1) Researchers purified and characterized a hyaluronidase enzyme from the venom of the freshwater stingray Potamotrygon motoro. 2) A two-step purification process involving gel filtration chromatography and ion exchange chromatography resulted in a 366-fold purification with a 79 kDa hyaluronidase enzyme. 3) The purified hyaluronidase had a pH optimum of 4.2 and maximum activity at 40°C. Its activity was inhibited by certain metal ions and heparin.

1. Toxicon 51 (2008) 1060–1067
A hyaluronidase from Potamotrygon motoro (freshwater
stingrays) venom: Isolation and characterization$
Marta R. Magalha˜ esa
, Nelson Jorge da Silva Jr.a
, Cirano J. Ulhoab,Ã
a
Centro de Estudos e Pesquisas Biolo´gicas, Departamento de Biologia, Universidade Cato´lica de Goia´s, 74.605-010, Goiaˆnia, GO, Brazil
b
Departamento de Cieˆncias Fisiolo´gicas (ICB), Universidade Federal de Goia´s, 74.001-940 Goiaˆnia, GO, Brazil
Received 13 September 2007; received in revised form 21 December 2007; accepted 28 January 2008
Available online 2 February 2008
Abstract
Freshwater stingrays (Potamotrygon motoro) are known to cause human accidents through a sting located in its tail. In
the State of Goia´ s, this accident happens especially during the fishing season of the Araguaia River. The P. motoro venom
extracted from the sting presented hyaluronidase activity. The enzyme was purified by gel filtration on Sephacryl S-100
and ion-exchange chromatography on SP-Sepharose. A typical procedure provided 376.4-fold purification with a 2.94%
yield. The molecular weight of the purified enzyme was 79 kDa as estimated by gel filtration on Sephacryl S-100. The Km
and Vmax values for hyaluronidase, using hyaluronic acid as substrate, were 4.91 mg/ml and 2.02 U/min, respectively. The
pH optimum for the enzyme was pH 4.2 and maximum activity was obtained at 40 1C. The hyaluronidase from P. motoro
was shown to be heat instable, being stabilized by bovine albumin and DTT, and inhibited by Fe2+
, Mn2+
, Cu2+
and
heparin.
r 2008 Elsevier Ltd. All rights reserved.
Keywords: Freshwater stingrays; Potamotrygon motoro; Venom; Hyaluronidase; Properties
1. Introduction
Stingrays are found around the world in tempe-
rate and tropical seas. They are also found in
Atlantic rivers of tropical and temperate South
America, Equatorial Africa and, at least, one Indo-
Chinese river system, the Mekong river of Laos
(Caras, 1974). In spite of being not aggressive, from
the point of view of public health, stingrays are the
most significant venomous fish in the world
(Junghanss and Bodio, 2006). These fishes have
one or more stings at the base of their tails, which
have serrated edges and a very sharp tip. Its position
on the tail, certainly, is responsible for the
effectiveness of the defensive response when it is
stepped on its back or badly handled. In these cases,
a powerful strike blow of the tail towards the
stimulus causes the penetration of the sting into the
body of the victim. The sting is covered by an
epithelium that possesses great quantities of gland-
ular cells which produce venom when compressed
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www.elsevier.com/locate/toxicon
0041-0101/$ - see front matter r 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.toxicon.2008.01.008
$
Ethical statement: Cirano J. Ulhoa declares that all
procedures used in this study are in accordance with the Brazilian
College of Animal Experimentation (http://www.cobea.org.br).
All animals used were free again in the Crixa´ s-Ac-u´ River, their
natural habitat.
ÃCorresponding author. Tel.: +55 62 35211494;
fax: +55 62 35211190.
E-mail addresses: ulhoa@icb.ufg.br, ulhoa@icb1.ufg.br
(C.J. Ulhoa).

2. during the penetration, spreading their content into
the tissues of the victim (Castex and Loza, 1964;
Castex, 1965; Halstead, 1971).
