A comparative study on the electrophoretic patterns of snake venoms

Comp. Btochem. Physiol. Vol. 102B, No. 1, pp. 103-109, 1992

0305..0491/92 $5.00 +0.00 © 1992 Pergamon Press Ltd

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A COMPARATIVE STUDY ON THE ELECTROPHORETIC PATTERNS OF SNAKE VENOMS NGET-HONGTAN and GNANAJOTHYPONNUDURA! Department of Biochemistry, University of Malaya, Kuala Lumpur, Malaysia (Fax: 603 757 3661)

(Received 27 August 1991) Abstract--1. Examination of the polyacrylamide gel electrophoretic (PAGE) and SDS--PAGEpatterns of snake venoms shows that these patterns are useful for species differentiation (and hence identification) for snakes of certain genera but have only limited application for snakes from some other genera, due either to the marked individual variations in the venoms or the lack of marked interspecifie differences within the same genus. 2. There is no substantial intersubspecific difference in the electrophoretic patterns of the venoms. 3. In general there are no common characteristics in the electrophoretic patterns of the venom at the generic level because of the wide variations in the electrophoretic patterns of venoms of snakes within the same genus. 4. At the familial level, the venoms of Elapidae exhibited SDS-PAGE patterns distinct from those of

Crotalidae.

INTRODUCTION Many authors have examined the electrophoretic patterns of snake venoms (Chippaux et al., 1982; Glenn and Straight, 1977; Johnson, 1968; Meier, 1986; Minton and Weistein, 1986; Rael et al., 1984; Suttnar et aL, 1988; Taborska, 1971; Willemse, 1978). While some authors (Bertke et al., 1966; Foote and MacMahon, 1977; Tan and Gnanajothy, 1991) have shown that electrophoretic patterns could be used for the taxonomy of snake, other authors doubted the taxonomic content of venom electrophoretic patterns as there are substantial individual variations in these patterns (Suttnar et al., 1988; Willemse, 1978). Recent studies, however, showed that many snake venoms indeed exhibited common characteristics at species, genus and family level (Bernadsky et al., 1986; Tan and Gnanajothy, 1990a, b, c; Tan and Tan, 1988; Tan et al., 1989), and that notwithstanding individual variation, snake venom biochemical composition is useful for snake species differentiation. In this study, we examined the PAGE and SDS-PAGE patterns of 284 venom samples from 117 taxa (23 genera) of snakes from the families of Viperidae and Elapidae so as to examine the individual variation in venom electrophoretic patterns as well as the usefulness of the patterns for differentiation and taxonomy of snake species. MATERIALS AND

METHODS

Snake venoms A total of 284 venom samples from 117 taxa of snakes (23 genera) from both the families of Viperidae and Elapidae were used in this investigation. The venom samples were obtained from Miami Serpentarium Laboratories (Salt Lake City, USA), Latoxan (Rosans, France), Ventoxin (Frederick, USA), Ophidia Venin (Tavannes, Switzerland), Quality Venoms for Medical Research (Florida, USA), Sigma Chemical Company (St Louis, USA). Dr R. D. G. Theakston (Liverpool, UK), Australian Reptile Park (Gosford, Australia), Venom Supplies (Whyalla, Australia), CBPB I02/I--H

South East Asian Venom Institute (Kuala Lumpur, Malaysia) and Snake and Venom Institute (Penang, Malaysia). Most of the venom samples are pooled samples and were shipped airmail and arrived within 2 weeks at the laboratory. The venom samples used are indicated in the various figures.

Other materials Reagents and materials for electrophoresis including the buffers, tetramethylethylene-diamine (TEMED), amido black 10B, N-N'-bis-acrylamide, acrylamide, fl-mereaptoethanol and trichloroacetic acid were obtained from Sigma Chemical Company (St Louis, USA).

Polyacrylamide gel electrophoresis Both polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE was conducted according to the discontinuous system of Laemmli (1970) as modified by Studier (1973) using a 10% running gel, and Amido Black 10B as staining solution. RESULTS AND DISCUSSION

Electrophoretic patterns of Vipera venoms Figure 1A and B shows the PAGE and SDS-PAGE patterns of the Vipera venoms. The venoms generally did not exhibit marked individual variation in these patterns except for V. palaestinae venom samples, which exhibited minor variation in their PAGE patterns. It is noted that V. palaestinae and V. xanthina venoms exhibited similar PAGE and SDS-PAGE patterns. Venoms of other species of Vipera exhibited characteristic eleetrophoretic patterns that are useful for species differentiation of snake species within the same genus, Vipera. There is no intersubspecific difference among the various taxa of V. aspis. V. lebetina and V. russelli venoms.

