Comp. Biochem. Physiol. Vol. 102B, No. 3, pp. 515-520, 1992 0305-0491/92 $5.00 + 0.00 Printed in Great Britain © 1992 Pergamon Press Ltd

CHARACTERIZATION OF THE SNAKE VENOMS FROM SEVEN BRAZILIAN SPECIES OF B O T H R O P S BY FPLC

ANION-EXCHANGE CHROMATOGRAPHY

LUCIANA C. C. LEITE,* M. FATIMA D. FURTADO,~ TANIA C. CORREA* and ISAIAS RAW*

*Biotechnology Center and ~Snake Venoms Laboratory, Instituto Butantan, 05504 S~o Paulo, SP, Brazil (Fax: 011-815-1505)

(Received 11 November 1991)

Abstract - - l . The elution profiles and the caseinolytic, myotoxic, coagulant and hemorrhagic activities of the venoms of seven Bothrops species fractionated on a Mono-Q FPLC column were analyzed.

2. Each venom separated into 16-20 peaks, with good reproducibility and the activities were concentrated in virtually discrete regions of the chromatogram.

3. There is a considerable overlap of active proteins in the different species venoms and our results indicate that a venom pool with the species B.jararaca, B.jararacussu, B. moojeni, B. neuwiedi and B. atrox venoms would contain the major active proteins determined in the seven species.

INTRODUCTION

Snake venoms are a complex mixture of toxins, enzymes and biologically active peptides with varying composit ions among different families, genera and species. In Brazil, 90% of ophidic accidents are caused by the genus Bothrops (Brasi l--Ministrr io da Safide, 1987). The clinical and laboratory symptoms of bothropic poisoning described by Rosenfeld (1971) are characterized mainly by local lesions and blood clotting disturbances.

The local effects provoked by Bothrops venoms were initially ascribed to their proteolytic enzymes (Brazil, 1914; Rosenfeld, 1971). At present, the pathogenicity of local actions is attributed to a series of factors of which the hemorrhagic and myotoxic effects are well-documented. The extensive hemor- rhagic area observed in bothropic poisoning (Jorge and Ribeiro, 1990) is a consequence of the action of hemorrhagic factors present in these venoms (Mandelbaum et al., 1988). Myotoxins are considered to be involved in muscular necrosis (Gutierrez and Lomonte, 1989; Moura da Silva et al., 1991). The enzyme that promotes the conversion of fibrinogen into fibrin with thrombin-like activity is present in most venoms of the bothropic species (Nahas et aL, 1979).

We studied the venoms of the seven most common Bothrops species in Brazil, by fractionation on Mono- Q columns, with regards to the proteolytic, myotoxic, thrombin-like coagulant and hemorrhagic activities.

MATERIALS AND METHODS

Venoms

Lyophilized snake venom from the species: B. alternatus, B. atrox, B. cotiara, B. jararaca, B. jararacussu, B. moojeni

tTo whom correspondence should be addressed at: Centro de Biotecnologia, Instituto Butantan, Av. Dr. Vital Brazil 1500, 05504 S~o Paulo, SP, Brazil,

and B. neuwiedi were produced at the Snake Venom Labora- tory of Instituto Butantan, and contained a pool of venom collected from 30 snakes of the same species with different ages, sexes and origin. Immediately before chromatography the lyophilized powder was resuspended in 20 mM Tris buffer, pH 8.2, to a final concentration of 10 mg/ml and clarified through a 0.22 #m membrane.

Chromatography

The samples (0.5ml, containing 5mg of venom) were injected into a Mono-Q HR-5 column (Pharmacia, Uppsala, Sweden) equilibrated with 20mM Tris buffer, pH, 8.2. Elution was carried out with a linear gradient up to 0.35 M NaC1 in 35 min, and then up to 1 M NaC1 in 5 more min, following the method of Dyr et al. (1983). Six venom samples from each species were fractionated and the hom- ologous peaks from the six samples of a same species were pooled, dialysed against PBS (0.15 M, pH 7.2) and stored at -20°C for activity determination.