South American freshwater stingrays are included in
the Potamotrygonidae family, which comprise three
valid genera: Plesiotrygon, Paratrygon and Potamo-
trygon, the last being more diversified, with 19
described species (Charvet-Almeida et al., 2002;
Carvalho et al., 2003). In accidents provoked for
freshwater stingrays, the victim complains of intense
pain, relating it with burning. Around the wounded
spot appears erythema and edema, characterizing the
first phase of envenomation. Then it develops a central
necrosis causing, in the affected area, tissue flabbiness
and formation of a pale pink deep ulcer, well cut,
which evolves slowly, being a peculiar characteristic
of this kind of envenomation (Castex, 1965; Haddad
et al., 2004; Cook et al., 2006; Clark et al., 2007).
Few studies about the toxic activities of fresh-
water stingrays venom have been developed. The
lack of data is mainly due to the difficulty to extract
venom, and it is very difficult and dangerous to
capture the animals. The amount of venom is very
low, and likewise it is thermolabile (Haddad et al.,
2004). The first study about the biochemistry and
pharmacology properties of stingrays venom was
carried out by Russell and Van Harreveld (1954),
which demonstrated cardiovascular effects of Ur-
obatis helleri venom. Rodrigues (1972) isolated an
active principle of freshwater stingray Potamotrygon
motoro venom with cholinergic activity on ileum of
guinea pigs and hypotensive activity when managed
by intravenous injection in rats. Russell (1953)
indicated the presence of polypeptides of high
molecular mass, serotonin and enzymatic activity
of phosphodiesterase and 50
-nucleotidase in marine
stingray venom. Recently, we have detected 50
-
nucleotidase, phospholipase, acid phosphatase,
hyaluronidase, caseinolytic, gelatinolytic and elasti-
nolytic activities in P. motoro venom obtained from
animals of Crixa´ s-Ac-u´ River (Goia´ s, Brazil) (Ma-
galha˜ es, 2001). Caseinolytic, gelatinolytic and hya-
luronidase activities were identified in Potamotrygon
falkneri venom (Haddad et al., 2004). In a
comparative study of Potamotrygon scobina and
Potamotrygon orbignyi venoms, Magalha˜ es et al.
(2006) identified significant edematogenic and noci-
ceptive response and necrosis in both venoms.
Conceic-a˜ o et al. (2006) isolated a vasoconstrictor
peptide from P. orbignyi venom with 1001.52 Da.
Barbaro et al. (2007), comparing the extracts from
the tissue of marine and freshwater stingrays
Dasyatis guttata and P. falkneri, observed edemato-
genic, gelatinolytic, caseinolytic and fibrinogenoly-
tic activities in both extracts. Nociceptive activity
was verified in both tissue extracts; however,
P. falkneri presented a two-fold higher activity than
D. guttata tissue extract. Lethal, dermonecrotic,
myotoxic and hyaluronidase activities were ob-
served only in the tissue extract of P. falkneri.
Hyaluronidases (EC 3.2.1.35) are enzymes that
naturally cleave hyaluronic acid, which is a major
component of the extracellular matrix of vertebrates
(Kreil, 1995). These enzymes are not toxic by
themselves, but can enhance local systemic envenoma-
tion by increasing the absorption and diffusion rates
of the venom through the victim’s tissues since it
catalyzes the hydrolysis of the glucosaminoglycans,
this being called the spreading factor (Duran-Reynals,
1936). Hyaluronidase enzyme has been reported in
venom of snakes, scorpions, bee, stonefish, lizards and
spiders (Owen, 1983; Tu and Hendon, 1983; Poh
et al., 1992; Kemparaju and Girish, 2006; Morey
et al., 2006; Nagaraju et al., 2007).
Recently, hyaluronidase activity was reported in
the freshwater stingrays’ crude venom (Magalha˜ es,
2001; Haddad et al., 2004; Barbaro et al., 2007). In
the present study, we show the results of purification
and characterization of hyaluronidase enzyme from
P. motoro venom.
2. Materials and methods
2.1. Venom and animals
Specimens of P. motoro were collected from
Crixa´ s-Ac-u´ River (Goia´ s, Brazil). The entire sting
was removed with bistouries, lyophilized and
scraped. The collected material was macerated and
dissolved in phosphate buffer 50 mM, pH 7.0,
containing 0.15 M NaCl and immediately centri-
fuged at 5000g for 10 min. Venom was stored at
À20 1C until use.
