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doi:10.1016/j.to

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Toxicon 51 (2008) 1060–1067

www.elsevier.com/locate/toxicon

A hyaluronidase from Potamotrygon motoro (freshwaterstingrays) venom: Isolation and characterization$

Marta R. Magalhaesa, Nelson Jorge da Silva Jr.a, Cirano J. Ulhoab,�

aCentro de Estudos e Pesquisas Biologicas, Departamento de Biologia, Universidade Catolica de Goias, 74.605-010, Goiania, GO, BrazilbDepartamento de Ciencias Fisiologicas (ICB), Universidade Federal de Goias, 74.001-940 Goiania, 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 Goias, 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 2.02U/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 inAtlantic rivers of tropical and temperate SouthAmerica, Equatorial Africa and, at least, one Indo-

e front matter r 2008 Elsevier Ltd. All rights reserved

xicon.2008.01.008

tement: Cirano J. Ulhoa declares that all

in this study are in accordance with the Brazilian

al Experimentation (http://www.cobea.org.br).

d were free again in the Crixas-Ac- u River, their

ng author. Tel.: +55 62 35211494;

11190.

sses: ulhoa@icb.ufg.br, ulhoa@icb1.ufg.br

Chinese river system, the Mekong river of Laos(Caras, 1974). In spite of being not aggressive, fromthe point of view of public health, stingrays are themost significant venomous fish in the world(Junghanss and Bodio, 2006). These fishes haveone or more stings at the base of their tails, whichhave serrated edges and a very sharp tip. Its positionon the tail, certainly, is responsible for theeffectiveness of the defensive response when it isstepped on its back or badly handled. In these cases,a powerful strike blow of the tail towards thestimulus causes the penetration of the sting into thebody of the victim. The sting is covered by anepithelium that possesses great quantities of gland-ular cells which produce venom when compressed

.

ARTICLE IN PRESSM.R. Magalhaes et al. / Toxicon 51 (2008) 1060–1067 1061

during the penetration, spreading their content intothe tissues of the victim (Castex and Loza, 1964;Castex, 1965; Halstead, 1971).

South American freshwater stingrays are included inthe Potamotrygonidae family, which comprise threevalid genera: Plesiotrygon, Paratrygon and Potamo-

trygon, the last being more diversified, with 19described species (Charvet-Almeida et al., 2002;Carvalho et al., 2003). In accidents provoked forfreshwater stingrays, the victim complains of intensepain, relating it with burning. Around the woundedspot appears erythema and edema, characterizing thefirst phase of envenomation. Then it develops a centralnecrosis causing, in the affected area, tissue flabbinessand formation of a pale pink deep ulcer, well cut,which evolves slowly, being a peculiar characteristicof this kind of envenomation (Castex, 1965; Haddadet al., 2004; Cook et al., 2006; Clark et al., 2007).

Few studies about the toxic activities of fresh-water stingrays venom have been developed. Thelack of data is mainly due to the difficulty to extractvenom, and it is very difficult and dangerous tocapture the animals. The amount of venom is verylow, and likewise it is thermolabile (Haddad et al.,2004). The first study about the biochemistry andpharmacology properties of stingrays venom wascarried out by Russell and Van Harreveld (1954),which demonstrated cardiovascular effects of Ur-

obatis helleri venom. Rodrigues (1972) isolated anactive principle of freshwater stingray Potamotrygon

motoro venom with cholinergic activity on ileum ofguinea pigs and hypotensive activity when managedby intravenous injection in rats. Russell (1953)indicated the presence of polypeptides of highmolecular mass, serotonin and enzymatic activityof phosphodiesterase and 50-nucleotidase in marinestingray venom. Recently, we have detected 50-nucleotidase, phospholipase, acid phosphatase,hyaluronidase, caseinolytic, gelatinolytic and elasti-nolytic activities in P. motoro venom obtained fromanimals of Crixas-Ac- u River (Goias, Brazil) (Ma-galhaes, 2001). Caseinolytic, gelatinolytic and hya-luronidase activities were identified in Potamotrygon

falkneri venom (Haddad et al., 2004). In acomparative study of Potamotrygon scobina andPotamotrygon orbignyi venoms, Magalhaes et al.(2006) identified significant edematogenic and noci-ceptive response and necrosis in both venoms.Conceic- ao et al. (2006) isolated a vasoconstrictorpeptide from P. orbignyi venom with 1001.52Da.Barbaro et al. (2007), comparing the extracts fromthe tissue of marine and freshwater stingrays