Due to variations in column performance during the experiments (a progressive increase in column back-pressure was observed), venom from B. jararaca was used as a standard for each set of chromatographic analysis with the different species. The retention times of peaks obtained for the venoms of the other species were normalized in relation to those of B. jararaca. Peaks from the venoms of the different species, with retention time within the standard deviations were considered homologous and a unified numeric system was established.

Proteolytic activity was measured using casein as a sub- strate (Hammarsten, Merck), according to Lomonte and Gutirrrez (1983). Samples contained 40/tg of protein. Activity was expressed as the increase in optical density at 280 nm after 30 min × 100 at 37°C.

Myotoxic activity was determined by means of the serum activity of creatine phosphokinase (CPK) induced by 20/~g of protein in 100 #1 of PBS injected i.m. in mice weighing 18-20g. Blood samples were collected from the orbital plexus 3 hr after inoculation. Sera were immediately assayed for CPK activity (Boehringer Mannheim, GmbH Diagnos- tica). CPK activity was expressed as U/1 of serum, and one unit converts 1/zmol of the substrate per min.

Thrombin-like activity was assayed by recording the clotting times of 0.4 ml of bovine fibrinogen (2 mg/ml), prepared by the method of Denson (1972) incubated at 37°C

CBPB I02/3~F 5 1 5

516 LUCIANA C. C. LErrE et al.

with I00 #1 of chromatographed venom fraction containing 20 #g of protein. The thrombin-like activity is expressed by the coagulant index which equals the inverse of clotting time x 1000.

Hemorrhagic activity was measured by the method of Kondo et al. (1960) with some modifications. Mice weighing 18-20 g were inoculated i.d. with 10 #g of venom protein in 0.1 ml of PBS. Animals were sacrificed with chloroform 2 hr after inoculation and the hemorrhagic area measured (cmZ).

Protein determination of each fractionated peak was calculated by integration of peak area and these values were used for the determination of the total activity of each fraction.

RESULTS AND DISCUSSION

Snake venom from seven bothropic species were submitted to chromatography on a Mono-Q column. The chromatographic patterns shown in Fig. l(a-g) are representative of the chromatograms obtained. From sixteen to twenty peaks were resolved and fractionated, depending on the species. The retention time of the corresponding homologous peaks from the same species were within a standard deviation of 5-10%.

In comparing the chromatographic patterns ob- tained for the protein distribution in the venoms of each species, it is possible to separate two groups: B. alternatus and B. cotiara (Fig. If, e), which have most of their proteins eluting between the retention times of 20 and 30 min, while the other species proteins elute mostly between 10 and 20 min.

The four different biological activities separated by this procedure are concentrated in virtually discrete regions of ionic strength in the chromatograms. Proteins with caseinolytic, myotoxic, coagulant and hemorrhagic activities, eluted sequentially in this order, with some overlap. This could indicate that proteins with the same function have similar charge distribution.

Caseinolytic activity

It was possible to separate two to four peaks with caseinolytic activity in each species. In all venoms tested peaks 1 and 4 contained most of the activity, with exception of B. jararaca (Fig. la), which had high activity in peak 15. B. jararaca and B. jarara- cussu venoms (Fig. 1 a, b) exhibited this activity some- what dispersed over the chromatogram, while for the other species it concentrates in the 3-15 min retention time interval. B. alternatus and B. cotiara (Fig. le, f) have significantly lower activity than the other species' venoms, in agreement with that determined in the crude venoms (Furtado et al., 1991).

Myotoxic activity

Two or three peaks with this activity were fraction- ated from the venoms of each species. Most of the activity was detected in peak 4, with the exception of B. jararaca in peak 3 and B. moojeni in peak 5 (Fig. la, c), which is in accordance with the work of Moura da Silva et al. (1991). The regions around peaks 10 and 15-17 have lower activity. In B. jararacussu and B. moojeni venoms myotoxic activity separated into two peaks (Fig. lb, c), which is in agreement with the work of other groups (Moura da Silva et al., 1991; Lomonte et al., 1990b).

