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Governo do Estado de São Paulo

Secretaria de Estado da Saúde

Coordenação dos Institutos de Pesquisa

Instituto Butantan — São Paulo — SP — Brasil

MEMÓRIAS

INSTITUTO BUTANTAN

Volume 54, número 2, 1992

São Paulo, SP — Brasil

1992

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MEMÓRIAS do INSTITUTO BUTANTAN.

(Secretaria de Estado da Saúde).

São Paulo, SP — Brasil, 1918 —

1991, 53 (1, Supl. 1,2)

1992, 54 (1,2)

ISSN 0073-9901

MIBUAH

CDD 614.07205

Solicita-se permuta/Exchange desired

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10 11 12 13

Mem. Inst. Butantan

V. 54, n. 2, 1992

SUMÁRIO/CONTENTS

Structural and functional characteristics of toxins purified from the venom of

the Brazilian scorpion Tityus serrulatus Lutz and Mello,

Características estruturais e funcionais de toxinas purificadas no veneno do es¬

corpião brasileiro Tityus serrulatus Lutz and Mello.

LD. POSSANI, RL. FLETCHER JR., M. FLETCHER, G.S. RODE, J.

MOCHCA-MORALES, S. LUCAS, F.V. CORONAS, A.C. ALAGON, B.M,

MARTIN . 35

Herpetofauna das dunas interiores do Rio São Francisco: Bahia: Brasil. V. Duas

novas espécies de Apostolepis (Ophidia, Colubridae).

Herpetofauna of palaeoquaternary sand dunes of the middie São Francisco Ri-

ver: Bahia: Brazil. V. Two new species of Apostolepis (Ophidia, Colubridae).

Miguel Trefaut RODRIGUES. 53

Malformações em ninhadas de caiçaca, Bothrops moojeni (Serpentes, Vi-

peridae).

Abnormalities in litters of the pit viper, Bothrops moojeni (Serpentes, Viperidae).

Denis Vieira de ANDRADE, Augusto Shinya ABE . 61

COLETÂNEA DE RESUMOS DE TRABALHOS PUBLICADOS PELOS PES-

OUISADORES DO INSTITUTO BUTANTAN (1991) . 69

BOLETIM DE BIOTECNOLOGIA

Purificação industrial do toxóide tetânico por gel filtração

Industrial purification of tetanus toxoids by gel filtration

S.M.A. PRADO, M.D.C. VANCETTO, F, FRATELLI, M.M. PRAL, J.M. OLI¬

VEIRA, H.G. HIGASHI. 11

Produção de plasma antitetânico hiperimune, de origem eqüina.

Production of antitetanus hyperimune equine plasma.

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R, GUIDOLIN, C.R CARICATI, S.M.A. PRADO, M.D.C. VANCETTO, F.

FRATELLI, A.K. NISHIKAWA, H.G. HIGASHI . 17

índice do v. 54

MINI-SIMPÓSIO: SISTEMA COMPLEMENTO;

pesquisas de interface . 1

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Mem. Inst. Butantan

V. 54, n. 2, p. 35-52, 1992

STRUCTURAL AND FUNCTIONAL CHARACTERISTICS OF

TOXINS PURIFIED FROM THE VENOM OF THE BRAZILIAN

SCORPION Tityus serrulatus LUTZ AND MELLO.*

Lourival D. POSSANP' +

Paul L. FLETCHER JR.^

Maryann FLETCHER^

Gustavo S. RODE^

Javier MOCHCA-MORALES^

Sylvia LUCAS^

Fredy V. CORONAS^

Alejandro C. ALAGON^

Brian M. MARTIN'^

ABSTRACT: Venom from the scorpion Tityus serrulatus collected in four

distinct geographical localities of Brazil was studied by gel electropho-

resis, immunoelectrophoresis, Sephadex G-50 gel filtration and ion ex-

change chromatography. Lethality tests in mice were aiso conducted.

None of these methods revealed a significant difference among these

samples of venom. The Instituto Butantan venom of T. serrulatus has

shown constant and reproducible biochemical and pharmacological

properties, in our laboratories, for 18 years. The venom has several poly-

peptides toxic in mice, among the major toxic components are toxin gam-

ma, III-8 and IV-5, which affect sodium channel permeability. The

molecular weight of these toxins is in the order of 7,000. One of the

^ Departamento de Bioquímica, Centro de Investigaoión sobre Ingeniería Genética y Biotecnologia, Universidad Na¬

cional Autónoma de México, Apartado Postal 510-3, Cuernavaca 62271, MÉXICO.

^Department of Microbiology and Immunology East Caroiina University School of Medicine, Greenville, North Caro-

lina 27858-4354. USA.

^Instituto Butantan, Av. Vital Brasil, 1.500, SSo Paulo, SP 05503-900 BRASIL

^National Institute of Mental Health, Section on Molecular Neurogenetics, Clinicai Neuroscience Branch., ADAM-

HA, Building 10 3D16, Bethesda Maryland, 20892 USA.

Author to whom correspondence should be addressed:

Departamento de Bioquímica, Centro de Investigación sobre Ingeniería Genética y Biotecnologia, UNAM, Apartado

Postal 510-3 Cuernavaca 62270 MÉXICO Telephone (73)172799, FAX: (73)172388.

• Pari of this article was used for the master of Sciences degree of G.S. Rode, Facultad de Químicas, UNAM, (1984).

Recebido para publicação em 9.10.1990 e aceito em 11.6.1992.

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POSSANI, L.D. et al. Structural and functional characteristics of toxins purified from the venom of

the Brazilian scorpion Tityus serrulatus Lutz and Mello. Mem. Inst. Butantan, v. 54, n. 2, p. 35-52,

1992.

minor components is toxin II-9A, a shorter peptide (approx. 4,200 mol.

weight) that modifies potassium pernneability in axons. At least four ad-

ditional minor toxic components were aiso purified with very similar amino

acid sequences to the major components. Tityus venom was shown to

exert an important secretagogue effect on guinea pig pancreas.

KEYWORDS; Scorpion toxins, T serrulatus, pancreatitis, amino acid se-

quence.

INTRODUCTION

Toxins from the venom of the Brazilian scorpion Tityus serrulatus Lutz and Mel¬

lo have been purified and studied by several independent groups of investi-

gators^®'^^'^'^^'^^, and several reviews'^'^® have been written. Tityustoxin and toxin

gamma are the most thoroughly characterized. Toxin gamma was the first toxin

for which a considerable part of the N-terminal amino acid sequence was

reported^^'^^ and the only one, thus far, for which the total amino acid sequence

has been determined^^e ^ brief summary of the different types of toxins present

in the venom of T. serrulatus was reviewed by Possani^®, Pharmacological and

neurochemical studies were initially performed with TityustoxinIn recent

years, both Tityustoxin and toxin gamma have been more extensively studied, and

have been shown to affect Na * channel permeability of a variety of tissues^^'^^

(and review)2°. Initial work conducted by Bartholomew^ reported that stings by

scorpions of the genus Tityus can cause acute pancreatitis in humans.

AIso the effect of the venom from T. serrulatus was demonstrated to cause

pancreatitis in dogs^^. Pioneer work by Novaes et aF^. performed with tityustoxin

has shown a clear effect of this purified toxin on the pancreatic secretion of the

rat. Our preliminary experiments have shown several purified toxins to be active

in secretory discharge of zymogen proteins of pancreatic lobules^^. In the past

it has been suggested that some of the differences found in the venom of T. ser¬

rulatus could be due to heterogeneity from scorpions of the same species col-

lected in different geographical localities of BraziP'*. There is need for a definitive

study on the different toxic components present in the venom of T. serrulatus used

at the Butantan Institute (Brazil). The purpose of this communication is to present

the analysis of the venom of T. serrulatus from scorpions collected at different

locations in Brazil by Butatan Institute, and at the same time to report some Chem¬

ical and physiological charecteristics of toxins isolated from this venom.

MATERIALS AND METHODS

Venom Source.

Venom from T. serrulatus was obtained from the Instituto Butantan, São Pau¬

lo, Brazil. The scorpions were collected in several geographical localities of the

State of Minas Gerais (Santa Barbara, Corrego Novo and Cataguases) and from

the State of São Paulo (Aparecida do Norte). In Minas Gerais scorpions often are

found in elevated places, mountains covered by low vegetation and grasses or

in cracks and crevices of eroded land. They are aiso found in wooden support

for the railroad track. In São Paulo they were mostiy collected near houses.