2.2. Estimation of protein concentration
Protein concentrations were determined by the
method of Lowry et al. (1951) using bovine serum
albumin (BSA) as a standard.
2.3. Assay of hyaluronidase enzyme activity
Hyaluronidase enzyme activity was determined
by the method described by Ferrante (1956),
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3. modified by Poh et al. (1992). The assay mixture
contained 200 ml acetate buffer 0.2 M, pH 6.0,
containing 0.15 M NaCl, 50 ml hyaluronic acid
(0.5 mg/ml in acetate buffer) and 50 ml enzyme in
acetate buffer. The mixture was incubated for
15 min at 37 1C and the reaction was stopped by
the addition of 500 ml of 2.5% (w/v) acetyltrimethy-
lammonium bromide in 2% (w/v) NaOH. After
10 min, the absorbance of each reaction mixture was
read at 400 nm. Specific activity was expressed as
National Formulary Units (NFU), which is defined
as the amount of enzyme required to hydrolyze
0.255 mg of the hyaluronic acid per minute.
2.4. SDS-polyacrylamide gel electrophoresis
SDS-PAGE (12%) was carried out under dena-
turing conditions according to the method described
by Laemmli (1970). After electrophoresis, gel was
silver stained as described by Blum et al. (1987).
Molecular weight standards from 97.4 kDa (phos-
phorylase B) and 66 kDa (BSA) were used.
2.5. Enzyme purification
The crude P. motoro venom (0.38 mg) was loaded
on a Sephacryl S-100 column (2.5 Â 48 cm) pre-
viously equilibrated with 50 mM phosphate buffer,
pH 6.0, containing 100 mM NaCl, and eluted with
the same buffer at a flow rate of 40 ml/h. Fractions
of 2.0 ml were collected and monitored at 280 nm.
Fractions showing the highest hyaluronidase activ-
ity were pooled, dialyzed and applied directly onto a
SP-Sepharose column (1.5 Â 13 cm) equilibrated
with 20 mM phosphate buffer, pH 6.0, and eluted
at a flow rate of 60 ml/h. Fractions of 3.0 ml were
collected and monitored at 280 nm. The column was
washed with the same buffer and eluted with a
linear gradient of 0–1.0 M NaCl. Fractions contain-
ing hyaluronidase activity were pooled, dialyzed,
lyophilized and stored at À20 1C.
2.6. Molecular weight determination
The molecular weight of the purified hyaluroni-
dase was estimated by gel filtration chromatography
according to the method of Andrews (1962) on
calibrated columns (2.5 Â 48 cm) of Sephacryl S-
100, using 50 mM phosphate buffer, pH 6.0 (con-
taining 100 mM NaCl), at a flow rate of 40 ml/h.
Void volume (Vo) of the column was determined by
using blue dextran (1 mg/ml in equilibration buffer).
Ovalbumin (43 kDa), chymotrypsinogen A (25 kDa)
and ribonuclease A (13.7 kDa) were used as
standard proteins for obtaining the calibration
curve. A calibration curve was obtained by plotting
Ve/Vo (KAV) against their respective logarithmic
molecular weights.
2.7. Enzyme characterization
The effect of pH on enzyme activity was
determined by varying the pH of the reaction
mixtures using 100 mM phosphate–citrate buffer
(pH 2.5–7.0). The effect of temperature on enzy-
matic activity was determined at pH 4.2, in the
range of 20–50 1C. The effect of temperature on
enzyme stability was determined after preincubation
at 20, 30 and 40 1C for 5–30 min. The effects of
metallic ions and some compounds on hyaluroni-
dase activity were determined after preincubation at
4 1C for 15 min. Km was determined from the
Michaelis–Menten plot using Origin 7.0 program
by measuring the initial rate of hyaluronic acid
hydrolysis using a range of 2.5–25 mg/ml.