B. jararaca myotoxic activity fractionated into two peaks (Fig. la), although four peaks have been characterized previously (Moura da Silva et al., 1991). Both B. atrox and B. neuwiedi venoms exhib- ited three myotoxins (Fig. lg, d), which adds to previous work in which one myotoxin had been isolated from the Columbian B. atrox species (Lomonte et al., 1990b) and one from the Brazilian B. neuwiedi (Moura da Silva et al., 1991). This chomatographic procedure enables us to fractionate and detect myotoxic activity in the venoms B. alternatus and B. cotiara (Fig. le, f) previously not detected by others (Moura da Silva et al., 1990, 1991; Lomonte et al., 1990a). The venoms of B. jararacussu, B. neuwiedi, B. alternatus and B. jararaca (Fig. lb, d, f, a) have higher total myo- toxic activities (in this order). In the other species, this activity was much lower.

Coagulant activity

This method fractionated from one to three peaks with fibrinogen coagulant activity from each venom defining five different regions, the three main ones being around peaks 12, 17 and 20. B. atrox, B. moojeni and B. jararaca venoms exhibit high coagulant activity, which is in agreement with the work of several authors (Stocker and Barlow, 1976; Holleman and Weiss, 1976; Banerjee et al., 1960).

Hemorrhagic activity

It was possible to separate two or three peaks containing hemorrhagic activity from the venoms of each species, with most of the activity around peaks 14, 16, 21, 25 and 27. The venoms of B. cotiara, B. jararaca, B. alternatus and B. neuwiedi had higher hemorrhagic activity which was all concentrated in peaks 21, 25 and 27 (Fig. le, a, f, d). The activity was lower for the other species and more spread over the chromatogram. The venom of B. cotiara, which showed low hemorrhagic activity when measured in the crude venom (Furtado et al., 1991), for unknown reasons exhibited three peaks with strong hemor- rhagic activity when fractionated (Fig. le). The hem- orrhagic components of the venoms of the species B. neuwiedi and B. jararaca have been well- studied: two hemorrhagic factors (NHFa and NHF6) have been isolated from the venom of B. neuwiedi (Mandelbaun et al., 1984) and three (HF 1, HF 2 and HF3) from the venom of B. jararaca (Mandelbaum et al., 1976, 1982, 1988).

The present results demonstrate that the method used allows fractionation of venom protein with high resolution and good reproducibility. It is an efficient technique for the initial purification of several pro- teins with different activities. It has the advantage of requiring smaller amounts of protein than the con- ventional processes. The pattern of active protein distribution can be used to establish the relationship between Bothrops species (Furtado, in preparation).

There were several protein peaks resolved by this method which have the same retention time and exhibit the same type of activity in the venoms of different species. It is likely that these peaks contain the same or homologous proteins. In this context there is a considerable overlap of proteins in the different venoms.

A 2

8 0

A

28

0

A2

80

.o

~

.0

.0

.---

0 ~

• ~

."

~,

----

---~

. ~

'I~

'

~ '

' '

I~

~ "~

'~~_

--~_

~-~

-'-:[

3 . ..

......

......

......

.. :::

:-'.'.

:'.'0

~

oq

~ -.

~

,~

..~

0"

_ ~

o!

s-I

: ~.

o o

o o

o o

o $

~o

~ ~

~ ~

" X

---."

X

CA

SE

INO

LY'Ir

lC A

CT

IVIT

Y(U

NIT

S)

x.-..

..-K

C

AS

EIN

OLY

TIC

A

CT

IVIT

Y (

UN

ITS

I o

~)

x- ..

.. x

C

AS

EIN

OLY

TIC

A

CT

IVIT

Y (

UN

ITS

) 0

0""

"~)

MY

OT

OX

IC A

CT

IVIT

Y

(UN

ITS

) x

10 3

0--.

.0

MY

OTO

XIC

AC

TIV

ITY

(UN

ITS

) xl

O 3

.b

0

0 ...

.... 0

M

YO

TOX

IC

AT

IVIT

Y(U

NIT

S)

x 10

3

..