Dasyatis guttata and P. falkneri, observed edemato-genic, gelatinolytic, caseinolytic and fibrinogenoly-tic activities in both extracts. Nociceptive activitywas verified in both tissue extracts; however,P. falkneri presented a two-fold higher activity thanD. 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 thatnaturally cleave hyaluronic acid, which is a majorcomponent of the extracellular matrix of vertebrates(Kreil, 1995). These enzymes are not toxic bythemselves, but can enhance local systemic envenoma-tion by increasing the absorption and diffusion ratesof the venom through the victim’s tissues since itcatalyzes the hydrolysis of the glucosaminoglycans,this being called the spreading factor (Duran-Reynals,1936). Hyaluronidase enzyme has been reported invenom of snakes, scorpions, bee, stonefish, lizards andspiders (Owen, 1983; Tu and Hendon, 1983; Pohet al., 1992; Kemparaju and Girish, 2006; Moreyet al., 2006; Nagaraju et al., 2007).

Recently, hyaluronidase activity was reported inthe freshwater stingrays’ crude venom (Magalhaes,2001; Haddad et al., 2004; Barbaro et al., 2007). Inthe present study, we show the results of purificationand characterization of hyaluronidase enzyme fromP. motoro venom.

2. Materials and methods

2.1. Venom and animals

Specimens of P. motoro were collected fromCrixas-Ac- u River (Goias, Brazil). The entire stingwas removed with bistouries, lyophilized andscraped. The collected material was macerated anddissolved in phosphate buffer 50mM, pH 7.0,containing 0.15M NaCl and immediately centri-fuged at 5000g for 10min. Venom was stored at�20 1C until use.

2.2. Estimation of protein concentration

Protein concentrations were determined by themethod of Lowry et al. (1951) using bovine serumalbumin (BSA) as a standard.

2.3. Assay of hyaluronidase enzyme activity

Hyaluronidase enzyme activity was determinedby the method described by Ferrante (1956),

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modified by Poh et al. (1992). The assay mixturecontained 200 ml acetate buffer 0.2M, pH 6.0,containing 0.15M NaCl, 50 ml hyaluronic acid(0.5mg/ml in acetate buffer) and 50 ml enzyme inacetate buffer. The mixture was incubated for15min at 37 1C and the reaction was stopped bythe addition of 500 ml of 2.5% (w/v) acetyltrimethy-lammonium bromide in 2% (w/v) NaOH. After10min, the absorbance of each reaction mixture wasread at 400 nm. Specific activity was expressed asNational Formulary Units (NFU), which is definedas the amount of enzyme required to hydrolyze0.255 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 describedby Laemmli (1970). After electrophoresis, gel wassilver 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 (0.38mg) was loadedon a Sephacryl S-100 column (2.5� 48 cm) pre-viously equilibrated with 50mM phosphate buffer,pH 6.0, containing 100mM NaCl, and eluted withthe same buffer at a flow rate of 40ml/h. Fractionsof 2.0ml were collected and monitored at 280 nm.Fractions showing the highest hyaluronidase activ-ity were pooled, dialyzed and applied directly onto aSP-Sepharose column (1.5� 13 cm) equilibratedwith 20mM phosphate buffer, pH 6.0, and elutedat a flow rate of 60ml/h. Fractions of 3.0ml werecollected and monitored at 280 nm. The column waswashed with the same buffer and eluted with alinear gradient of 0–1.0M NaCl. Fractions contain-ing hyaluronidase activity were pooled, dialyzed,lyophilized and stored at �20 1C.

2.6. Molecular weight determination

The molecular weight of the purified hyaluroni-dase was estimated by gel filtration chromatographyaccording to the method of Andrews (1962) oncalibrated columns (2.5� 48 cm) of Sephacryl S-100, using 50mM phosphate buffer, pH 6.0 (con-taining 100mM NaCl), at a flow rate of 40ml/h.Void volume (Vo) of the column was determined byusing blue dextran (1mg/ml in equilibration buffer).

Ovalbumin (43 kDa), chymotrypsinogen A (25 kDa)and ribonuclease A (13.7 kDa) were used asstandard proteins for obtaining the calibrationcurve. A calibration curve was obtained by plottingVe/Vo (KAV) against their respective logarithmicmolecular weights.