Venom used for immunization at the Butantan Institute is normally obtained from

scorpions of different areas, mainly from Santa Barbara and Corrego Novo, but for

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Brazilian scorpion Tityus serrulatus Lutz and Mello. Mem. Inst. Butantan, v. 54, n. 2, p. 35-52, 1992.

this specific work, special care was taken to keep the venom separate for each

area (see above). Venoms were dhed in vacuum and kept refrigerated. Venom

from T discrepans was obtained from animais collected in the mountains sur-

rouding Caracas City, in Venezuela. The venom was lyophilized and kept at -20°C.

The dhed venoms were dissolved in water and centrifuged 18,000 x g, for 10 min.

The supernatants were directiy used for comparative studies of whole venoms

or for fractionation and characterization of toxic components.

Source of Chemicals.

Sephadex G-50 was obtained from Pharmacia Fine Chemicals (Uppsala,

Sweden) and carboxymethyl-cellulose resins (CM-32) and diethylaminoethyl cel-

lulose resins (DE-32) were from Whatman Inc., (Clifton, New Jersey). Spectrapor

type 3 dialysis tubing was obtained from Spectrum Medicai Industries (Los An¬

geles, CA.). Radiolabeled lodoacetic acid carne from New England Nuclear (Boston,

Mass.), while dithiothreitol (DTT) was from Calbiochem Behring Corp. (La Joila,

CA). Most reagents for high performance liquid chromaography were from Pierce

Chemical Co. (Rockford, Illinois) and Chemicals and solvents for amino acid se-

quencing were from Beckman Instruments Inc. (Paio Alto, CA.), or from the sources

indicated^®'^L23 Additional Chemicals were analytical grade reagents purchased

from the following sources; paraformaldehyde, Eastman Kodak Co., (Rochester,

NY); glutaraldehyde, Electron Microscopy Sciences (Fort Washington, PA); osmi-

um tetroxide, Ernest F. Fullam, (Latham, NY); magnesium uranyl acetate, K&K

Laboratories (Plainview, NY); epoxy resin, LX-112, Ladd Res. Industries (Burling¬

ton, VT); acrylamide and other reagents for gel electrophoresis, BioRad Labora¬

tories (Richmond, CA).

Animais.

Adult albino mice (CD1 strain), approximately of 20 g body weight were used

for lethality tests. Male albino Dunkin-Flartiey guinea pigs weighing 250-350 g

were used for microscopy studies.

Source of Anti-venom.

Florse anti-venom was obtained from Butantan Institute. The anti-venom (whole

serum, undigested) was separated from the blood of horses immunized against

the standard venom of T. serrulatus.

Lethality Tests.

Lethality tests in mice were conducted as described^^. LD^q values were graphi-

cally determined from percentage of death mice versus logarithm of venom concentration.

Basically, aliquots were injected intraperitoneally into 20 g mice that were observed

for toxic effects. Among the symptoms of toxicity are: hyperexcitability, lacrimation,

apnea, partial paralysis of the rear limbs, diarrhea, respiratory arrest and death.

Gel Electrophoresis and Immunoelectrophoresis.

The gel electrophoresis system of Reisfeld et al.^^. was used for comparison

of venoms and assess purity of samples. Immunodiffusion and immunoelectropho¬

resis were carried out in 1% agarose gels'^, and precipitates were visualized af-

ter dye staining ot dhed and washed gels‘*.

Protein quantification.

Uniess otherwise stated, protein content was determined spectrophotometri-

cally, assuming that one unit at 280 nm (1 cm pathiength) is equivalent to 1 mg/ml

protein concentration.

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the Brazilian scorpion Tityus serrulatus Lutz and Mello. Mem. Inst. Butantan, v. 54, n. 2, p. 35-52,

1992.

Separation Procedures.

Separation of soluble venom was basically performed according to previous

publication by our group^^. Briefly, the procedure includes a size exclusion chro-

matography and two consecutive steps of weak cation exchange chromatogra-

phy through carboxymethyl-cellulose (CM-cellulose) resins. The first separation

is performed in a column (0.9 x 200 cm) of Sephadex G-50 (médium), equilibrat-

ed with 20 mM ammoniumm acetate buffer, pH 4.7, from which four main frac-

tions are obtained, Each one of the toxic fractions (II to IV) are further separated

in CM-Cellulose resins at two different pH's. The first CM-Cellulose column (0.9

X 30 cm) is developed in ammonium acetate buffer, pH 4.7. In this step toxic com-

ponents 11-11, III-8 and 111-10, and IV-5 are separated, corresponding to the main

toxic components of the venom^^.

Each one of these components is subsequentiy purified by rechromatography

of the dialyzed fractions through a second CM-Cellulose column equilibrated with

50 mM sodium phosphate buffer, pH 6.0. A linear gradient from 0 M to 0.5 M

NaCI in the same buffer is used to elute the toxic proteins in homogeneous form.

Enzymatic digestion mixtures of reduced and alkylated proteins used for ami-

no acid sequencing were chromatographically separated into their component pep-

tides by reverse-phase high performance liquid chromatography (HPLC) on

octadecylsilane columns developed with linear gradients up to 60% acetonitrile

(containing 0.1% trifluoracetic acid). Isolated fractions were evaporated to dry-

ness under vacuum in a Savant Speed-Vac Concentrator prior to amino acid anal-

ysis and sequenator appiication.

Hyaluronidase Purification.

Hyaluronidase was assayed by the method of Tolksdorf et a!?^. The enzyme

was purified from the venom of T. serrulatus by three consecutive steps: Sepha¬

dex G-50 gel filtration, ion-exchange chromatography through diethylaminoethyl-

cellulose (DE-Cellulose) resins and affinity chromatography using Sepharose-

concanavalin A. The hyaluronidase activity elutes in the first fraction of the Sepha¬

dex G-50 column (see Resuits). This fraction was loaded in a DE-Cellulose column,

after dialysis against 50 mM TRIS-HCL buffer, pH 7.6. To the unbound material

containing the hyaluronidase activity saits were added to give the final concen-

tration of 1 mM CaCI, 1 mM MnCI and 1.0 M NaCI, in the same TRIS-HCI buffer,

pH 7.6. This material was chromatographed through a Sepharose-concanavalin

A column (10 ml) equilibrated with the same buffer mentioned above. Elution was

obtained with stepwise gradients of the equilibration buffer containing 0.04 M

and 0.5 M alpha-methyl-mannoside. Most of the enzyme was eluted with the se¬

cond buffer (0.5 M mannoside).

Amino Acid Composition and Sequence Determination.

Amino acid composition was obtained with acid hydrolysates of purified pro¬

teins analyzed with a Durrum/Dionex D-502 automatic amino acid analyzer, as

described^T32 p^jor to amino acid sequence determination the toxins were either

reduced and carboxymethylated or pyridylethylated, as described^^^^s.s’, Deter¬

mination of half-cystine was obtained after performic acid oxidation^'*. Tryptophan

content was deduced from sequence data. Carboxypeptidase digestionn for the

determination of carboxyl-terminal amino acid residues was carried out in 50 mM

sodiumm acetate buffer, pH 3.5, with carboxypeptidase P (Boehringer Mannheim

Biochemicals, Indianapolis, IN). Fragmentation of reduced and alkylated toxins

by enzymatic cleavage with protease V8 from Staphylococcus aureus and/or tryp-

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POSSANI, L.D. et al. Structural and functional characteristics of toxins purified from the venom of

the Brazilian scorpion Tityus serrulatus Lutz and Mello. Mem, Inst. Butantan, v. 54, n. 2, p. 35-52,

1992.

sin was obtained as described previously^^ Peptides were separated by HPLC as

mentioned above.

Primary sequence determination by automatic Edman degradation^^ was car-

ried out either on a Beckman 890M (Paio Alto, CA) microsequencer or on

polybrene-treated fiber disks in an automated gas phase sequenator (Mod. 470A,

Applied Biosystems, Foster City, CA), equipped with an online PTH-analyzer, as

described^’'^^.

Light and Electron Microscopy.

Pancreatic lobules were prepared according to the method described by Scheele

and Palade^®.

They were immersed in Karnovsky's fixative''® (1% formaldehyde-2%

glutaraldehyde in 0.1M sodium cacodylate, pH 7.4) and diced into 1.0 mm cubes,

and allowed fo fix overnight at 4°C.

Tissues were postfixed 3 hours at 4°C in 1% osmium tetroxide in 0.1M sodium

cacodylate, pH 7.4. Following en bloc stain in 0.5% magnesium uranyl acetate

overnight, tissue blocks were dehydrated and embedded in LX-112 epoxy resinai.