..

.

, ,

..

. -_

;~

~,

~

-.

~,

[-I-

--1

7

CO

AG

ULA

NT

AC

TIV

ITY

(C

. I.)

x

102

I-I-

--17

C

OA

GU

LAN

T A

CTI

VIT

Y

(C.Z

.)

x I0

s'

~--

"~

CO

AG

ULA

NT

AC

TIV

ffY (

C. I

.)

x 10

2

i !

! !

&

,_.

- ~

~,

o ~

~ ~,

~

, ,,

,

,

0--

-~

HE

MO

RR

AO

IC

AC

TIV

ITY

(

0m

2)

~)-

---~

H

EM

OR

RA

GIC

A

CT

IVIT

Y

(cm

~ )

0

....

0

HE

MO

RR

AG

IC

AC

TIV

ITY

(cm

2)

-.j

o

z

"n

.~-

oo

A2

80

A

28

0

A2

80

I ,

n ,

0 ~

~ O

~ ~

0

....

....

....

....

. .~

"::"

0 o~

o o

o o

o o

o o

o o

x--.

.--x

C

AS

EIN

OLY

TIC

A

CT

IVIT

Y

(UN

ITS

) x .

....

x

CA

SE

INO

LYTI

C

AC

TIV

ITY

(U

NIT

S)

X--

---X

CA

SE

INO

LYT

IC

AC

TIV

ITY

(UN

ITS

) "~

, ,

. ~

,

o

m

o 4~

0

0..

--.0

M

YO

TO

XIC

A

CT

IVIT

Y (

UN

ITS

) x

103

(3-.

-.0

MY

OTO

XIC

AC

TIV

ITY

(U

NIT

S)

x 10

3 0

....

.. -0

M

YO

TO

XIC

A

TIV

ITY

(U

NIT

S)

x 10

3

' '

;0

'--

'-.

,~

r~

~,

' '

,:,,

,~

& ~

[~"-

-'-[

~ C

OA

GU

LAN

T A

CT

IVIT

Y

(C.

T.)

X 10

9

[~--

-.=

~

CO

AG

ULA

NT

AC

TIV

ITY

(C

. Z

.)

x 10

2 [~

'-'~

] C

OA

GU

LAN

T

AC

TIV

ITY

(C

. Z.

) x

lO E

' '

;- ~o

~,

$

- -

,~

~,

~ '-

,-.

~,

;o

P

b

(m

• o

is,

b ~.

•

"

O--

---O

HE

MO

RR

AG

IC

AC

TIV

ITY

(

cm 2

) 0

----

~

HE

MO

RR

AG

IC

AC

TIV

ITY

(

cm 2

) 0

----

~

HE

MO

RR

AG

IC

AC

TIV

ITY

(c

m 2

)

FPLC characterization of Bothrops venoms 519

1.Q

OO

o.6

~" o.4

RETENTION

[~] i19 / -~ ,150

? 21 r,

, i / i

TIME (mini

Fig. l(g)

-3O

-18

-12 o

-25

o v -

- 2 0 _ 14 6

-15 ~ >

~ t

. J

O ,1I

.s 8

2 .5

Fig. l(a-g). Chromatography of the snake venoms of (a) B. jararaca, (b) B. jararacussu, (c) B. moojeni, (d) B. neuwiedi, (e) B. cotiara, (f) B. alternatus and (g) B. atrox on Mono-Q FPLC column. Venom samples (0.5 ml, containing 5 mg protein) dissolved in 20 mM Tris buffer pH 8.2 were eluted at 0.5 ml/min flow rate with a linear NaCI gradient up to 0.35 M in 35 rain and up to 1 M in five more minutes in the same buffer. Total caseinolytic (× . . . . . × ), myotoxic (O .... O), coagulant (I-7 . . . . . I-q) and hemorrhagic

( A - - - A ) activities of the indicated fractions are expressed.

E • 2 .0 o

>- I.-

3 -1.g ~

U

0 • I.(1 ,~ ¢e

o

-0.5 = ?