2.7. Enzyme characterization

The effect of pH on enzyme activity wasdetermined by varying the pH of the reactionmixtures using 100mM phosphate–citrate buffer(pH 2.5–7.0). The effect of temperature on enzy-matic activity was determined at pH 4.2, in therange of 20–50 1C. The effect of temperature onenzyme stability was determined after preincubationat 20, 30 and 40 1C for 5–30min. The effects ofmetallic ions and some compounds on hyaluroni-dase activity were determined after preincubation at4 1C for 15min. Km was determined from theMichaelis–Menten plot using Origin 7.0 programby measuring the initial rate of hyaluronic acidhydrolysis 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 theSephacryl S-100 gel filtration chromatography,which fractionated P. motoro venom gland extractinto two peaks of proteins (Fig. 1A). Fractions withhyaluronidase activity were pooled and concen-trated by lyophilization. Only 3.62% of the activityloaded onto the column was recovered in the pooledfraction. The second step involved the SP-Sepharoseion-exchange chromatography and resolved intoone peak of protein. The peak containing hyalur-onidase activity was eluted with a linear gradient ofNaCl (Fig. 1B). In this step 80% of the enzymeloaded onto the column was recovered. A summaryof the purification procedure is given in (Table 1).The enzyme was purified to 366.4-fold with ayield of 2.90%, having a specific activity of 1.33�108NFU/minmg of protein. SDS-PAGE showedthat the enzyme migrated as a single band (Fig. 2).Molecular weight of the hyaluronidase was esti-mated by gel filtration on Sephacryl S-100 usingstandard protein molecular weight markers and itwas found to be approximately 79 kDa (Fig. 3).

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250A

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)

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FU/m

L (-

----

--)

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0.00

0.01

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0.03

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250

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)

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Hya

luro

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se a

ctvi

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FU/m

L (-

----

-)

NaC

l (0

- 1.0

M)

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� 48 cm) was eluted with 50mM phosphate buffer, pH 6.0, containing 100mM NaCl at a flow rate of 40ml/h, and 2ml 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� 13 cm) was equilibrated with 20mM phosphate buffer, pH

6.0, at a flow rate of 60ml/h, and 2ml fractions were collected. The column was washed with the same buffer and eluted with a linear

gradiet of 0–1.0M 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� 103 3.63� 105 1 100

Sephacryl S-100 0.69 5� 103 7.24� 106 19.9 3.62

SP-Sepharose 0.03 4� 103 1.33� 108 366.4 2.90

M.R. Magalhaes et al. / Toxicon 51 (2008) 1060–1067 1063

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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.

0.051.0

1.2

1.4

1.6

1.8

2.0

Mol

ecul

ar W

eigh

t Log

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� 48 cm) of Sephacryl S-100, using

50mM phosphate buffer, pH 6.0 (containing 100mM NaCl), at

a flow rate of 40ml/h. Void volume (Vo) of the column was

determined by using blue dextran (1mg/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. Magalhaes et al. / Toxicon 51 (2008) 1060–10671064

3.2. Biochemical characterization

The pH activity profile of purified hyaluronidasewas determined in a pH range from 2.5 to 7.0 using

phosphate/citrate buffer. The enzyme had a typicalbell-shaped profile covering a broad pH range andan optimal pH of 4.2 (Fig. 4A). The influence oftemperature on hyaluronidase activity was deter-mined between 4 and 50 1C at pH 4.2. The optimaltemperature for hyaluronidase activity was 40 1Cand the activity decreased significantly above 40 1C(Fig. 4B). The enzyme was stable for at least 30minwhen incubated at 20 and 30 1C, but lost 70% of theactivity at 40 1C (Fig. 4C). The effect of varying con-centrations of hyaluronic acid on the initial velocityof the hyaluronidase showed a typical hyperbolicsaturation curve (Fig. 5). The Km (4.91 mg/ml) andVmax (2.02U/min) values were calculated from theMichaelis–Menten plot.

The activity of the purified hyaluronidases wastested in presence of metal ions and some chemicalcompounds (Table 2). No considerable effect wasobserved with Ca2+, Mg2+, Zn2+ and Hg2+,whereas Ni+, Fe2+ and Cu2+ reduced activity by25% approximately. b-Mercaptoethanol had aslight effect on enzyme activity, whereas heparin(0.05 IU) inhibited hyaluronidase activity by 20%.