For light microscopy, sections 0.5 /xm in thickness were stained with 1% tolui-

dine blue in 1% sodium borate^' and photographed in a Zeiss Standard 16 micro-

scope equipped with a Zeiss MC63a photomicrographic camera.

For electron microscopy, thin sections of 800 A were stained with alkaiine lead

citrate^°, then examined and photographed in a JEOL 1200EX electron microscope.

RESULTS

The five different venom samples obtained from T. serrulatus collected in

Aparecida do Norte, Cataguases, Corrego Novo, Santa Barbara and the standard

Butantan venom (mainly a mixture of Santa Barbara and Corrego Novo) were as-

sayed for toxicity in mice and for enzymatic activities. They were analyzed by elec-

trophoresis in beta-alanine-urea-acetate gels and immunoelectrophoresis for

comparativa purposes. They were each separated under identical conditions by

gel filtration and ion-exchange chromatography. Lethal dose (LD^q determinations

performed with CD1 albino mice, by intraperitoneal injections, gave essentially the

same resuits for each of the venom samples. For example, the venom from Santa

Barbara has a LD^q of 21 ± Vg/20 g mouse weight, while the standard venom

gave 22 ± Vg/20 g mouse weight. The only enzymatic activity found in all 5

samples of venom was hyaluronidase, which was further characterized (see be-

low). The resuits of electrophoresis separation of venom from scorpions collect¬

ed in different geographical localities are presented in Fig. 1. The same amount

of protein (100 ^g) was appiied in each cylindrical tube for polyacrylamide gel

separation. At least 14 components are visible when stained with coomassie blue;

numbers 5, 10, 11, 12, 13 and 14 being the more intensely colored. The most

basic components with faster eletrophoretic mobility correspond to toxic peptides.

The venoms have very similar electrokinetic properties. A slight variation in the

realtive intensity of certain colored bands might be present. Compare for exam¬

ple, band 2 and 10 of lane c with the others. In Fig. 2 are the immunoelectrophoretic

analysis of the different venoms using horse anti-venom as the serum source. The

longitudinal slot contained the serum, while the venoms were placed inthe lateral

Wells. Two plates were run in parallel. The resuits of Fig. 2A show no appreciable

immunological differences among the venoms. Fig. 2B shows the result of T. ser-

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POSSANI, L.D. et al. Structural and functional characteristics of toxins purified from the venom of

the Brazilian scorpion Tityus serrulatus Lutz and Mello. Mem. Inst. Butantan, v. 54, n. 2, p. 35-52,

1992.

a b c d e

Fig.l. Gel electrophoresis of T. serrulatus venonns.

T. serrulatus venom from scorpions.collected in Corrego Novo and Santa Barbara

(a), Santa Barbara(b), Corrego Novo(c), Aparecida do Norte(d) and Cataguases(e)

was appiied to cylindrical gels of polyacrylamide (100 ug soluble venom in each la¬

ne) and run in the beta-alanine-acetate-urea electrophoresis system of Reisfeld

eíal^^. Proteins and peptides run towards the cathode. A least 14 bands are visible;

the most intensevely colored are number 5 and 10 to 14. R. designates origin of run-

ning gel, and T designates tracking dye.

ruiatus venom and a control from another species of the same genus of scorpion

Tityus discrepans). This figure (2B) shows a marked difference between T. serru¬

latus venom and T. discrepans collected in Venezuela. Fractionation of soluble

venom by gel filtration on Sephadex G50 separates the venom from T. serrulatus

into four main components (I to IV), see Fig. 3A, aiso Possani et al.^^ Very simi¬

lar resuits were observed with venoms obtained in different geographical areas

(data not shown). In Fig. 3 (B to D) are the resuits of further fractionation of the

toxic fractions II, lll and IV in CM-Cellulose columns.

Ten toxic sub-fractions were obtained; 11-9 to 11-11, 111-7 to 111-10, IV-5, IV-6 and

IV-8, all toxic for mice. As discussed further below the toxic sub-fractions (ten)

are not necessarily all different.

By this ion exchange separation method no appreciable differences were found

among venoms of T. semu/afus collected in different geographical localities (data

not shown). Final separation of pure polypeptides requires an additional chroma-

tographic step. Usually a CM-Cellulose column equilibrated and run in 50 mM

sodium-phosphate buffer, pH 6.0 is used. Dialyzed components 11-11, III-8, 111-10,

and IV-5 were purified under these conditions to homogeneity.

Separation was obtained with a sait gradient from 0 to 0.5 M NaCI (see exam-

ple in Fig. 3E).

During this chromatography small contaminants are eliminated; and the major

toxic component is the main fraction of the chromatogram. The amino acid com-

POSSANI, L.D. et al. Structural and functional characteristics of toxins purified from the venom of

the Brazilian scorpion Tityus serrulatus Lutz and Mello, Mem. Inst. Butantan, v. 54, n. 2, p. 35-52,

1992.

position of the major toxins is compiled in Table I. Toxin 11-11 (aiso called gamma

toxin^^, and toxin 111-10 are identical.

Recoveries obtained during these separation procedures are indicated in the

legend of Fig. 3.

The homogeneity of toxic peptides was assessed by several criteria; shape of

ion exchange columns (symmetrical peaks), gel electrophoresis^^, amino acid

composition, profile of Sephadex G50 separation of reduced and alkylated tox-

Fig.2. Immunoelectrophoresis of venoms from T.serrulatus.

PANEL A: Venom from scorpions collected in 5 different places were appiied in aga-

rose gel in 25 mM TRIS-Glycine buffer, pH 8.3 for electrophoresis and immunodif-

fusion, according to King eí a/17 . Wells contained 100 fig (in 10 ul) of each venom:

Corrego Novo and Santa Barbara (1), Santa Barbara (2), Corrego Novo(3), Apareci¬

da do Norte (4) and Cataguases (5). In the central slots 50 ix\ of horse anti-serum

were used for immudiffusion.

PANEL B: Comparison of standard venom of T. serrulatus (6) and venom from T.

discrepans (7) appiied in the same conditions as in panei A.

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POSSANI, L.D. et al. Structural and functional characteristics of toxins purífied from the venom of

the Brazilian scorpion Tityus serrulatus Lutz and Mello. Mem. Inst. Butantan, v. 54, n. 2, p. 35-52,

1992.

Fig.3. Chromatographic separatíon of T. serrulatus venom.

A: Sephadex G-50 gel filtration of 160 mg (in 1.4 ml) of soluble venom in 20 mM

ammonium acetate buffer pH 4,7. Column of 0.9 x 200 cm run at flow rate of 15

ml/h and collected in 2 ml volume-fraction. Horizontal bars indicated pooled frac-

tions (l-IV). T means toxic components. Final recovery was in the order of 83% as

follows: fraction I (14%), 11 (32%), III (20%), IV (14.5%) and side tubes (2,5%).

B; CM-cellulose column separation of fraction II (approx. 42 mg) from Fig. 3A in

the same buffer 20 mM ammonium acetate, pH 4.7, eluted yyith a linear gradient

(250 ml each) from O to 0.55 M NaCI. Column (0.9 x 30 cm) was run at 30 ml/h,

and tube-fractions of 2.5 ml were collected. Toxic fractions 9, 10 and 11 correspond

respectively to 3.4, 2.4 and 24.1% of total protein recovered.

C: Same as in B for fraction III (26 mg) of Fig.3A Toxic fractions 8,9 and 10 corres¬

pond respectively to 15.5., 6.3 and 16.4% of protein recovered. Fractions 6 and 7

(toxic) were pooled because they were not well separated, and correspond to 32,4%

of total material recovered.

D: Same as in B fraction IV (19 mg) of Fig.3A. Toxic fractions 5, 6 and 8 correspond

respectively to 10.5, 5.9 and 4.9% of material recovered.

E: Final purification of gamma toxin from sub-fraction 11-11 of Fig 3B (10 mg) in 50

mM phosphate buffer pH 6.0, Column, ion exchanger, flow rate, tube-fractions and

sait gradient similar to Fig. 3B. Purified toxin is approximately 84% of the material

appiied to the column.

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POSSANI, L.D. et al. Structural and functional characteristics of toxins purified from the venom of

the Brazilian scorpion Tityus serrulatus Lutz and Mello. Mem. Inst. Butantan, v. 54, n. 2, p. 35-52,

1992.

Fig.4. Light and electron micrographs of guinea pig pancreas.

