�ed for “resolution” of 0.1 Da/channel; the simulated isotope pattern 96-well plates.2 e standard2 assay mixture contained 200 L model was used with the spectrum blur width parameter of buffer (10 mM tris-HCl, 10 mM CaCl and 100 mM NaCl, set to 0.2 Da and the minimum intensity ratios between successive peaks were 20 (le and right). e deconvoluted pH 8.0), 20 L of substrate (4-nitro-3-octanoyloxy-benzoic acid), 20 L of water, and 20 L of Bbil-TX2 in a �nal volume of spectrum was then smoothed (2 3 channels, Savitzky Golay smooth) and the mass centroid values obtained using 80 of 260 L. Aer adding Bbil-TX (20 g), the mixture was incu- % the peak top and a minimum peak width at half height of 4 batedforupto40minat37 C, with the reading of absorbance × channels. at intervals of 10 min. e∘ enzyme activity, expressed as the % initial velocity of the reaction ( ), was calculated based on the increase of absorbance aer 20 min. e optimum pH 2.7. Analysis of Tryptic Digests. e protein was reduced and temperature of the PLA were determined by incubating (DTT 5 mM for 25 min to 56 C) and alkylated (Iodoac- the enzyme in four buffers of different pH values (4–10) etamide 14 mM for 30 min) prior∘ to the addition of trypsin and at different temperatures,2 respectively. e effect of (Promega�s sequencing grade modi�ed). Aer trypsin addi- substrate concentration (0.1, 0.2, 0.3, 0.5, 1, 2, 5, 10, 20, and tion (20 ng/ L in ambic 0.05 M), the sample was incubated 30 mM) on enzyme activity was determined by measuring for 16 hr at 37 C. To stop the reaction, formic acid 0.4 was the increase of absorbance aer 20 min. e inhibition of added and the ∘ sample centrifuged at 2500 rpm for 10 min. PLA activity by crotapotins from Crotalus durissus cascavella e pellet was discarded and the supernatant dried in a% speed and DAII-2 from Didephis albiventris serum was determined vac.

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