Short communication
Kinetic characterization of gyroxin, a serine protease from
Crotalus durissus
terri
fi
cus
venom
Camila M. Yonamine
a,*, Marcia Y. Kondo
b, Maria A. Juliano
b, Marcelo Y. Icimoto
b, Gandhi R. Baptista
c,
Tetsuo Yamane
d, Vitor Oliveira
b, Luis Juliano
b, Antônio J. Lapa
a, Maria Teresa R. Lima-Landman
a,
Mirian A.F. Hayashi
a,*aDepto Farmacologia, UNIFESP, 04044-020 São Paulo-SP, Brazil
bDepto Biofísica, UNIFESP, 04044-020 São Paulo-SP, Brazil
cInstituto de Ciências do Mar
eUFC, 60165-081 Fortaleza-CE, Brazil
dUniversidade do Estado do Amazonas, Escola Superior de Ciências da Saúde
eINCT, 69065-001 Manaus, AM, Brazil
a r t i c l e
i n f o
Article history: Received 13 April 2012 Accepted 26 July 2012 Available online 3 August 2012
Keywords: Gyroxin Crotalus Kinetic Enzymatic Substrate Specificity
a b s t r a c t
This work describes for thefirst time the characterization of the enzymatic features of gyroxin, a serine protease fromCrotalus durissus terrificusvenom, capable to induce barrel rotation syndrome in rodents. Measuring the hydrolysis of the substrate ZFR-MCA, the optimal pH for proteolytic cleavage of gyroxin was found to be at pH 8.4. Increases in the hydrolytic activity were observed at temperatures from 25C to 45C, and increases of NaCl concentration up to 1 M led to activity decreases. The preference of gyroxin for Arg residues at the substrate P1 position was also demonstrated. Taken together, this work describes the characterization of substrate specificity of gyroxin, as well as the effects of salt and pH on its enzymatic activity.
Ó2012 Elsevier Masson SAS.
Gyroxin is a serine protease from the venom ofCrotalus durissus terrificus, and corresponds to about 2% of the protein content of the crude venom [1]. The intravenous injection of gyroxin in mice produces temporary episodes characterized by opisthotonos and rotations around the long axis of the animal through a still unknown mechanism[2]. Besides these several known biological effects, very little is known about the enzymatic characteristics of gyroxin and its substrate specificity. In this work we performed kinetic studies of gyroxin to characterize the effects of pH, temperature, salt and peptide substrate specificities. The crude venom ofC. d. terrificuswas kindly provided by Dr. E.B. Oliveira (Dept. Biochemistry and Immunology, USP, Ribeirão Preto, Brazil), and gyroxin was purified by fractionation as described previously[3]. The primary sequence of pure gyroxin was confirmed by MSfingerprint analysis in a ESI-TRAP spectrometer after trypsin digestion (data not shown).
Fig. 1(AeC) shows the effects of pH, salt and temperature on gyroxin
activity using the synthetic substrate ZFR-MCA. For binding and catalysis of substrates titrated groups E1and E2, the pKe1 and pKe2
values of 7.3 0.1 and 9.50.1, respectively, were found. The optimal pH for proteolytic cleavage was at pH 8.4 (Fig.1A). This result is in line with what was described for acuthrombin-A and -C serine-like proteases from the snake Agkistrodon acutus, in which the optimum point for the arginineeesterase activity was determined to
be at pH 7.5, using BAEE (benzoyl-arginine ethyl ester) and TAME (tosyl arginine methyl ester) as substrates[4]. Gyroxin hydrolytic activity diminished with increasing concentration of NaCl up to 1 M, when 50% of residual activity was achieved (Fig. 1B). We believe that this effect was due to the charge shielding that interferes with the interaction of enzyme and substrate, mostly determined by a strong ionic interaction involving the Arg residue at the P1 position of substrate, as also described for other enzymes[5]. The activity of leucurobin (a thrombin-like enzyme fromBothrops leucurusvenom) was also decreased in the presence of Naþand Kþions, when the
substrate D-Phe-Pro-Arg-pNA was used. At ion concentration of 0.08 M, the residual activity was around 62% for Kþ
and 50% for Naþ
. At a concentration of 0.15 M, the activity was reduced to about 51% for Kþand 39% for Naþion[6]. Gyroxin hydrolytic activity increased
with temperature from 25 to 45C (Fig. 1C). The intrinsic fl uores-cence spectra of gyroxin at pH 3e10 are shown inFig. 1D. The
maximum intensity of fluorescence at
l
max ¼ 335 nm in pH 3*Corresponding authors. Tel.:þ55 11 5576 4447.
E-mail address:mhayashi@unifesp.br(M.A.F. Hayashi).
Contents lists available atSciVerse ScienceDirect
Biochimie
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b i o c h i
0300-9084Ó2012 Elsevier Masson SAS. http://dx.doi.org/10.1016/j.biochi.2012.07.020
Biochimie 94 (2012) 2791e2793
Open access under the Elsevier OA license.
