POLYACRYLAMIDE GEL ELECTROPHORESIS AS A TOOL FOR THE TAXONOMIC IDENTIFICATION OF SNAKES FROM THE ELAPIDAE AND VIPERIDAE FAMILIES

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POLYACRYLAMIDE GEL ELECTROPHORESIS AS A TOOL FOR THE TAXONOMIC IDENTIFICATION OF SNAKES FROM THE ELAPIDAE AND VIPERIDAE FAMILIES

A. M. SOARES, L. H. ANZALONI PEDROSA , M. R. M. FONTES , R. J. DA SILVA , J. R. GIGLIO

1 Department of Biochemistry, School of Medicine, University of São Paulo-USP, Ribeirão Preto, State São Paulo, Brazil; 2 Department of Physics and Biophysics, São Paulo State University-UNESP, Botucatu, State São Paulo, Brazil; 3 Center for the Study of Venoms and Venomous Animals - CEVAP, Botucatu, State São Paulo, Brazil.

ABSTRACT. Polyacrylamide gel electrophoresis (PAGE) for basic proteins may be a useful tool for the characterization of whole snake venoms and for the taxonomic classification of snakes of the Elapidae and Viperidae families. However, due to the close proximity of molecular weights among toxins bearing different net charges, sodium dodecyl sulfate (SDS) - PAGE was not able to provide an efficient differentiation. This article reports the electrophoretic analysis of several venoms from the genera Micrurus, Bothrops, Bothriopsis, Crotalus and Lachesis and shows a typical and distinctive electrophoretic profile for each species, with intraspecific and geographic variations. Even in cases in which extreme morphological similarities were present, such as between B. jararacussu and B. pirajai ("Bahia jararacuçu"), differentiation could be evidenced by PAGE. This simple and sensitive procedure may be applied to similar cases involving basic toxins.

KEY WORDS: snake venoms, basic toxins, polyacrylamide gel electrophoresis, Elapidae, Viperidae.

INTRODUCTION

Brazilian venomous snakes belong to the Elapidae family (Micrurus) and Viperidae family, subfamily Crotalinae (Bothrops, Crotalus, Lachesis, Bothriopsis and Porthidrium) (2). Individual variability in the composition of these venoms depends on geographic distribution, age, sex, feed and temperature(1,8).

Taxonomy of these snakes is usually based on morphological aspects. This paper focuses on polyacrilamide gel electrophoresis (PAGE) for basic proteins (9) as an auxiliary technique for: 1. the detection of inter and intraspecific qualitative variability; 2. the classification of genera and species of snakes from the Elapidae and Viperidae families. Thus, 2 elapidic and 13 viperidic venoms were analyzed by basic PAGE, from which characteristic electrophoretic patterns could be obtained (Figure 1A). However, this kind of differentiation could not be seen by SDS-PAGE due to overlapping of those toxins with similar Mr.

In the elapidic venoms of Micrurus frontalis frontalis and Micrurus lemniscatus carvalhoi, it was possible to demonstrate the presence of some basic components different from those of the Viperidae family, including one with an electrophoretic migration close to that of crotamine (C1) from Crotalus durissus terrificus (Figure 1A). In the Viperidae family, the venoms from Bothrops alternatus, B. cotiara, B. jararaca, Bothriopsis bilineata and Lachesis muta muta presented toxins whose basicity was lower than those of the other analyzed venoms. In B. asper venom, a basic toxin (Ap1) was identified as myotoxin II (5). Similarly, M1 (Figure 1B) was identified as myotoxin II from B. moojeni (10).

Despite the similarities between B. jararacussu and B. pirajai, their venoms show characteristic PAGE bands, J1 and P1, referred to as bothropstoxin-I (Bth TX-I) and piratoxin-I (Pr TX-I) isolated from these venoms, respectively (4,7).

In addition, only quantitative differences could be detected regarding the composition of the venoms as a function of age of B. alternatus, B. jararaca., B. moojeni and B. neuwiedi, although qualitative differences have also been reported for B. asper (3) and C. d. terrificus (6). Intraspecific variability was easily detected in C. durissus terrificus venom in which crotamine (C1) may be absent (Figure 1B).

For B. neuwiedi from the State of São Paulo (SP), 12 samples from adult snakes were individually examined. Two basic toxins (N6 and N7) showed a high intraspecific variability, appearing or not in different samples. Three of the samples showed other less basic components which were not present in the nine remaining samples (Figures 2A and 2B). The B. neuwiedi venom pool from the State of São Paulo (BnSP) also showed myotoxins (Figures 2A and 2B) different from those of the State of Minas Gerais (BnMG). Two myotoxins (N6 and N7) from BnSP were isolated (results not shown) which are absent in those from BnMG. For B. alternatus, an intraspecific variation of basic toxins (An1) from 12 young and adult animals was also observed (Figure2C).