4. Discussion

In this study with P. motoro venom extract, wefound that two-step fractionation on Sephacryl S-100 column and SP-Sepharose column resulted inthe purification of a protein with hyaluronidaseactivity. The final yield of 2.90% obtained andactivity of 4� 103NFU/ml will signify the difficultassociated with working with this enzyme. Similarresults were described by Xu et al. (1982) workingwith hyaluronidase from Agkistrodon acutus snakevenom. However, Poh et al. (1992) recovered 57%hyaluronidase from Synanceja horrida stonefishvenom and Pessini et al. (2001) recovered 43.6%hyaluronidase from Tityus serrulatus scorpionvenom.

Polyacrylamide gel electrophoresis showed thathyaluronidase purified from P. motoro migrated as asingle band with an estimated molecular mass of79 kDa, and exists as monomer. Most of thehyaluronidase described in the literature appearsas a monomer and varies considerably betweenorganisms. Molecular weight of hyaluronidase fromHeterometrus fulvipus (Ramanaiah et al., 1990) wasin a similar range, while proteins with 33, 52 and116 kDa have been described from A. acutus (Xuet al., 1982), T. serrulatus (Pessini et al., 2001) and

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500

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Hya

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200400600800

1000120014001600180020002200

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020

30

40

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(20°C) (30°C) (40°C)

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e ac

tivity

(%)

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 (N

FU/m

in)

5 10 15 20 25Hyaluronic 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. Magalhaes et al. / Toxicon 51 (2008) 1060–1067 1065

Streptococcus agalactiae (Ozegowski et al., 1994),respectively.

The optimal pH for the enzyme activity (4.2) wassimilar to that found for hyaluronidase from avariety 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). Theoptimum temperature was found to be 37 1C at pH4.2, and it is in agreement with hyaluronidase fromPalamneus gravimanus (Morey et al., 2006) andHippasa partita (Nagaraju et al., 2007). Thermo-stability is considered an important and usefulcriterion for enzyme characterization. The hyalur-onidase from P. motoro was stable for at least30min when incubated at 20 and 30 1C, but retainedonly 30% of the activity after incubation at 40 1C.

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Table 2

Inhibitor effects in the activity of P. motoro hyaluronidase

Compound (10mM) 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 (0.05UI) 3425.43778.00 79.5 20.5

M.R. Magalhaes et al. / Toxicon 51 (2008) 1060–10671066

The purified hyaluronidase from P. motoro

showed Michaelis–Menten-type kinetics with hya-luronic acid as substrate. The Km of 4.91 mg/mlindicates that the enzyme has comparatively highaffinity for hyaluronic acid compared with otherhyaluronidases. This value was substantially lowerthan 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, aconcentration as low as 10mM of some metal ionscould affect enzyme activity. The poor inhibition byHg2+ and b-mercaptoethanol, compounds that usual-ly react with cystein, led us to hypothesize about theabsence of these amino acids in the catalytic site of theenzyme. Similar results are found by hyaluronidasefrom P. gravimanus (Morey et al., 2006).

P. motoro purified enzyme is inhibited by heparinas described by hyaluronidase from A. acutus

venom (Xu et al., 1982), H. fulvipus scorpion venom(Ramanaiah et al., 1990) and P. gravimanus (Moreyet al., 2006).

In conclusion, this study presents the firstpurification of a hyaluronidase from P. motoro

sting. This enzyme shows similar characteristics asenzymes from venom of different organisms andexhibited high affinity for hyaluronic acid. Furtherstructural and functional analyses might provide aninsight for the better understanding of the role ofthis enzyme in envenomation by P. motoro.

Acknowledgments

This work was supported by a biotechnologyresearch grant to C.J.U. (CNPq, CAPES and

FUNAPE/UFG). M.R.M. was supported by Uni-versidade Catolica de Goias (CEPB). The authorsthank Dr. Joao Luiz da Costa Cardoso and Dr.Vidal Haddad Jr. (Hospital Vital Brazil, InstitutoButantan), and Dra Katia Cristina Barbaro (La-boratorio de Imunopatologia, Instituto Burantan)for valuable suggestions during this study.

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