Electrophoretic patterns of other Viperinae venoms Figure 2A and B shows the PAGE and SDS-PAGE patterns of the other Viperinae venoms tested. PAGE patterns of all three Bitis species and

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Fig. 1. Polyacrylamide gel electrophoretic (A) and SDS-PAGE (B) patterns of Vipera venoms using 10% gel at pH 8.8. The proteins migrated from cathode (top) to anode (bottom). Line assignments are: (1)-(3) V. a. ammodytes; (4) 1I. aspis; (5) V. a. aspis; (6) V. aspis atra; (7) V. aspisfracisredi; (8) V. berus; (9)-(10) V. lebetina schweizeri; (1 I)-(12) V. lebetina turanica; (13) V. latastei gaditana; (14) V. latastei latastei; (15)-(17) V. russelli russelli; (18)-(20) V. russellisiamensis; (21)-(23) V. palaestinae; (24)-(25) II. xanthina. of E. carinatus exhibited marked intraspecific variation. B. arietans and B. gabonica venoms also exhibited marked intraspecific variation in the S D S - P A G E patterns. Other venoms examined did not exhibit intraspecific variation. Venom electrophoretic patterns are useful for species identification for the Viperinae venoms of C. rhombeatus, C. cerastes, E. carinatus, E. macmahonii and P. persicus but are not useful for the Bitis venoms tested.

Agkistrodontini venoms examined. The venoms' electrophoretic patterns are generally useful for the differentiation of the species examined, except between A. contortrix and A. piscivorus venoms, which exhibited similar P A G E and S D S - P A G E patterns. Also, there is no intersubspecific difference among the taxa of A. contortrix and A. piscivorus. Electrophoretic patterns of the Bothrops venoms

Electrophoretic patterns of venoms o f the Agkistrodon and o f related species of the tribe Agkistrodontini. Figure 3A and B shows the P A G E and S D S - P A G E patterns of the venoms of Agkistrodon and some related species belonging to the same tribe Agkistrodontini. There is no marked individual variation in the electrophoretic patterns of all

Figure 4A and B shows the P A G E and S D S - P A G E patterns of the Bothrops venoms tested. Except for B. atrox, B. jararaca and B. nummifera venoms, other Bothrops venoms tested exhibited marked individual variation in the P A G E patterns. There is, however, no marked individual variation in the S D S - P A G E patterns of the Bothrops venoms. It is noted that in general there are no marked

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I !I liiiiiiAii iiiR iiiiiiiiiiiii"r iiniiii iiiiiii Fig. 2. Polyacrylamide gel electrophoretic (A) and SDS-PAGE (B) patterns of other Viperinae venoms using 10% gel at pH 8.8. The proteins migrated from cathode (top) to anode (bottom). Line assignments are: (1)--(4) Bitis arietans; (5)-(7) B. gabonica gabonica; (8)-(10) B. gabonica rhinoceros; (11)-(14) B. narsicornis; (15)--(16) Causus rhombeatus; (17)-(19) Cerastes cerastes; (20) C. vipera; (21) Echis carinatus sochureki; (22) E. carinatus leucogaster; (23) E. carinatus sochureki; (24) E. carinatus leakyi; (25)-(26) Eristicophis macmahoni; (27)--(28) Pseudocerastes persicus.

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Fig. 3. Polyacrylamide gel electrophoretic (A) and SDS-PAGE (B) patterns of Agkistrodontini venoms using 10% gel at pH 8.8. The proteins migrated from cathode (top) to anode (bottom). Line assignments are: (1)-(5) A. contortrix contortrix; (6)-(8) A. c. laticinctus; (9)-(13) A. c. mokasen; (14) A. c. pictigaster; (15)-(19) A. piscivorus piscicvorus; (20)-(23) A. p. conanti; (24)-(26) A. p. leucostoma; (27)-(31) A. b. bilineatus; (32)-(34) Calloselasma rhodostoma; (35)-(36) Deinagkistrodon acutus; (37)-(40) Gloydius blomhoffi blomhoffi; (41) G. b. ussuriensis; (42) Hypnale hypnale. interspecific differences in the electrophoretic patterns of Bothrops venoms and as such the as patterns are of limited use for the differentiation of the species of Bothrops. Electrophoretic patterns o f the Crotalus and Sistrurus venoms