Therefore, the electrophoretic characterization of basic proteins is an accessible and efficient method for the taxonomic classification of snake venoms of the Elapidae and Viperidae families, as well as for the quality control of these venoms for scientific purposes.

FIGURE 1. PAGE at 10% (w/v) gel for basic proteins at pH 4.5, 25 mA, 95-143 V, for 2h15min. Marker: basic fuchsin. Samples of 20µg/50µl 10% (v/v) glycerol. The gel was stained for 20 min with 0.2% (w/v) Coomassie Brilliant Blue G-250 and distained by 7% (v/v) acetic acid. (A). Venoms of the Viperidae and Elapidae families. (B). Quantitative age-dependent variations.

FIGURE 2. PAGE under the same conditions as in Figure 1. Intraspecific variability of basic toxins is shown. A and B: B. neuwiedi [venoms collected from the left (L) and right (R) glands]; C: B. alternatus [venoms collected from young (1-4) and adult (5-12) snakes].

ACKNOWLEDGEMENTS

The authors gratefully acknowledge Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support and O. A. B. Cunha and C. A. Vieira for technical assistance.

Received 02 October 1997

Accepted 10 November 1997

• 01 ASSAKURA MT., FURTADO MF., MANDELBAUM FR. Biochemical and biological differentiation of the venoms of the lancehead vipers (Bothrops atrox, Bothrops asper, Bothrops marajoensis and Bothrops moojeni). Comp. Biochem. Physiol., 1992, 102B, 727-32.
• 02 CAMPBELL JA, LAMAR WW. The venomous reptiles of Latin America. Ithaca: Cornell University Press, 1989, 425p.
• 03 GUTIÉRREZ JM., CHAVES F., BOLAÑOS R. Estudio comparativo de venenos de ejemplares recién nacidos y adultos de Bothrops asper Rev. Biol. Trop., 1980, 28, 341-51.
• 04 HOMSI-BRANDEBURGO MI., QUEIROZ LS., SANTO-NETO H., RODRIGUES-SIMIONI L., GIGLIO JR. Fractionation of Bothrops jararacussu snake venom: partial chemical characterization and biological activity of Bothropstoxin. Toxicon, 1988, 26, 615-27.
• 05 LOMONTE B., CARMONA E. Individual expression patterns of myotoxin isoforms in the venom of the snake Bothrops asper Comp. Biochem. Physiol., 1992, 102B, 325-9.
• 06 LOMONTE B., GENE JA., GUTIÉRREZ JM., CERDAS L. Estudio comparativo de los venenos de serpiente cascavel (Crotalus durissus durissus) de ejemplares adultos y recién nacidos. Toxicon, 1983, 21, 379-84.
• 07 MANCUSO LC., CORREA MM., VIEIRA CA., CUNHA OAB., LACHAT, JJ., SELISTRE DE ARAÚJO HS., OWNBY C., GIGLIO JR. Fractionation of Bothrops pirajai snake venom: isolation and characterization of piratoxin-I, a new myotoxic protein. Toxicon, 1995, 33, 615-26.
• 08 MEIER J., STOCKER K., SVENDSEN LG., BROGLI M. Chromogenic proteinase substrates as possible tools in the characterization of Crotalidae and Viperidae snake venoms. Toxicon, 1985, 23, 393-7.
• 09 REISFELD RA., LEWIS WJ., WILLIAMS DF. Disk electrophoresis of basic proteins and peptides on polyacrylamide gels. Nature, 1962, 195, 281-3.
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  10 SOARES AM., RODRIGUES VM., HOMSI-BRANDEBURGO MI., TOYAMA MH., LOMBARDI FR., ARNI RK., GIGLIO JR. A rapid procedure for the isolation of myotoxin II from Bothrops moojeni (Caissaca) venom: biochemical characterization, myonecrotic activity and crystallization. Toxicon, 1998. (in press).

CORRESPONDENCE TO:

J. R. GIGLIO - Departamento de Bioquímica, Faculdade de Medicina-USP, CEP 14.049-900, Ribeirão Preto, São Paulo, Brasil.

Publication Dates

• Publication in this collection
  26 Nov 1998
• Date of issue
  1998

History

• Received
  02 Oct 1997
• Accepted
  10 Nov 1997