1 ,5,

Taking into account the presence of proteins of higher activities in the different species, we can observe that only a few venoms would represent the activity present in all the species venoms: B. jararacussu, B. jararaca and B. atrox could cover for caseinolytic activity in any of these species; B. jararacussu and B. moojeni (or B. neuwiedi) would cover for myotoxic activity; B. moojeni and B. jararaca may cover for coagulant activity; and B. jararca, B. jararacussu and B. neuwiedi would cover for hemorrhagic activity. Considering the active protein distribution, we can observe that B. jararaca, B. jararacussu, B. moojeni, B. neuwiedi and B. atrox contain all of the active proteins determined in any of the species studied here.

Common antigens have been demonstrated in the venoms of Bothrops snakes by several immuno- chemical methods and by biological assays (Dias da Silva et al., 1989). These findings have been confirmed for hemorrhagic factors (Mandelbaun et al., 1988), myotoxins (Moura da Silva et al., 1990; Lomonte et al., 1990) and coagulant activity (Rosenfeld and Kelen, 1966).

Bothropic antivenin produced at Instituto Butantan, S~.o Paulo, Brazil, is obtained by immuniz- ation of horses with a pool of venoms from five Bothrops species (Raw et al., 1991), as suggested by cross-immunization studies of the venoms of the different species (Dias da Silva et al., 1989). It is interesting to note that, although a strict correlation has not been established between activity and immunogenicity, the pool of venoms which contains the major active proteins identified in the seven species' venoms (B. jararaca, B. jararacussu, B. moojeni, B. neuwiedi and B. atrox ), is very similar to the pool selected at Insti tuto Butantan

(B. alternatus, B. jararaca, B. jararacussu, B. moojeni and B. neuwiedi) which was based on immuno- crossreactivity.

Acknowledgements--We are indebted to Eva M. A. Kelen, Martha M. Tanizaki, Paulo Lee Ho and Luis R. Camargo Goncalves for critical reading of the manuscript. This project was supported by FINEP. TCC is a fellow from FUNDAP.

REFERENCES

Banerjee R., Devi A. and Sarkar N. (1960) Isolation and purification of a coagulant from snake venom of the species Bothropsjararaca and the study of its properties. Thromb. Diath. Haemorrh. 5, 296-303.

Brasil--Minist6rio da Sa6de (1987) Centro de Documenta- qao do Minist6rio da Sa6de: Manual de diagnostico e tratamento de acidentes ofidicos.

Brazil V. (1914) La defense contre rophidisme. 2nd edn. Pocai & Weiss, S~o Paulo.

Denson K. W. E. (1972) The preparation of general reagents and coagulation factors. In Human Blood Coagulation, Heamostasis and Thrombosis (Biggs R.), pp. 587-675. Blackwell, Oxford.

Dias da Silva W., Guidolin R., Raw I., Higashi H. G., Caricati C. P., Morais J. F., Lima S. R., Yamagushi I. K., Nishikawa A. K., Stcphano M. A., Marcelino J. R., Pinto J. R. and Santos M. J. (1989) Cross-reactivity of horse monovalent antivenoms to venoms of ten Bothrops species. Mere. Inst. Butantan 51, 153-168.

Dyr. J. E., Fortova H., Vodrazka Z. and Komalik F. (1983) Fast protein liquid chromatography of Echis carinatus venom J. Chromat. 261, 163-165.

Furtado M. F. D., Colleto G. M. D. D. and Dias da Silva W. (1991) Controle da qualidade dos venenos animais e dos correspondentes antivenenos. I - - Padronizaq~o dos m~todos de ensaio das atividades

520 LUCIANA C. C. LEITE et al.

bioquimicas e farmacol6gicas dos venenos de algumas esp~cies do genero Bothrops e Crotalus usando amostras secas a temperatura ambiente ou liofilizadas. Mere. Inst. Butantan 53, 149-159.

Gutierrez J. M. and Lomonte B. (1989) Local tissue damage induced by Bothrops snake venoms. A review. Mere. lnst. Butantan 51, 211-223.