Panei A: left panei (control): Light micrograph of normal guinea pig pancreas show-

ing exocrine cells (arrows) arranged in alveolar units of acini around a central lumen

(L). Densely stained zymogen granules (Z) are clustered in abundance around the

apical portions of acínar cells. These cells characteristically have large numbers of

zymogen granules prior to stimulation by cholinergic or peptidergic exocrine secreta-

gogues. Another portion of the picture shows an Islet of Langerhans (IL). Bar igual 10;x.

Panei A: right panei (venom): Light micrograph of guinea pig pancreas after a lethal

dose of the Brazilian scorpion Tityus serrulatus whole venom. The animal died two

hours postinjection. Zymogen granules are almost completely depleted due to their

discharge from stimulation by the venom secretagogues. Bar igual ^0^l.

Panei B: Upper panei (control): Electron micrograph of normal guinea pig pancreas

24 hours after a control injection of Krebs-Ringer Bicarbonato. Densely stained zymo¬

gen granules (Z) are packed at the apical portions of acinar cells prior to stimulation

by natural secretagogues. The acinar lumen (L) is contracted with numerous microvilli

and a small amount of secretory material characteristic of resting secretion. In general,

both the Golgi complexes and the endoplasmic reticuium (ER) are normal in appear-

ance. Bar igual V

Panei B: Lower panei (venom): Electron micrograph of guinea pig pancreas from an

animal injected with a lethal dose of venom from the Brazilian scorpion, Tityus ser¬

rulatus. At death, 2.8 hours post-injection, the zymogen granules had been discharged

in response to stimulation by the venom secretagogues. The acinar lumen (L) is dis-

tended with densely stained crystailine secretory material. The two cells at right of

picture have rough ER cisternae grossiy dilated with secretory proteins and may have

sustained some structural damage. Numerous apical vesicles (V) present in the cells

at bottom and upper left are evidence that exocytosis had occurred. Bar igual V

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POSSANI, L.D. et al. Structural and functional characteristics of toxins purified from the venom of

the Brazilian scorpion Tityus serrulatus Lutz and Mello. Mem. Inst. Butantan, v. 54, n. 2 , p. 35-52,

1992.

ins, and amino acid sequence. Table II summarizes the resuits of amino acid se-

quence determination of all the toxic components thus far analyzed. AIso includ-

ed for comparative purposes are data from the literature. It should be noted that

toxin II-9A and II-9B were originally separated after reduction and alkylation of

fraction 11-9 (ref.^°). Using a different strategy we were abie to obtain toxin II-9A

in homogeneous form to show the effect on channels ® and Valdivia, H.H.

and Possani, L.D,, unpublished. Complete amino acid sequence of toxin gamma

(11-11) was obtained after HPLC of enzymatically cleaved toxin^’. Toxin III-8 se¬

quence was aiso obtained after several cleavages of reduced and alkylated pep-

tide, including carboxypeptidase time course hydrolysis^®.

Purified toxins have been used to verify the mechanism of action in a variety

of preparations "''■le gnd reviews'°''‘''2°. As wili be discussed below two classes

of toxins were found in the venom of T. serrulatus, long Chain polypeptides (ap-

proximately 60-62 amino acids), which impair functioning of Na*' channels, and

short-chain peptides (approximately 40 amino acid residues), which modify potas-

sium channels from a variety of excitable membranes. Still another known effect

of T, serrulatus venom is the secretagogue effect on pancreas^’''2'22.25 por this

reason the resuits of morphology experiments in guinea pigs reported in Fig. 4A

(light microscope) and 4B (electron microscope) were performed. It is clearly

shown in Fig. 4A (left picture for control) that zymogen granules in pancreatic

acinar cells are abundant in the apical areas of these cells. The tissue displays

its typical cytological organization, while in Fig. 4A (right picture for animais in-

jected with venom) the zymogen granules are reduced in number or even absent

in some acinar cells. Referring of the higher magnifications of the electron micro-

graphs in Fig. 4B the acinar tissue demonstrates some of the distinctive charac¬

teristics of normal apical acinar cell organization with abundant electron-dense

zymogen granules, compact acinar lumen with distinctive microvilli, and stacks

of endoplasmic reticuium (ER) cisternae. In Fig. 4B (lower part, venom-treated

pancreas) the apical region is largely devoid of mature zymogen granules, and

a few granules of reduced size remain. The discharge of zymogen granules is evi-

dent from the abundance of crystailine and amorphous secretory content in this

lumen. Fig. 4B shows in addition to the lack of zymogen granules that the ER

cisternae are greatly distended with proteins vectorially destined for discharge.

Apical vesicles are evidence of recycling of zymogen granule membrane after ex-

ocytotic release in response to venom-secretagogue stimulation. Other micrographs

(not shown) demonstrate luminal surfaces partially effaced with loss of microvilli

on part of the acinar plasmalemma.

The only enzymatic component isolated in homogeneous form from this ve¬

nom was the hyaluronidase, which was non-toxic. For this purpose we have used

three chromatographic separation steps (see Material and Methods). The enzyme

activity is very stable in the whole venom, but during purification, especially in

the last step, the activity is lost within a few hours.

The enzyme was obtained in pure form, as judged by polyacrylamide gel elec-

trophoresis, and its molecular weight was calculated to be 42,000, with the aid

of SDS polyacrylamide gels^^ (data not shown).

DISCUSSION

T. serru/aftvs venom from the Instituto Butantan (São Paulo, Brazil) used by our

group during the last 18 years has always given very consistent biochemical and

pharmacological resuits.

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POSSANI, L.D. et al. Structural and functional characteristícs of toxins purified from the venom of

the Brazilian scorpion Tityus serrulatus Lutz and Mello. Mem. Inst. Butantan, v. 54, n. 2, p. 35-52,

1992.

Numerous batches of venom obtained from the Butantan Institute during the peri-

od of 1973 to 1990 have been used in our laboratories, initially for purification of

several toxins^ and characterization of gamma toxin^^''^. Since 1981 the strategy

we have followed for purification and Chemical characterization of toxins is very similar

to the one presentiy described^^'^!, which we assume is the most convenient one.

At least in our hands the venom from the Butantan Institute is highly appreciated

because of its constant and reproducible biochemical and pharmacological behavior.

One of the main objectives of this communication is to compare venom obtained

from T. serru/aíusscorpion of different geographical localities of Brazil; at least from

regions not considered to be "restricted areas", as mentioned by other authors''°'34

A carefui observation of our resuits (Fig. 1 to 3) indicates that there are no signifi-

cant differences in the venom of T. serrulatuscoWecXed in topological areas greater

than 400 Km. The distance of Santa Barbara, Corrego Novo and Cataguazes from

Belo Horizonte, the capital of the State of Minas Gerais, is 100, 400 and 460 Km

respectively, while the distance of Aparecida do Norte from the capital of São Paulo

State is 200 Km. Lethality tests conducted with venom from scorpions of different

localities, using 10 different doses of venom, in 10 different groups of mice (strain

GDI, i.p.) gave essentially the same value (see Resuits), which in turn is the same

or very similar to the value initially reported by Bücherr, and by our group^^, of 25

figl20 g mouse weight. Electrophoretic separation of venoms by the system of Reis-

feld et aP^, suggested aiso that there was no appreciable difference in these ven¬

oms. It is worth mentioning that Reisfeld's method is capable of discriminating small

differences (charge groups) in scorpion toxins. If sodium dodecyl sulfate gel elec-

trophoresis is used according to the technique of Laemmli^^, most of the toxins

comigrate in a single broad band. Thus, we conclude that the beta-alanine-acetate-

urea gel system is better suitedforverification of scorpion toxin purity, and isa good

criterion for judging homogeneity of sample. Futhermore, immunoelectrophoresis

is an adequate technique for verification of heterogeneity of samples; at least, it

can discriminate mixtures of antigens or possible immunodominant antigens. This

was the case found (Fig. 2B) when comparing venom from another related species

of scorpion (Tityus discrepans) coWected in Venezuela. Finally, the chromatograph-

ic separation by Sephadex G-50 and CM-Cellulose shows that these venoms are

very similar in composition (data not shown).

The separation procedure described here for the isolation of mammalian tox¬

ins from the venom of T. serrulatus allows the identification of 10 sub-fractions

toxic to mice. However, fraction 11-11 (originally denominated toxin gamma^^ and

111-10 are identical components, as demonstrated by amino acid analysis (Table

I) and by amino acid sequence (Table II).