3. Results
3.1. Purification of hyaluronidase
A two-step protocol was standardized for hyalur-
onidase purification. The first step involved the
Sephacryl S-100 gel filtration chromatography,
which fractionated P. motoro venom gland extract
into two peaks of proteins (Fig. 1A). Fractions with
hyaluronidase activity were pooled and concen-
trated by lyophilization. Only 3.62% of the activity
loaded onto the column was recovered in the pooled
fraction. The second step involved the SP-Sepharose
ion-exchange chromatography and resolved into
one peak of protein. The peak containing hyalur-
onidase activity was eluted with a linear gradient of
NaCl (Fig. 1B). In this step 80% of the enzyme
loaded onto the column was recovered. A summary
of the purification procedure is given in (Table 1).
The enzyme was purified to 366.4-fold with a
yield of 2.90%, having a specific activity of 1.33 Â
108
NFU/min mg of protein. SDS-PAGE showed
that the enzyme migrated as a single band (Fig. 2).
Molecular weight of the hyaluronidase was esti-
mated by gel filtration on Sephacryl S-100 using
standard protein molecular weight markers and it
was found to be approximately 79 kDa (Fig. 3).
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4. ARTICLE IN PRESS
20
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0
50
100
150
200
250
Absorbanceat280nm()
Fraction number
HyaluronidaseactivityNFU/mL(-------)
0
0.00
0.01
0.02
0.03
0.04
0.05
0
50
100
150
200
250
0.0
0.2
0.4
0.6
0.8
1.0
Absorbanceat280nm()
Fraction number
HyaluronidaseactvityNFU/mL(------)
NaCl(0-1.0M)
30 40 50 60 70 80
10 20 30 40 50
Fig. 1. Isolation of hyaluronidase from P. motoro venom. (A) Elution profile from Sephacryl S-100 chromatography. The column
(2.5 Â 48 cm) was eluted with 50 mM phosphate buffer, pH 6.0, containing 100 mM NaCl at a flow rate of 40 ml/h, and 2 ml fractions were
collected. Protein elution was monitored at 280 nm (———) and hyaluronidase activity at 400 nm (- - - - - -). Fractions having the
hyaluronidase activity (dotted line) were pooled, concentrated and applied onto SP-Sepharose columns for further fractionation. (B)
Elution profile from SP-Sepharose column chromatography. The column (1.5 Â 13 cm) was equilibrated with 20 mM phosphate buffer, pH
6.0, at a flow rate of 60 ml/h, and 2 ml fractions were collected. The column was washed with the same buffer and eluted with a linear
gradiet of 0–1.0 M NaCl.
Table 1
Summary of purification of hyaluronidase from P. motoro venom
Purification step Total protein (mg) Total activity
(NFU/ml)
Specific activity
(NFU/mg)
Purification (fold) Yield (%)
Crude venom 380 138 Â 103
3.63 Â 105
1 100
Sephacryl S-100 0.69 5 Â 103
7.24 Â 106
19.9 3.62
SP-Sepharose 0.03 4 Â 103
1.33 Â 108
366.4 2.90
M.R. Magalha˜es et al. / Toxicon 51 (2008) 1060–1067 1063

5. 3.2. Biochemical characterization
The pH activity profile of purified hyaluronidase
was determined in a pH range from 2.5 to 7.0 using
phosphate/citrate buffer. The enzyme had a typical
bell-shaped profile covering a broad pH range and
an optimal pH of 4.2 (Fig. 4A). The influence of
temperature on hyaluronidase activity was deter-
mined between 4 and 50 1C at pH 4.2. The optimal
temperature for hyaluronidase activity was 40 1C
and the activity decreased significantly above 40 1C
(Fig. 4B). The enzyme was stable for at least 30 min
when incubated at 20 and 30 1C, but lost 70% of the
activity at 40 1C (Fig. 4C). The effect of varying con-
centrations of hyaluronic acid on the initial velocity
of the hyaluronidase showed a typical hyperbolic
saturation curve (Fig. 5). The Km (4.91 mg/ml) and
Vmax (2.02 U/min) values were calculated from the
Michaelis–Menten plot.
