R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c i n a T r o p i c a l 2 2 ( 4 ) : 1 7 1 - 1 7 5 , O u t - D e z , 1 9 8 9 .
A R T I G O S
S O U T H A M E R I C A N R A T T L E S N A K E V E N O M : I T S H E M O L Y T I C P O W E R
É d im o G a rc ia d e L im a -*, P a u lo In á c io da C o s ta1 a n d C a rlo s J u lio L a u re2
T h e h e m o l y t i c p o w e r o f r a t t l e s n a k e v e n o m (Crotalus durissus terrificus) w a s S t u d i e d . A h i g h p e r c e n t a g e o f s a m p l e w i t h n e g a t i v e h e m o l y t i c p o w e r w a s d e t e c t e d w h e n s h e e p r e d b l o o d c e l l s w e r e u s e d . A l a r g e n u m b e r o f v e n o m s w i t h h e m o l y t i c p o w e r , t h o u g h w i t h a l o w h e m o l y s i s p e r c e n t a g e , w e r e d e t e c t e d w h e n l i q u i d , r e c e n t l y e x t r a c t e d v e n o m w a s u s e d . W h e n c r y s t a l l i z e d v e n o m w a s u s e d u n d e r t h e s a m e e x p e r i m e n t a l c o n d i t i o n s , a h i g h e r p e r c e n t a g e o f p o s i t i v i t y f o r h e m o l y s i s w a s o b t a i n e d . W h e n t h e r e s u l t s o b t a i n e d o n a g a r p l a t e s w e r e c o m p a r e d t o t h o s e o b t a i n e d i n t e s t t u b e s , a l a r g e n u m b e r o f a n i m a l s w i t h a h i g h e r p e r c e n t a g e o f h e m o l y s i s w e r e d e t e c t e d , t h o u g h t h i s v a l u e w a s n o t p r o p o r t i o n a l t o t h e n u m b e r o f a n i m a l s s h o w i n g p o s i t i v e p l a t e h e m o l y s i s . W h e n t h e h e m o l y t i c p o w e r o f t h e s e v e n o m s w a s t e s t e d o n h u m a n r e d b l o o d c e l l s , a l a r g e p e r c e n t a g e o f a n i m a l s w i t h v e n o m s h a v i n g a l o w h e m o l y t i c p o w e r w a s a l s o d e t e c t e d . H e m o l y t i c p o w e r w a s m u c h g r e a t e r w h e n h u m a n r e d b l o o d c e l l s w e r e t e s t e d w i t h c r y s t a l l i z e d v e n o m . T h e p r e p a r a t i o n o f r e d b l o o d c e l l s a l s o h a d a n i m p o r t a n t e f f e c t a n d t h e u s e o f r e d b l o o d c e l l s f r o m d e f i b r i n a t e d b l o o d i s r e c o m m e n d e d . W e c o n c l u d e t h a t r a t t l e s n a k e v e n o m h a s h e m o l y t i c p o w e r t h a t i n c r e a s e s w h e n t h e v e n o m i s c r y s t a l l i z e d . R e d b l o o d c e l l s s h o u l d b e p r o p e r l y p r e p a r e d f o r t h e l y s i s r e a c t i o n s . W e s u g g e s t t h a t t h e l y t i c p o w e r o f t h e v e n o m i s r e l a t e d t o
v e n o m c o n c e n t r a t i o n a n d t o t h e p u r i t y o f i t s f r a c t i o n s .
K ey-w ords: R a t t l e s n a k e v e n o m . H e m o l y t i c p o w e r .
Studies have dem onstrated the p roteolytic pro perties o f snake venom 4 5 7 8 12 15 ^ proteinase
p resent in the v en om o f a snake o f the genus C r o t a l u s has b een found to be able to induce this p henom en on in the vascular sy stem o f m ice injected intram uscularly with a subletal d ose, the effect being dose-dependent 13 . Cellular lesion s and ev e n m em brane rupture w ere d etected b y electron m icroscop y. S om e investiga
tors6 11 dem onstrated that som e ven om s can increase erythrocyte m em brane perm eability and can also act on leu cocytes.
Because of its enzymatic action on cell mem brane phospholipids, phospholipase can alter mem brane permeability, causing indirect hemolysis by the formation o f lysophosphatides. Rattlesnake venom has been shown to have a hemolytic action on the red
blood cells of man and other animals91 4 .
1. D e p artm e n t o f P arasitology, M icrobiology an d Im m unology.
2 . D e p artm e n t o f B iochem istry.
C orresp o n d en ce to: D r. E dim o G a rc ia de Lim a, F a c u lty o f medicine o f R ibeirão P reto /U S P . 14049 Ribeirão Preto, SP. Brasil.
Recebido para publicação em 15/06/89.
The objective of the present study was to demonstrate the hemolytic power “ in vitro” induced by whole rattlesnake venom, using freshly collected and crystallized venom and sheep and' human red blood cells.
M A T E R IA L A N D M E T H O D S
The material used in the present study consisted of liquid venom, i.e. venom immediately extracted from the animal, and dry, crystallized but notlyophyli- sed venom, venom fractions and sheep and human red blood cells diluted in incomplete phosphate buffered
solution (PBSI): (N a C l, 41.0g; K H2PO4, l.Og;
N a2H P 0 4; 12H20 , 1.0g; K C 1,1 .0g; H 20 , 5000 ml),
pH 7.4.
Venom was extracted from C r o t a l u s d u r i s s u s t e r r i f i c u s snakes by the technique of G arcia Lima and Laure3. The venom was immediately placed on steri- lesed blood-agar (rabbit blood) plates, which were incubated for 24 hours at 37°C. Hemolysis was read with E E L colorimeter with a 540nm filter. The amount
of liquid venom used was p \ , corresponding to 1.9
mg of crystallized venom.