Fractions II and III from Sephadex G50 column (Fig. 3A) are not well separat-

ed, hence the subsequent ion exchange chromatography of both fractions in CM-

Cellulose columns at pH 4.7 (Fig. 3B and 30 provided two chromatographic peaks

corresponding to the same toxin. The major toxic components in the venom of

T. serrulatus are toxin gamma, III-8 and IV-5. They have in the order of 60-62

amino acid residues (Table I), with a calculated molecular weight of 6,500 to 7,000.

Toxic sub-fraction III-7 is aIso a major component of the venom, but it is heavily

contaminated (data not shown), and stili further purification is required. Among

the minor components are toxins 11-9, 11-10, III-9, IV-6 and IV-8. Toxin 11-10 has

an amino acid sequence identical to toxin gamma up to residue 46 (Table II), al-

ready sequenced. Similarly, component II-9B is equivalent to III-8 up to residue

30 (Table II). Chemical differences between these two pairs of components pos-

sibly are at the C-terminal region of the molecule.

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POSSANI, L.D. et al. Structural and functional characteristics of toxins purified from the venom of

the Brazilian scorpion Tityus serrulatus Lutz and Mello. Mem. Inst. Butantan, v. 54, n. 2, p. 35-52,

1992.

Minor components III-9 and IV-8 were not studied, and component IV-6 is iden-

tical to IV-5 up to residue number 30 (Table II). Moreover, the difference in chro-

matographic behaviour may be due to a deamination occuring at an Asn (or Gin).

Component II-9A is a minor component, initially found by direct sequence3°, with

sequence homology to Noxiustoxin, a K* channel blocking peptide isolated from

Centruroides noxius scorpion venom^. Rechromatography of component 11-9,

produced toxin 1, which blocks K" channels of squid giant axons^.

Other authors have studied venom from T. serrulatus and found similar toxins

named with different nomenclatura as the one we have proposed, The first toxin

purified was Tityustoxin by the group of Prof. Diniz''^ in Brazil. This toxin is simi¬

lar in amino acid composition to our component IV-5 (see review^°). The purifi-

cation procedure of Toledo and Neves^^ confirms the presence of more than one

toxin in the venom of T. serrulatus, but presented no amino acid sequence data.

Based on amino acid composition and N-terminal amino acid (lysine) we have

surmised that toxin TsTx-l^"^ is the toxin gamma of our work, while toxin TsTx-ll is

a new toxin in this venom^^. It does not correspond with any of the toxins we

are describing or to the ones purified by Sampaio et a!?^.

The group of Prof. Rochat in France has sequenced a toxin® almost identical

to gamma toxin®'', which they have called toxin VII. The only difference is at the

cysteine at position 61, which they report to be amidated. The publication by Sam¬

paio et aP^ reports the purification of five toxins from T. serrulatus. Considering

the amino acid sequence reported®®, toxin T1VIII is similar to toxin gamma®^, to¬

xin T2III1 is possibly the same as IV-5 of our work. Toxins T1VI and T1IV®® seem

to be toxins previousiy unreported in this venom.

Concerning the pharmacological effects of venom or purified toxins from T.

serrulatus, the reviews by Diniz'° and recentiy by Freire-Maia and Campos''^ sum-

marize the main resuits. It is clear that most of the action of the toxic peptides

is through impairment of ion channel (sodium and potassium) funcion. Among

the most significant works thus far published are the effects of gamma toxin and

Tityustoxin on neuroblastoma cells (NIE 115), rat brain synaptosome membranes,

chick heart sarcolemma and other membrane preparations (see review by Laz-

dunski et al.^°). Our group was abie to show that gamma toxin affects the peak

permeabillyty of Na+ in cardiac cells, like the beta scorpion toxins'^', while toxin

IV-5 modifies Na+ channel gating like the North African alpha scorpion toxins'®.

Squid giant axon potassium channels are blocked by toxin II-9A (toxin 1) of T.

serru/afus venom®. Experimental pancreatitis in dogs caused by T. serrulatus was

reported by Machado etai^^, and T. trinitatis induced pancreatitis in humans by

Bartholomew®. Pioneer work with purified scorpion toxin from T. serrulatus (tit¬

yustoxin) on the pancreatic secretion of the rat was published by Novaes et al.^^.

We have demonstrated the effects of whole venom from T. serrulatus and pu¬

rified peptides (III-8, 111-10, and IV-5) in rodents (mouse, rat and guinea pig) in

vitro and in v/vo'®. Morphological alterations due to secretagogue effects of who¬

le venom (Fig 4) and its peptide components are apparent by both light and elec-

tron microscopy of pancreatic acinar cells. Biochemical measurements of

pancreatic secretion were carried out by both radioisotopic pulse-chase experi-

ments as well as determinations of enzymatic activity discharged'®. These stu-

dies indicate that the venom from T. serrulatus causes extensive discharge of

secretory proteins in vivo and in vitro.

Flumans stung by scorpions of genera Tityus and Centruroides in North and

South America develop potentially lethal symptoms from action of the venom on

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POSSANI, L.D. et al. Structural and functional characteristics of toxins purified from the venom of

the Brazilian scorpion Tityus serrulatus Lutz and Mello. Mem. Inst. Butantan, v. 54, n. 2, p. 35-52,

1992.

peripheral nerves and vital organs'°'’'‘^'2°'25,4i ^ included in a myriad of distress factors

is the epigastric pain of acute pancreatitis^'^^'^^'^®'^®. This pain is accompanied be

elevated serum amylase associated with clinicai signs of acute pancreatitis'^'^^'^®.

There are occasions when the pancreatitis alone is severely debilitating and represents

a complication that can extend the recovery of these patients considerably. When

the individuais are nottreated promptiy with appropriateantiserum the clinicai course

is aiways aggravated and prolonged, particularly in children under age

The only non toxic protein isolated from T. serru/afus venom, in pure form, is

the enzyme hyaluronidase that we have described here. This enzyme is known

to be ubiquitous in most scorpion venoms''’2T39_ and was initially reported to be

present aiso in T. serrulatus venom^^.

The enzyme is of high molecular weight (approx. 42,000) and is likely to be

a glycoprotein, since it binds concanavalin A. We have originally described a similar

enzyme in the venom of Centruroides limpidus Hmpidus^.

CONCLUSIONS

1. Venom from the scorpion T. serru/afus collected in several distinct geographi-

cal localities of Brazil does not show detectable differences in biochemical

and pharmacological properties.

2. The venom from Butantan Institute, in our hands, has proved to provide reproducible

resuits over a period of 18 years.

3. The venom contains two classes of mammalian peptide toxins, long-chain neu-

rotoxins with 60 to 62 amino acid residues, which block sodium channel

of excitable membranes (variety of tissues and sources), and short chain

neurotoxins (approx. 40 amino acids) which block potassium permeability

(squid giant axons and rabbit skeletal muscle).

4. The diversity of toxic peptides in the venom of T. serrulatus venom confirms

similar finding with Centruroides species of the North American continent,

and Androctonus, Leiurus and Buthus species of África and Asia.

5. We confirm data of a potent secretory effect of venom from T. serrulatusin pancreas.

RESUMO; O veneno de escorpiões Tityus serrulatus coletados em qua¬

tro localidades geográficas distintas do Brasil foi estudado por eletrofo¬

rese em gel, imunoeletroforese, filtração em Sephadex G-50 e cro-

matografia de intercâmbio iônico. Também se conduziram experiên¬

cias de letalidade em camundongos. Nenhum destes métodos revelou

diferenças significativas entre as amostras de veneno. O veneno de T.

serrulatus do Instituto Butantan tem demonstrado propriedades bioquí¬

micas e farmacológicas constantes e reproduzíveis em nossos labora¬

tórios por cerca de 18 anos. O veneno possui vários polipeptídeos tóxicos

ao camundongo. Entre os componentes tóxicos que aparecem em maior

concentração no veneno estão toxina gama, III-8 e IV-5, as quais modi¬

ficam a permeabilidade de sódio em canais iônicos. O peso molecular

destas toxinas é da ordem de 7,000. Um dos componentes que apare¬

cem em menor concentração no veneno é a toxina II-9A, um pequeno

peptídeo (aproximadamente de 4,200 de peso molecular) que modifica

a permeabilidade do potássio em axons. Pelo menos outros 4 compo¬

nentes que aparecem em baixa quantidade no veneno também foram

purificados com seqüência de aminoácidos muito semelhantes aos com-

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POSSANI, L.D. et al. Structural and functional characteristics of toxins purified from the venom of

the Brazilian scorpion Tityus serrulatus Lutz and Mello. Mem. Inst. Butantan, v. 54, n. 2, p. 35-52,

1992.

ponentes gama, 111-8 e lV-5. O veneno de Tityus serrulatus exerce um

efeito secretagôgo importante em pâncreas de cobaias.