increased up to
l
max¼340 nm at pH 9, indicating that the trypto-phan residues are probably located in a highly stable hydrophobic ambience in acid pH and more hydrophilic ambience in basic pH. Circular dichroism (CD) spectra at different pH ranging from 6 to 9 confirmed this profile (Fig. 1E). Although these results did not show a significant change in the overall structure, it probably indicates that the observed pKa at the activity pH curve are real microscopic constants, due to titration of specific residues involved in the cata-lytic activity of the enzyme. The hydrolyis of peptidyl-MCA in 50 mM TriseHCl pH 8.0 buffer at 37C were monitored atl
ex¼380 nm andl
em¼460 nm. Gyroxin kinetic parameters determined using ZFR-MCA substrate were calculated by MichaeliseMenten equationusing Grafit 5.0 software (Erithacus Software Horley, Surrey, UK) and the values were determined askcat¼0.083 s 1,Km¼53.5
m
M and kcat/Km¼1.55 mM 1s 1. Gyroxin is a serine protease with the abilityto cleave with high hydrolysis rate the peptide bond adjacent to Arg residue at P1 site of the FRET substrate Abz-KLXSSKQ-EDDnp, where X is any amino acid residue. The hydrolysis of the FRET peptides was quantified by measuring thefluorescence of Abz at
l
em¼420 nm, following excitation atl
ex¼320 nm, in a Hitachi F-2500spectro-fluorimeter. The peptide cleaved with the highest initial velocity by gyroxin under conditions of maximum activity contained Arg at the P1 position, with a single cleavage at the ReS bond, as determined by
HPLC and characterized by MALDI-TOF and electron-spray mass spectrometry, and this value of initial velocity was considered 100% (relative rate). The relative rates for the hydrolysis by giroxin substrates of the peptide series Abz-KLXSSKQ-EDDnp containing substitutions with other amino acids at X position were then calculated (in percentage) based on the values of initial velocity
pH
6 7 8 9 10 11
relative activity (%)
0 20 40 60 80 100
salt (mM)
0 200 400 600 800 1000
relative activity (%)
0 20 40 60 80 100
T(ºC)
15 20 25 30 35 40
relative activity (%)
0 20 40 60 80 100
pH
2 4 6 8 10 12
WL max
334 336 338 340
A
B
C
D
E
nm
180 200 220 240 260
E
lli
pti
ci
ty
(
m
de
gr
ee
)
-6 -4 -2 0 2 4
pH 6
pH 7
pH 8
pH 9
Fig. 1.Influence of pH, salt and temperature on gyroxin enzyme activity, and the pH effect on gyroxin structure. The influence of pH (A), salt (B) and temperature (C) on gyroxin [30mM] hydrolytic activity was measured using ZFR-MCA [3 mM] substrate. (A, B, C) The Z position is carbobenzoxy, and MCA ([7-amino-4-methyl]coumarin) is thefluorescence
donor. The hydrolysis of peptidyl-MCA was monitored atlex¼380 nm andlem¼460 nm. Enzymatic assays were performed using the standard assay conditions described for serinoproteases[7]. The pH dependency profile for intrinsicfluorescence change (D) and CD spectra (E) of gyroxin is shown. (D) Representativefluorescence spectrum profiles of gyroxin [0.2mM] intrinsic maximumfluorescence intensity (lem¼340 nm) at each pH determined using a Hitachi F-2500 spectrofluorimeter (Tokyo, Japan). (E) CD spectra were
recorded on a Jasco J-810 spectropolarimeter with a Peltier system for controlling cell temperature. The absorbance spectra of gyroxin [2mM] were collected in the far-UV range (190e260 nm) using a 1 cm path length cell. Both assays were carried out in the standard buffer [15 mM TriseHCl, 5 mM MES, 5 mM acetic acid, 5 mM glycine] at 37C, and the pH
was adjusted with 0.1 N NaOH.
aa
R A M K H V F Q L N Y T S G P I W D E
Relative rate (%)
0 10 20 30 60 80 100
Fig. 2.Characterization of substrate subsite specificity of gyroxin [0.3mM] by
hydro-lysis of the FRET peptide series Abz-KLXSSKQ-EDDnp [10 mM] in 50 mM TriseHCl pH
8.0 buffer at 37
C. The X of the peptide series Abz-KLXSSKQ-EDDnp represents different amino acids in the X position, Abz (ortho-aminobenzoic acid) is thefl uo-rescence donor and EDDnp ([N-(2,4-dinitrophenyl)-ethylenediamine]) is thefl uores-cence acceptor. The hydrolysis of the FRET peptides was quantified by measuring the
fluorescence of Abz atlem¼420 nm, following excitation atlex¼320 nm, in a Hitachi F-2500 spectrofluorimeter. The peptide with Arg at the P1 position was cleaved with the highest initial velocity by gyroxin, with a single cleavage at the ReS bond, and this
value of initial velocity was considered 100% (relative rate). The relative rates for the hydrolysis of the peptide series Abz-KLXSSKQ-EDDnp containing substitutions with other amino acids at X position were then calculated (in percentage) based on the values of initial velocity observed for each substitution.
C.M. Yonamine et al. / Biochimie 94 (2012) 2791e2793
observed for each substitution (Fig. 2). Interestingly, the thrombin-like enzyme leucurobin isolated from the venom ofB. leucuruswas also shown to present amidase activity against chromogenic substrates containing an Arg residue at P1 site[6]. This work is the
first to elucidate the substrate specificity, and the salt and pH influences on gyroxin enzyme activity. These data will be of utmost importance for further investigations aiming to elucidate the bio-logical mechanism of actions underlying the barrel rotation syndrome triggered by gyroxin.
Acknowledgement
The authors acknowledge CAPES, CNPq and FAPESP forfinancial support, Dr. E.B. Oliveira (USPeRibeirão Preto) for the kind supply
ofC. d. terrificusvenom, and Dr. J.E. Oliveira and Dr. M.A.P. Camillo (CB-IPEN) for the support on gyroxin purification.
References
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