Figure 5A and B shows the P A G E and S D S - P A G E patterns of the rattlesnake (Crotalus and Sistrurus) venoms. The rattlesnake venoms examined generally exhibited marked interspecific differences

but not marked individual variation in their electrophoretic patterns, and as such the electrophoretic patterns are useful for differentiation of species of snakes from these genera. There are no marked intersubspecific differences among Crotalus venoms examined. Electrophoretic patterns o f the Trimeresurus venoms

Figure 6A and B shows the P A G E and S D S - P A G E patterns of the Trimeresurus venoms tested. The venoms did not exhibit marked individual

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Fig. 4. Polyacrylamide gel electrophoretic (A) and SDS-PAGE (B) patterns of Bothrops venoms using 10% gel at pH 8.8. The proteins migrated from cathode (top) to anode (bottom). Line assignments are: (1)-(2) B. alternatus; (3) B. asper; (4)-(5) B. atrox; (6) B. bilineatus; (7) B. cotiara; (8)-(11) B. jararaca; (12) B. lansbergi; (13)-(14) B. jararacussu; (15) B. moojeni; (16) B. nasuta; (17)-(18) B. nummifera; (19) B. pradoi; (20) B. neuweidi; (21)-(23) B. schlegeli.

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Fig. 5. Polyacrylamide gel electrophoretic (A) and SDS-PAGE (B) patterns of Crotalus and Sistrurus venoms using 10% gel at pH 8.8. The proteins migrated from cathode (top) to anode (bottom). Line assignments are: (1)-(5) C. adamanteus; (6)-(9) C. atrox; (10)-(11) C. cerastes; (12)-(13) C. d. durissus; (14)-(16) C. d. terrificus; (17) C. d. totonacus; (18)-(20) C. basiliscus; (21)-(23) C. m. molossus; (24)-(26) C. r. tuber; (27)-(30) C. h. atricaudatus; (31)-(34) C. h. horridus; (35)-(37) C. scutulatus; (38) C. viridis cereberus; (39) C. v. concolor; (40)-(41) C. v. helleri; (42) C. v. lutosus; (43) C. v. oreganus; (44)-(45) C. v. viridis; (46)--(47) S. cutenatus; (48)-(50) S. m. barbouri. variation in the electrophoretic patterns, except for T. stejnegeri venoms. Venom electrophoretic patterns are useful for differentiating species of snakes from the Trimeresurus as there are marked interspecific differences in the patterns. Eleetrophoretic patterns o f the Bungarus venoms

Figure 7A and B shows the PAGE and S D S - P A G E patterns of the Bungarus venoms examined. There are some individual variations in the 5

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PAGE patterns of B. candidus and B. multicinctus venoms but no marked interspecific differences in their electrophoretic patterns. This is in contrast to the observations by Bon and Saliou (1983) who reported that venoms from B. multicinctus, B. caeruleus and B. fasciatus possessed characteristic electropboretic patterns that allowed the identification of the venoms by the patterns. The discrepancy is probably due to the differences in methodologies and resolution of the methods employed. _,

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Fig. 6. Polyacrylamide gel electrophoretic (A) and SDS-PAGE (B) patterns of Trimeresurus venoms using 10% gel at pH 8.8. The proteins migrated from cathode (top) to anode (bottom). Line assignments are: (1)-(4) T. flavoviridis ; (5)-(6) T. elegans ; (7)-(8) T. okinavensis ; (9)-(11) T. wagleri ; (12)-(14) T. albolabris ; (15)--(16) T. mucrosquamatus; (17)-(18) T. stejnegeri; (19) T. tokarensis; (20) T. purpureomaculatus; (21) T. sumatranus.