The activity of the purified hyaluronidases was
tested in presence of metal ions and some chemical
compounds (Table 2). No considerable effect was
observed with Ca2+
, Mg2+
, Zn2+
and Hg2+
,
whereas Ni+
, Fe2+
and Cu2+
reduced activity by
25% approximately. b-Mercaptoethanol had a
slight effect on enzyme activity, whereas heparin
(0.05 IU) inhibited hyaluronidase activity by 20%.
4. Discussion
In this study with P. motoro venom extract, we
found that two-step fractionation on Sephacryl S-
100 column and SP-Sepharose column resulted in
the purification of a protein with hyaluronidase
activity. The final yield of 2.90% obtained and
activity of 4 Â 103
NFU/ml will signify the difficult
associated with working with this enzyme. Similar
results were described by Xu et al. (1982) working
with hyaluronidase from Agkistrodon acutus snake
venom. However, Poh et al. (1992) recovered 57%
hyaluronidase from Synanceja horrida stonefish
venom and Pessini et al. (2001) recovered 43.6%
hyaluronidase from Tityus serrulatus scorpion
venom.
Polyacrylamide gel electrophoresis showed that
hyaluronidase purified from P. motoro migrated as a
single band with an estimated molecular mass of
79 kDa, and exists as monomer. Most of the
hyaluronidase described in the literature appears
as a monomer and varies considerably between
organisms. Molecular weight of hyaluronidase from
Heterometrus fulvipus (Ramanaiah et al., 1990) was
in a similar range, while proteins with 33, 52 and
116 kDa have been described from A. acutus (Xu
et al., 1982), T. serrulatus (Pessini et al., 2001) and
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Fig. 2. SDS-PAGE of the purified P. motoro hyaluronidase.
(Lane 1) Molecular weight markers. (Lane 2) Crude P. motoro
venom. (Lane 3) Purified enzyme after Sephacryl S-100 chroma-
tography. (Lane 4) Purified enzyme after SP-Sepharose chroma-
tography.
0.05
1.0
1.2
1.4
1.6
1.8
2.0
MolecularWeightLog
KAV
Hyaluronidase
Ovoalbumin
Chymotrypsinogen
Ribonuclease
0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50
Fig. 3. Determination of molecular mass of the purified
hyaluronidase from the venom of P. motoro by gel filtration
chromatography. The molecular weight of the purified hyalur-
onidase was estimated by gel filtration chromatography on
calibrated columns (2.5 Â 48 cm) of Sephacryl S-100, using
50 mM phosphate buffer, pH 6.0 (containing 100 mM NaCl), at
a flow rate of 40 ml/h. Void volume (Vo) of the column was
determined by using blue dextran (1 mg/ml in equilibration
buffer), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and
ribonuclease A (13.7 kDa). A calibration curve was obtained by
plotting Ve/Vo against their respective logarithmic molecular
weights.
M.R. Magalha˜es et al. / Toxicon 51 (2008) 1060–10671064

6. Streptococcus agalactiae (Ozegowski et al., 1994),
respectively.
The optimal pH for the enzyme activity (4.2) was
similar to that found for hyaluronidase from a
variety of organisms. The optimal pH for hyalur-
onidase activity is usually in the range of 3.5 and 6.5
(Xu et al., 1982; Poh et al., 1992; Ozegowski et al.,
1994; Morey et al., 2006; Nagaraju et al., 2007). The
optimum temperature was found to be 37 1C at pH
4.2, and it is in agreement with hyaluronidase from
Palamneus gravimanus (Morey et al., 2006) and
Hippasa partita (Nagaraju et al., 2007). Thermo-
stability is considered an important and useful
criterion for enzyme characterization. The hyalur-
onidase from P. motoro was stable for at least
30 min when incubated at 20 and 30 1C, but retained
only 30% of the activity after incubation at 40 1C.