UNITERMOS: Toxinas de escorpião, Tityus serrulatus, pancreatite, se-

qüência de aminoácidos.

ACKNOWLEDGEMENTS

Dr. Jorge Villegas is acknowledged for providing facilities for two of us (L.D.P.

and RLE.) during collection of scorpions Tityus discrepans o\ Caracas (Venezue¬

la); and Prof. M.A. Gonzalez-Sponga for identification of the species collected.

Technical assistance of Mr. Pedro Sauceda Ramirez and Mercedes Sifuentes Valenzuela

during preparation of the art work is recognized.

REFERENCES

1. ALAGON, A.C., POSSANI, L.D. Utilizacion de cromatografia por afinidad para

la purificacion de enzimas de venenos animales. In: HUITRON, C., ed. Bi¬

otecnologia de enzimas. México: UNAM, 1983. p. 125-135.

2. ALAGON, A.C., POSSANI, L.D., ERICKSON, B.W, Isolation and characteriza-

tion of several proteins from Tityus serru/afus scorpion venom. In: INTER¬

NATIONAL SYMPOSIUM ON ANIMAL PLANT AND MICROBIAL TOXINS,

5, 1976, San José, Costa Rica. Abstracts... p. 35.

3. ALAGON, A.C., POSSANI, L.D., ERICKSON, B.W. Isolation and characteriza-

tion of several proteins from Tityus serrulatus scorpion venom. Toxicon,

Suppl. 1, p. 609-618, 1978.

4. AXELSEN, N.H., CLAESSON, M.H., HARDT, R, RANLOV, R, ROPKE, C. A low-

dose immunization schedule for the production of ALG in rabbits. Scand.

J. Immunol, v.l, p. 109-113, 1972.

5. BARTHOLOMEW, C. Acute scorpion pancreatitis in Trinidad. Br. Med. J., v.l,

p. 666-668, 1970.

6. BECHIS, G., SAMPIERI, R, YUAN, P.M., BRANDO, T., MARTIN, M.R, DINIZ,

C.R., ROCHAT, H. Amino acid sequence of toxin VII, a beta-toxin from the

venom of the scorpion Tityus serrulatus. Biochem. Biophys. Res. Commun.,

V.122. p. 1.146-1.153, 1984.

7. BUCHERL, W. Classification, biology, and venom extraction of scorpions. In:

BUCHERL, W., BUCKLEY, E.E., ed. Venomous animais and their venoms,

III. Venomous vertebrates. New York: Academic Press, 1971. p. 317-147.

8. CARBONE, E., PRESTIPINO, G., WANKE, E., POSSANI, L.D., MAELICKE, A.

Selective action of scorpion neurotoxins on the ionic currents of the squid

giant axon. Toxicon, Suppl. 3, p. 57-60, 1983.

9. CARBONE, E., WANKE, E.; PRESTIPINO, G., POSSANI, L.D., MAELICKE, A.

Selective blockage of voltage-dependent K" channels by a novel scorpi¬

on toxin. Nature, v 296, p. 90-91, 1982.

10. DINIZ, C.R. Chemical and pharmacologic aspects of Tityinae venoms. In: BETTINI,

S., ed. Arthropod venoms. Berlim: Springer-Verlag, 1978. p.379-394.

11. EDMAN, R, BEGG, G. A protein sequenator. Eur. J. Biochem., v. 1, p. 80-91, 1967.

12. FLETCHER, M.D., FLETCRIER JR., RL. Biochemical and ultrastructural changes

associated with scorpion venom induced pancreatitis. Digestive Dis., v. 29,

p. AIO, 1984.

SciELO

10 11 12 13 14 15

POSSANI, L,D. et al. Structural and functional characteristics of toxins purified from the venom of

the Brazilian scorpion Tityus serrulatus Lutz and Mello. Mem. Inst. Butantan, v. 54, n. 2, p. 35-52,

1992.

13. FLETCHER JR., RL, POSSANI, L.D., ALAGON, A.C. Amino terminal sequence

of the gamma-neurotoxin isolated from Tityus serrulatus scorpion venom.

In: INTERNATIONAL SYMPOSIUM ON ANIMAL PLANT AND MICROBIAL

TOXINS, 5, 1976, San José, Costa Rica. Abstracts... p. 46.

14. FREIRE-MAIA, L., CAMPOS, J.A. Pathophysiology and treatment of scorpion

poisoning. In: OWNBY, C.L., ODELL, G.V., ed. Natural toxins. Oxford: Per-

gamon Press, 1989. p. 139-159.

15. GOMEZ, M.V., DINIZ, C.R. Separation of toxic components from the Brazilian

scorpion Tityus serrulatus \jer\om. Mem. Inst. Butantan, v. 33, p. 899-902,

1966.

16. KARNOVSKY, M.J. A formaldehyde-glutaraldehyde fixative of high osmolarity

for use in electron microscopy. J. Cell Biol, v. 27, p. 137A, 1965.

17. KING, T.P., ALAGON, A.C., KWAN, J., SOBOTKA, A.K., LICHTEINSTEIN, LM.

Immunochemical studies of yellow-jacket venom proteins. Mol. Immunol,

V. 20, p. 297-308, 1978.

18. KIRSCH, G.E., SKATTEVOL, A., POSSANI, L.D., BROWN, A.M. Modification

of Na channel gating by an alpha scorpion toxin from T. serrulatus. J. Gen.

Physiol., V. 93, p. 67-83, 1989.

19. LAEMMLI, U.K. Cleavage of structural proteins during the assembly of the head

of bacteriophage T4. Nature, v. 227, p. 680-685, 1970.

20. LAZDUNSKI, M., FRELIN, C., BARHANIN, J., LOMBET, A., MEIRI, H., PAU-

RON, D., ROMEY, G., SCHMID, A., SCHWEITZ, H., VIGNE, R, VIJVERBERG,

FI.P.M. Polypeptide toxins astools to study voltage-sensitive Na* channels.

In: KAO, C.Y., LEVINSON, S.R., ed. Tetrodotoxin Saxitoxin and the Molecu¬

lar Biology of the Sodium Channel. (AnnaIsN. York Acad. Sei., 479, 1986,

p. 204-220).

21. LUFT, J.FI. Improvements in epoxy resin embedding methods. J. Biophys. Bi-

ochem. Cytol., v. 9, p. 409-414, 1961.

22. MACFIADO, J.C., SILVEIRA F°, J.F. Obtenção experimental da pancreatite hemor¬

rágica aguda no cão por veneno escorpiônico. Mem. Inst. Butantan, v. 40/41,

p. 1-9, 1976/77.

23. MARTIN, B.M., CARBONE, E., YATANI, A., BROWN, A.M., RAMIREZ, A.N,,

GURROLA, G.B., POSSANI, L.D. Amino acid sequence and physiological

characterization of toxins from the venom of the scorpion Centruroides limpidus

tecomanus Floffmann. Toxicon, v 26, p. 785-794, 1988.

24. MOORE, S. On the determination of cystine as eysteie acid. J. Biol. Chem.,

V. 238, p. 235-237, 1963.

25. NOVAES, G., CATANZARO, D.L., BERALDO, W.T., EREIRE-MAIA, L. Effect of

purified scorpion toxin (tityustoxin) on the pancreatic secretion of the rat.

Toxicon, V. 20, p. 847-853, 1982.

26. POSSANI, L.D. Strueture of scorpion toxins. In: TU, A.T, ed. Handbook of natural

toxins. New York: Marcei Dekker, 1984. v.2, p. 513-550.

27. POSSANI, L.D., ALAGON, A.C., FLETCHER JR., RL, ERICKSON, B.W. Purifi-

cation and properties of mammalian toxins from the venom of the Brazilian

scorpion Tityus serrulatus Lutz and Mello. Arch. Biochem. Biophys., v.180,

p. 394-403, 1977.

28. POSSANI, L.D., MARTIN, B.M., ELETCHER, M.D., FLETCHER JR, RL. Discharge

effect on pancreatic exoerine secretion produced by toxins purified from

Tityus serrulatus scorpion yenom. J. Biol. Chem., v. 266, p. 3.178-3.185, 1991.

29. POSSANI, L.D, MARTIN, B.M., MOCHCA-MORALES, J., SVENDSEN, I. Purification

and Chemical characterization of the major toxins from the venom

SciELO

10 11 12 13

POSSANI, L.D, et al. Structural and functional characteristics of toxins purified from the venom of

the Brazilian scorpion Tityus serrulatus Lutz and Mello. Mem. Inst. Butantan, v. 54, n. 2, p. 35-52,

1992.

of the Brazilian scorpion Tityus serrulatus Lutz and Mello. Carisberg Res.

