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 PLA
2
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 Marangoni
1
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; poncesoto@yahoo.com.ar
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
2
, 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. e partial protein sequence obtained was then submitted to BLASTp, with the search restricted to PLA
2
from snakes
and shows high identity values when compared to other PLA
2
s. PLA
2
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
2
activity required
Ca
2+
and in the presence of Cd
2+
, Zn
2+
, Mn
2+
, and Mg
2+
it was reduced in the presence or absence of Ca
2+
. Crotapotin from
Crotalus durissus cascavella rattlesnake venom and antihemorrhagic factor DA2-II from Didelphis albiventris opossum 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
Viperidae snakes are represented in South America by Cro-
talus, Bothrops, Bothriopsis and Lachesis. Bothriopsis bilineata
is the endemic and rare bothropic snake species [1].
e envenomation is characterized by a generalized
in�ammatory state. e normal reaction to envenomation
involves a series of complex immunologic cascades that
ensures a prompt protective response to venom in humans
[2]. Although activation of the immune system during
envenomation is generally protective, septic shock develops
in a number of patients as a consequence of excessive or
poorly regulated immune response to the injured organism
[3]. is imbalanced reaction may harm the host through a
maladaptitive release of endogenous mediators that include
cytokines and nitric oxide.
PLA
2
s are abundant in snake venoms and have been
widely employed as pharmacological tools to investigate their
role in diverse pathophysiological processes. Viperid and
crotalid venoms contain PLA
2
s with the ability to cause
rapid necrosis of skeletal muscle �bers, thus being referred
to as myotoxic PLA
2
s [4]. Local in�ammation is a prominent