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2.0
0
500
1000
1500
2000
2500
3000
3500
4000
Hyaluronidaseactivity(NFU/ml)
pH
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
Hyaluronidaseactivity(NFU/ml)
Temperature (°C)
0
20
30
40
50
60
70
80
90
100
110
(20°C)
(30°C)
(40°C)
Relativeactivity(%)
Time
2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 10 20 30 40 50
5 10 15 20 25 30
Fig. 4. Characterization biochemistry of P. motoro hyaluronidase. (A) Profile of the pH optimum for hyaluronidase activity. (B) Profile of
the temperature optimum for hyaluronidase activity. (C) Temperature–stability profile of hyaluronidase activity purified.
0
0.6
0.8
1.0
1.2
1.4
1.6
1.8
V(NFU/min)
5 10 15 20 25
Hyaluronic acid (µg/mL)
Fig. 5. Michelis–Menten plot of the hyaluronidase activity with
the substrate hyaluronic acid. Experiments were performed at
40 1C and pH 4.2.
M.R. Magalha˜es et al. / Toxicon 51 (2008) 1060–1067 1065

7. The purified hyaluronidase from P. motoro
showed Michaelis–Menten-type kinetics with hya-
luronic acid as substrate. The Km of 4.91 mg/ml
indicates that the enzyme has comparatively high
affinity for hyaluronic acid compared with other
hyaluronidases. This value was substantially lower
than those reported for P. gravimanus (47.61 mg/ml)
(Morey et al., 2006), T. serrulatus (69.7 mg/ml)
(Pessini et al., 2001), S. agalactiae (81.9 mg/ml)
(Ozegowski et al., 1994) and S. horrida stonefish
(709 mg/ml) (Poh et al., 1992).
As reported from studies on other hyaluronidase, a
concentration as low as 10mM of some metal ions
could affect enzyme activity. The poor inhibition by
Hg2+
and b-mercaptoethanol, compounds that usual-
ly react with cystein, led us to hypothesize about the
absence of these amino acids in the catalytic site of the
enzyme. Similar results are found by hyaluronidase
from P. gravimanus (Morey et al., 2006).
P. motoro purified enzyme is inhibited by heparin
as described by hyaluronidase from A. acutus
venom (Xu et al., 1982), H. fulvipus scorpion venom
(Ramanaiah et al., 1990) and P. gravimanus (Morey
et al., 2006).
In conclusion, this study presents the first
purification of a hyaluronidase from P. motoro
sting. This enzyme shows similar characteristics as
enzymes from venom of different organisms and
exhibited high affinity for hyaluronic acid. Further
structural and functional analyses might provide an
insight for the better understanding of the role of
this enzyme in envenomation by P. motoro.
Acknowledgments
This work was supported by a biotechnology
research grant to C.J.U. (CNPq, CAPES and
FUNAPE/UFG). M.R.M. was supported by Uni-
versidade Cato´ lica de Goia´ s (CEPB). The authors
thank Dr. Joa˜ o Luiz da Costa Cardoso and Dr.
Vidal Haddad Jr. (Hospital Vital Brazil, Instituto
Butantan), and Dra Ka´ tia Cristina Barbaro (La-
borato´ rio de Imunopatologia, Instituto Burantan)
for valuable suggestions during this study.
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ARTICLE IN PRESS
Table 2
Inhibitor effects in the activity of P. motoro hyaluronidase
Compound (10 mM) Enzymatic activity (NFU/ml) Relative activity (%) Inhibition (%)
Control 4305.9176.9 100 0
CaCl2 3926.257114.14 91.2 8.8
FeSO4 3220.97122.12 74.8 25.2
HgCl2 3712.87111.9 86.2 13.8
MgCl2 4046.09773.00 93.9 6.4
CuSO4 3044.51719.6 70.7 29.3
MnSO4 2149.9719.62 49.9 50.1
ZnSO4 3656.6712.84 84.9 15.1
b-Mercaptoetanol 4278.85719.64 99.3 0.7
Heparin (0.05 UI) 3425.43778.00 79.5 20.5
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