Commun., tz 46, p. 195-205, 1981.

30. POSSANI, L.D;, MARTIN, B.M., SVENDSEN, I. The primary structure of Nox-

iustoxin: a K*channel blocking peptide from the venom of the scorpion

Cenfruro/des nox/us Hoffmann. Carisberg Res. Commun., v.47, p, 285-289,

1982.

31. POSSANI, LD., MARTIN, B.M., SVENDSEN, I., RODE, G.S., ERICKSON, B.W.

Scorpion toxins from Centruroides noxius and Tityus serrulatus. Biochem.

J., V.229, p. 739-750, 1985.

32. RAMIREZ, A.N., GURROLA, G.B., MARTIN, B.M., POSSANI, L.D. Isolation of

several toxins from the venom of the scorpion Centruroides limpidus

tecomanus Hoffmann. Toxicon, v.26, p. 773-783, 1988.

33. REISFELD, R.A., LEWIS, U.J., WILLIAMS, D.E. Disck electrophoresis of basic

proteins on polyacrylamide gels. Nature, v.l95, p. 281-283, 1962,

34. ROCHAT, H., BERNARD, R, COURAUD, F. Scorpion toxins: chemistry and mode

of action. In: CECCARELLI, B., CLEMENTI, F, ed.Advances in cytopharma-

cology. New York: Raven, 1979. v.3. p. 325-334.

35. SAMPAIO, S.V., LAURE, C.J., GIGLIO, J.R. Isolation and characterization of toxic

proteins from the venom of the Brazilian scorpion Tityus serrulatus. Toxi¬

con, V.2I, p. 265-277, 1983.

36. SCFIEELE, G.A., PALADE, G.E. Studies on the guinea pig pancreas. J. Biol.

Chem., V.250, p. 2.660-2.670, 1975.

37. TOLEDO, D., NEVES, A.G.A. Purification and partial characterization of a se-

cond toxin from the scorpion Tityus serrulatus. Comp. Biochem. PhysioL,

V.55B, p. 249-253, 1976.

38. TOLKSDORF, S., McCREADY, M.H., McCULLAGH, D.R., SCHWENK, E. The

turbidimetric assay of hyaluronidase. J. Lab. Clin. Med., v.34, p. 74-89,

1949.

39. TU, A.T. Scorpion venoms. In: TU, A.T., ed. Venoms: chemistry and molecular

biology. New York: John Wiley, 1977. p. 459-483.

40. VENABLE, J.FI., COGGESFIALL, R. A simplitied lead citrate stain for use in

electron microscopy. J. Cell Biol., v. 25, p. 407-408, 1965.

41. YATANI, A., KIRSCH, G.E., POSSANI, L.D., BROWN, A.M. Effects of New World

scorpion toxins on single-channel and whole cell cardiac sodium currents.

Amer. J. PhysioL, v. 254 (Heart Circ. PhysioL 23), p. H443-H451, 1988.

SciELO

10 11 12 13 14 15

POSSANI, L.D. et al. Structural and functional characteristics of toxins purified from the venom of

the Brazilían scorpion Tityus serrulatus Lutz and Mello. Mem. Inst. Butantan, v. 54, n. 2, p. 35-52,

1992.

TABLE 1

Amino Acid Composition of Toxins from the Venom of Tityus serrulatus'

Toxin

11-11

111-10

III-8

IV-5

Amino

Residues

Acid

per Mole

ASP

3.7 (4)

3.8 (4)

4.9 (5)

9.5 (10)

THR

0.9 (1)

1.1 (1)

1.9 (2)

SER

3.8 (4)

3.9 (4)

2.8 (3)

GLU

3.1 (3)

3.3 (3)

2.2 (2)

2.0 (2)

PRO

3.0 (3)

2.8 (3)

GLY

8.0 (8)

6.0 (6)

4.0 (4)

ALA

3.0 (3)

5.6 (6)

2.9 (3)

1/2CYS

8.0 (8)

6.3 (6-8)

6.5 (6-8)

7.0 (6-8)

VAL

1.8 (2)

2.0 (2)

1.9 (2)

1.8 (2)

MET

0.9 (1)

0.0 (0)

ILE

1.9 (2)

2.0 (2)

1.8 (2)

LEU

3.0 (3)

3.1 (3)

1.3 (1)

3.0 (3)

TYR

4.5 (5)

4.7 (5)

5.3 (5)

7.7(8)

PHE

1.0 (1)

1.1 (1)

2.9 (30)

0.0 (0)

HIS

1.0 (1)

2.7 (3)

1.0 (1)

LYS

5.5 (5)

6.2 (6)

6.9 (7)

6.7 (7)

ARG

2.6 (3)

2.8 (3)

1.0 (1)

0.0 (0)

TRP

3.0 (3)

2.0 (2)

Residues

Mol. Wt.

6686

6957

6918

*Taken from Possani et al.^^

SciELO

10 11 12 13 14 15

POSSANI, L.D. et al. Structural and functional characteristics of toxins purified from the venom of

the Brazilián scorpion Tityus serrulatus Lutz and Mello. Mem. Inst. Butantan, v. 54, n. 2, p. 35-52,

1992.

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SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

V. 54, n. 2, p. 53-59, 1992

HERPETOFAUNA DAS DUNAS INTERIORES DO RIO SÃO

FRANCISCO: BAHIA: BRASIL V. DUAS NOVAS ESPÉCIES DE

APOSTOLEPIS (OPHIDIA, COLUBRIDAE)

Miguel Trefaut RODRIGUES

RESUMO: São descritas duas novas espécies de colubrídeos do gênero

/\posfo/ep/s obtidas na região de dunas paleoquaternárias do rio médio

São Francisco. Apostolepis arenarius é caracterizado pelo número mais

baixo de ventrais até então conhecido para o gênero. Apostolepis gaboi

é diagnosticado pelo padrão de sete faixas longitudinais, pelo número

de escamas ventrais e subcaudais e pela escutelação cefálica.

UNITERMOS: Taxonomia, Reptilia, Colubridae, Apostolepis

INTRODUÇÃO

As dunas paleoquaternárias do médio rio São Francisco encontram-se no no¬

roeste do Estado da Bahia, encravadas em uma extensa área dominada por caa¬

tingas típicas. Os trabalhos de campo conduzidos na região têm mostrado que

a fauna de répteis local é variada e distinta da das caatingas que a circundam.

Até 0 momento 29 espécies de lagartos e 6 de anfisbenídeos são conhecidas

da área, sendo 20 espécies de répteis supostamente endêmicas dali, muitas de¬

las caracterizadas por adaptações ao habitat psamófilo. As publicações anterio¬

res sobre a fauna da área trataram da descrição de novos gêneros e espécies de

lagartos (Rodrigues, 5,6,7), cobras (Rodrigues, 8) e anfisbenídeos (Vanzolini, 10,11).

Uma descrição detalhada do habitat e do contexto ecológico e evolutivo em que

o trabalho se insere pode ser encontrada em Rodrigues (5). Descrevo aqui duas

novas espécies do gênero Apostolepis obtidas na região. A primeira delas, em

função do seu modo de vida passo a tratar por

Universidade de São Paulo, Instituto de Biociências, Departamento de Zoologia, Caixa Postal 20.520, 01498-970

— São Paulo, SP.

Recebido para publicação em 03.02.92 e aceito em 06.07.92.

SciELO

RODRIGUES, M.T. Herpetofauna das dunas interiores do Rio São Francisco: Bahia. Brasil, V, Duas

novas espécies de Apostolepis (Ophidia, Colubridae). Mem. Inst. Butantan, v. 54, n. 2, p. 53-59,

1992

Apostolepis arenaríus, sp. n

(Figuras 1 e 2)

Holótipo: MZUSP 10.027, fêmea, Brasil: Bahia: Alagoado (9° 29'S, 41° 21'

N) 5.ÍX.88, MRodrigues, número de campo 88.6819

Parátipos: MZUSP 10.028-10.030, demais dados como para o holótipo, MZUSP

10.289 Alagoado, Ba., 20 x 90.

Diagnose

Colorido de fundo vermelho tijolo com cinco faixas longitudinais estreitas

castanho-escuras. Quinta e sexta supralabiais em contato com a parietal. Ven-

trais 168-181; 24 a 31 pares de subcaudais.