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Fig. 7. Polyacrylamide gel electrophoretic (A) and SDS-PAGE (B) patterns of Bungarus venoms using 10% gel at pH 8.8. The proteins migrated from cathode (top) to anode (bottom). Line assignments are: (1)-(3) B. caeruleus; (4)-(6) B. eandidus; (7)-(10) B. fas¢iatus; (11)-(14) B. multicinctus. Electrophoretic patterns o f the Naja venoms

Figure 8A and B shows the PAGE and S D S - P A G E patterns of the Naja venoms. In general the Naja venoms exhibited an intense low molecular weight band in their SDS--PAGE patterns. While there are some minor intraspecific variations in the Naja venoms tested, generally there is no marked interspecific difference in the electrophoretic patterns and thus the patterns are not useful for species identification of snakes from the genus Naja. Electrophoretic patterns o f the Australian venoms Figure 9A and B shows the PAGE and S D S - P A G E patterns of the Australian elapid venoms. Generally, the venoms exhibited substantial interspecific differences in the electrophoretic patterns and there is no marked intraspecific variation in the patterns, though there are some minor individual

variations in the PAGE patterns of N. scutatus and N. ater venoms. The Australian elapid venoms examined thus possess species-specific characteristics in the electrophoretic patterns that allow identification of the species. The usefulness o f electrophoretic patterns o f the snake venom in species differentiation and taxonomy

The usefulness of electrophoretic patterns of snake venom in the differentiation of snake species and taxonomy depends on the degree of individual (intraspecific) variation as well as interspecific difference of the patterns, particularly within the same genus. Results of this study show that electrophoretic patterns of venom are indeed useful for species differentiation (and hence identification) for snakes of certain genera, in particular the Vipera, Crotalus, Trimeresurus, and the Australian elapids; but have

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Fig. 8. Polyacrylamide gel electrophoretic (A) and SDS-PAGE (B) patterns of Naja venoms using 10% gel at pH 8.8. The proteins migrated from cathode (top) to anode (bottom). Line assignments are: (1)-(4) N. nivea; (5)-(10) N. nigricollis; (11)-(12) N. mossambica pallid_a; (13)-(14) N. m. mossambica; (15)-(16) N. n. sputatrix; (17)-(21) N. n. kaouthia; (22)-(25) N. n. atra; (26)-(28) N. n. oxiana; (29)-(32) N. n. naja; (33)-(34) N. haje; (35)-(36) N. h. haje; (37)-(38) N. h. annulifera; (39)-(41) N. melanoleuca; (42) N. melanoleuca subfulva.

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Fig. 9. Polyacrylamide gel electrophoretic (A) and SDS-PAGE (B) patterns of Australian elapid venoms using 10% gel at pH 8.8. The proteins migrated from cathode (top) to anode (bottom). Line assignments are: (1)-(3) Austrelaps superbus; (4)-(9) Notechis scutatus; (10) N. ater serventyi; (1 I) N. ater ater; (12) N. ater humphreyi ; (13)-(14) Tropidechis carinatus ; (15)-(17) Pseudechis porphyriacus ; (18)-(20) P. colletti ; (21)-(25) P. australis; (26)-(28) P. guttatus; (29)-(32) Oxyuranus scutellatus; (33)-(34) O. microlepidotus; (35)-(37) Acantophis antarticus; (38) Hoplocephalus stephensii; (39) Pseudonaja textilis. only limited application for species from some other genera, due either to marked individual variations in the venoms (the genera Bitis, Bothrops, and Echis) or the lack of marked interspecific differences within the same genus (the genera Bungarus and Naja ). Our results also demonstrate that in general, there is no substantial intersubspecific difference in the venom electrophoretic patterns. Both P A G E and S D S - P A G E patterns are therefore not useful for differentiating between venoms of the subspecies. It is recognized that there are proven c o m m o n venom characteristics at the familial and generic levels, particularly the biological activities (Bernadsky et al., 1986; Tan and Gnanajothy, 1990a). However, our results show that in general there are no c o m m o n characteristics in the electrophoretic patterns at the generic level because of the wide variations in the electrophoretic patterns of venoms of snakes within the same genus. The only exception are venoms from the genus Naja, which exhibited similar electrophoretic patterns: very intense, highly basic protein bands in the P A G E patterns and very intense low molecular weight protein bands in the S D S - P A G E patterns. At the familial level, however, the venoms of Elapidae did exhibit S D S - P A G E patterns distinct from those of Crotalidae: generally, venoms of Elapidae exhibited intense low molecular weight protein bands, which were absent in S D S - P A G E patterns of most Crotalidae venoms.

Acknowledgement--This work was supported by a research grant, PJP 87/89, from the University of Malaya, Malaysia.

REFERENCES

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