Holleman W. H. and Weiss L. J. (1976) The thrombin-like enzyme from Bothrops atrox snake venom: properties of the enzyme purified by affinity chromatography of a p-aminobenzamidine substituted agarose. J. biol. Chem. 251, 1663-1669.

Jorge M. T. and Ribeiro L. A. (1990) Acidentes por serpentes perqonhentas do Brasil. Rev. Ass. Med. Brasil 36, 66-77.

Kondo H., Kondo S., Ikezawa K. and Murata R. (1960) Studies on the quantitative method for determination of hemorrhagic activity of Habu snake venom. Jpn J. Med. Sci. Biol. 13, 43-51.

Lomonte B. and Guti&rez J. M. (1983) La actividad proteolitica de los venenos de serpientes de Costa Rica sobre la caseina. Rev. Biol. trop. 31, 37-40.

Lomonte B., Furtado M. F., Rovira M. E., Carmona E., Rojas G., Aymerich R. and Guti~rrez J. M. (1990a) South American snake venom proteins antigenically related to Bothrops asper myotoxins. Braz. J. Med. BioL Res. 23, 427-435.

Lomonte B., Guti~rrez J. M., Furtado M. F., Otero R., Rosso J. P., Vargas O., Carmona E. and Rovira M. E. (1990b) Isolation of basic myotoxins from Bothrops moojeni and Bothrops atrox snake venoms. Toxicon 28, 1137-1146.

Mandelbaum F. R., Assakura M. T. and Reichl A. P. (1984) Characterization of two hemorrhagic factors isolated from the venom of Bothrops neuwiedi (Jararaca Pintada). Toxicon 22, 193-206.

Mandelbaum F. R., Reichl A. P. and Assakura M. T. (1976) Some physical and biochemical characteristics of HF 2, one of the hemorrhagic factors in the venom of Bothrops

jararaca. In Animal, Plant and Microbial Toxins (Edited by Ohsaka A., Hayashi K. and Sawai Y.), Vol. 1, pp. 111-121. Plenum Press, London.

Mandelbaum F. R., Reichl A. P. and Assakura M. T. (1982) Isolation and characterization of a proteolytic enzyme from the venom of the snake Bothrops jararaca (Jararaca). Toxicon 20, 955-972.

Mandelbaum F. R., Reichl A. P. and Assakura M. T. (1988) Hemorrhagic factors from the venoms of two species of Bothrops snakes. Anais do XII Simposio Anual da ACTESP sobre Toxinas proteicas, Publica~g.o ACIESP No. 57-1, Vol, 1.

Moura Da Silva A. M., Cardoso D. F. and Tanizaki M. M. (1990) Differences in distribution of myotoxic proteins in venoms from different Bothrops species. Toxicon 28, 1293-1301.

Moura da Silva A. M., Desmond H., Laing G. and Theakston R. D. G. (1991) Isolation and comparison of myotoxins isolated from venoms of different species of Bothrops snakes. Toxicon 29, 713-723.

Nahas L., Kamiguti A. S. and Barros M. A. R. (1979) Thrombin-like and Factor X--Activator com- ponents of Bothrops snake venoms. Thromb. Haemost. 41, 314-328.

Raw I., Guidolin R., Higashi H. G., Kelen E. M. A. (1991) Antivenins in Brazil: preparation. In Handbook o f Natural Toxins (Edited by Tu A. T.), Vol. 15, Chap. 18, pp. 557-581. Marcel Dekker, New York.

Rosenfeld G. (1971) Symptomatology, pathology and treatment of snake bites in South America. In Venomous Animals and their Venoms (Edited by Bucherl W., Buckey E. and Deulofeu V.), pp. 345-348. Academic Press, New York.

Rosenfeld G. and Kelen E. M. A. (1966) Cross- neutralization of the coagulant activity of some snake venoms by antivenins. Toxicon 4, 7-15.

Stocker K. and Barlow G. H. (1976) The coagulant enzyme from Bothrops atrox venom (Batroxobin). Meth. Enzym. 45, 214-223.