Descrição

Rostral visível de cima e quase tão longa quanto larga, subtriangular, com vér¬

tice voltado para trás e separando as nasais. Nasal inteira, separada da preocular,

mais longa do que alta e mais estreita posteriormente, inserida entre a primeira

e a segunda supralabiais, a prefrontal e a rostral. Narina na metade posterior da

nasal. Loreal e internasais ausentes.

Prefrontais grandes, maiores que a frontal, quase tão longas quanto largas, em

amplo contato mediano e em contato com as supralabiais. Supraocular menor

que a frontal e sobre o olho. Uma pré e uma postocular quase quadrangulares

e um pouco maiores que o olho. Frontal mais longa do que larga, indentando a

sutura posterior das prefrontais. Parietais muito longas, cerca de duas vezes mais

longas que a frontal e em contato com as supralabiais posteriores, com uma es¬

cama ligeiramente maior que as dorsais, a postocular, a supraocular e a frontal.

Seis supralabiais, quinta e sexta maiores, a terceira sob o olho; quinta e sexta

em contato com a parietal, eventualmente a quarta. Temporais ausentes fusiona¬

das à quinta e sexta supralabiais.

Fig. 1. Apostolepis arenaríus

2 3

5 6

10 11 12 13 14 15

RODRIGUES, MJ. Herpetofauna das dunas interiores do Rio São Francisco: Bahia. Brasil. V. Duas

novas espécies de Apostolepis (Ophidia, Colubridae). Mem. Inst. Butantan, v. 54, n. 2, p. 53-59,

1992.

Sinfisal pequena, triangular, um pouco mais longa que larga. Sete a oito infra-

labiais, 0 primeiro par em contato atrás da sinfisal. Dois pares de mentais seguin¬

do o contato posterior do primeiro par de infralabiais. 0 primeiro em contato com

as quatro primeiras infralabiais, o segundo apenas com a quinta e sexta.

Dorsais lisas, dispostas em 15 fileiras longitudinais sem redução. Fossetas api¬

cais ausentes. Ventrais largas, 168 a 181; 24 a 31 pares de subcaudais. Anal di¬

vidida.

Porção dorsal da cabeça predominantemente negra. Frontal, prefrontal e es¬

cudos anteriores em sua maioria amarelòs com algumas manchas negras irregu¬

lares. Primeira supralabial manchada de amarelo. Uma faixa estreita amarela pouco

após 0 olho ocupa parte da terceira e quarta supralabiais. Porção ventral da cabe¬

ça predominantemente amarela com manchas escuras difusas e irregulares so¬

bre as infralabiais.

Fig. 2: Apostolepis arenahus

Um colar amarelo nítido logo após a cabeça ocupa a largura de cinco esca¬

mas, e é seguido por uma faixa negra transversal que ocupa apenas a região me-

diodorsal e tem 2 a 3 escamas de largura. Deste último partem três linhas negras

longitudinais. A primeira corre ao longo da fileira vertebral, as outras duas na sex¬

ta fileira de dorsais. Abaixo destas, e de cada lado do corpo na altura da 41 fileira

de dorsais está presente uma linha nepra muito mais estreita que se estende co¬

mo as demais até a ponta da cauda. Apice da cauda caracteristicamente negro,

ventre branco uniforme.

Comprimento do corpo e da cauda (mm), respectivamente: Holótipo; 295-F28;

Parátipos: 151-F17, 172-F24 e 145-F14. Diâmetro do corpo do holótipo e paráti-

pos respectivamente 5,8mm, 4,6mm, 4,4mm, 3,8mm e 5,9mm.

A segunda espécie apresenta corpo muito mais delgado e também foi encon¬

trada na areia. Ela é descrita abaixo em homenagem a Gabriel Skuk que coletou

o único exemplar e prestou valiosa ajuda no campo.

SciELO

RODRIGUES, MJ. Herpetotauna das dunas interiores do Rio São Francisco: Bahia. Brasil. V. Duas

novas espécies de Apostolepis (Ophidia, Colubridae). Mem. Inst. Butantan, v. 54, n. 2, p. 53-59,

1992.

Sinfisal pequena, triangular, um pouco mais longa que larga. Sete a oito infra-

labiais, 0 primeiro par em contato atrás da sinfisal. Dois pares de mentais seguin¬

do o contato posterior do primeiro par de infralabiais. 0 primeiro em contato com

as quatro primeiras infralabiais, o segundo apenas com a quinta e sexta.

Dorsais lisas, dispostas em 15 fileiras longitudinais sem redução. Fossetas api¬

cais ausentes. Ventrais largas, 168 a 181; 24 a 31 pares de subcaudais. Anal di¬

vidida.

Porção dorsal da cabeça predominantemente negra. Frontal, prefrontal e es¬

cudos anteriores em sua maioria amarelos com algumas manchas negras irregu¬

lares. Primeira supralabial manchada de amarelo. Uma faixa estreita amarela pouco

após o olho ocupa parte da terceira e quarta supralabiais. Porção ventral da cabe¬

ça predominantemente amarela com manchas escuras difusas e irregulares so¬

bre as infralabiais.

Fig. 2: Apostolepis arenarius

Um colar amarelo nítido logo após a cabeça ocupa a largura de cinco esca¬

mas, e é seguido por uma faixa negra transversal que ocupa apenas a região me-

diodorsal e tem 2 a 3 escamas de largura. Deste último partem três linhas negras

longitudinais. A primeira corre ao longo da fileira vertebral, as outras duas na sex¬

ta fileira de dorsais. Abaixo destas, e de cada lado do corpo na altura da 41 fileira

de dorsais está presente uma linha nepra muito mais estreita que se estende co¬

mo as demais até a ponta da cauda. Apice da cauda caracteristicamente negro,

ventre branco uniforme.

Comprimento do corpo e da cauda (mm), respectivamente: Flolótipo: 295-F28;

Parátipos; 1514-17, 1724-24 e 1454-14. Diâmetro do corpo do holótipo e paráti-

pos respectivamente 5,8mm, 4,6mm, 4,4mm, 3,8mm e 5,9mm.

A segunda espécie apresenta corpo muito mais delgado e também foi encon¬

trada na areia. Ela é descrita abaixo em homenagem a Gabriel Skuk que coletou

0 único exemplar e prestou valiosa ajuda no campo.

10 11 12 13 14 15

Fig. 3; Apostolepis gaboi

Descricão

Rostral bem visível de cima, quase tão longa quanto larga, com vértice voltado

para trás e separando amplamente as nasais. Nasal inteira, separada da preocu-

lar; mais longa do que alta e mais estreita posteriormente; inserida entre a pri¬

meira e a segunda supralabiais, a prefrontal e a rostral. Narina na metade da

escama. Loreal e internasais ausentes. Prefrontais grandes, mais curtas que a fron¬

tal, quase tão longas quanto largas e em amplo contato mediano, em contato

com a segunda supralabial. Frontal ligeiramente mais longa do que larga, ultra¬

passando pouco o nível da margem anterior das supra-oculares e indentando li¬

geiramente a sutura com as prefrontais. Supra-oculares menores que a frontal

com sua margem posterior retilínea. Uma pré e uma postocular, a primeira muito

pequena, a segunda maior, em contato com a supra-ocular e com o tamanho

aproximado do olho. Parietais muito longas, cerca de duas vezes mais longas que

a frontal e em contato com a quinta e sexta supralabiais, com uma escama ligei¬

ramente maior que as dorsais, com a frontal, a supra-ocular e a postocular. Seis

supralabiais, a quinta e sexta maiores; a segunda e terceira atingem a órbita sen¬

do que a última está sob o olho. Temporais ausentes, respectivamente fusiona¬

das à quinta e sexta supralabiais.

SciELO

RODRIGUES, M.T. Herpetofauna das dunas interiores do Rio São Francisco: Bahia. Brasil. V. Duas

novas espécies de Apostolepis (Ophidia, Colubridae). Mem. Inst. Butantan, v, 54, n. 2, p. 53-59,

1992.

Apostolepis gaboi, sp. n.

(Figura 3)

Flolótipo: MZUSP 10.290, fêmea. Brasil: Bahia: Queimadas (10° 23'S,

42°30W), 12/x/90, Gabriel Skuk col. MRodrigues 907198.

Diagnose

Colorido dorsal ferrugem com 7 finas linhas longitudinais ao longo do corpo.

Quinta e sexta supralabiais em contato com a parietal. Nasal separada da preo-

cular., Ventrais 229; 31 pares de subcaudais.

10 11 12 13 14 15

Fig. 3: Apostolepis gaboi