L i m a E G , C o s t a P I , L a u r e C J . S o u t h A m e r i c a n r a t t l e s n a k e v e n o m : i t s h e m o l y t i c p o w e r . R e v i s t a d a S o c i e d a d e . B r a s i l e i r a d e M e d i c i n a T r o p i c a l l i : 1 7 1 - 1 7 5 , O u t - D e z , 1 9 8 9 .
The mixture was incubated for 24 hours and hemolysis readings were taken at 30 minute intervals. The remaining part of the extracted venom was allowed to dry at room temperature (22°C ) and weighed. A 1.0 mg amount was weighed for each venom sample from each snake and placed in 0.1 ml PBSI containing 0.2 ml 2.5% sheep red blood cells in PBSI. The mixtures were incubated for 4 hours at 37°C and PBSI was added to a final volume of 1.0 ml. Hemolysis readings were taken after 12 and 24 hours. Venom fractions
were obtained by the methods of Laure Gabilan2
and Conti1. Controls were made in PBSI.
extracted whole venom and incubated for 24 hours at 37°C. Hemolysis results are reported in Table 4.
Hemolysis of human red blood cells was also studied using crystallized venom which had been extracted 7 days before use (Table 4 and Figure 1).
A study to determine the effect of the red blood cell preparation, i.e. red blood cells washed with PBSI but preserved with heparin, or red blood cells simply obtained from defibrinated sheep blood is shown in Table 5 and Figure 2.
The results of the hemolytic power of venom fractions are shown in Table 6.
RESULTS
S h e e p r e d b l o o d c e l l s
Venom samples of 27 animals held in captivity under controlled temperature, humidity and feeding conditions were examined. Venoms were extracted under usepsis to avoid contamination by the operator or the environment and immediately added to a red blood cell suspension. The material (10 pi samples of liquid venom) was incubated at 37°C and hemolysis read. The results are shown in Table 1.
D ISC U SSIO N A N D C O N C LU SIO N S
Few studies have been carried out on the
hemolytic power of rattlesnake venom ( C r o t a l u s
d u r i s s u s t e r r i f i c u s ) , but the subject has been exten sively discussed.
Habermann® and O ’Connor and Peck' 1 repor ted that some venoms have the ability to increase the permeability of red blood cell and leucocyte membra nes. It is known that phospholipase, as an enzyme, has the ability to induce hemolysis in an indirect manner.
Hemolysis obtained with crystallized venom (1.0 mg whole venom with 0.5 ml diluted red blood cells) was also studied.
W hen plate hemolysis was compared with tube hemolysis the data shown in Table 2 were obtained.
The results obtained with plate hemolysis and tube hemolysis were related to the presence of crota- mine in venom, determined by paper electrophoresis, and are shown in Table 3.
H u m a n r e d b l o o d c e l l s
Human red blood cells were washed and diluted to 5% at the proportion of 0.5 ml per 4 pi recently
The venoms studied here were obtained from animals held in captivity using a technique^ that permits the extraction of venom free from environmen tal contamination. Whole venom and red blood cell mixtures, separate or incorporated into agar, were incubated at 37° for 24 hours for hemolysis determi nation. W hen the venom was used i n n a t u r a , i.e. still in the liquid state, with sheep red blood cells, the number of negative hemolyses (68.82% ) was much higher than the number of positive hemolyses (3.18%). The positive readings, however, were evident and distri buted over a percentage o f 10 to 60% . Only the venom of two animals showed a percentage of hemolysis higher than this limit (70% ). These results led us to
L i m a E G , C o s t a P I , L a u r e C J . S o u t h A m e r i c a n r a t t l e s n a k e v e n o m : i t s h e m o l y t i c p o w e r . R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c i n a T r o p i c a l 2 2 : 1 7 1 - 1 7 5 , O u t - D e z , 1 9 8 9 .
believe that the active hemolytic portion of the venom is diluted in liquid, recently extracted venom. Howe ver, even though hemolysis rates were low, hemolysis was shown to be present in the venoms of more than 40% of the animals examined (Table 1).
When the venoms were allowed to crystallize the incidence ofnegative hemolyses was low(10.65%), but the percentage of animals whose venoms showed hemolytic ability increased to 89.35%. These results indicate that crystallized venom can cause hemolysis of a greater concentration of its fractions into a smaller portion (Table 1).
L i m a E G , C o s t a P I , L a u r e C J . S o u t h A m e r i c a n r a t t l e s n a k e v e n o m : i t s h e m o l y t i c p o w e r . R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c i n a T r o p i c a l 2 2 : 1 7 1 1 7 5 , O u t - D e z , 1 9 8 9 .
venoms of some animals had no hemolytic power on agar plates but did show hemolytic power on material in tubes, and vice versa (Table 2). W hen the results of hemolysis on plates were correlated with the presence of crotamine in the venoms, crotamine was found not to have any interfering effect on hemolysis either on agar plates or in tubes (Table 3).
W hen the hemolytic power of whole venom on human red blood cells was studied, the following results were obtained: a high percentage of low hemolysis (89% ), a medium percentage of hemolysis (15 to 30%) in a small number of venoms, though the results were statistically very clear, and also a high percentage of hemolysis (35-100% ) in a small number of venoms (8%). These results show that the number of animals whose venoms had a hemolytic effect was small, though the effect was quite evident This may also have occurred because of the concentration of hemoly tic power in each venom (Table 4 and Figure 1).
W hen the hemolytic power of crystallized ve nom was tested on human red blood cells (Table 4 and Figure 1), we also detected a large number of venoms
(5 3.00%) with low hemolysis rates, 18 animals whose venoms had medium hemolytic power (15-30% ) and a larger number of venoms (29.00% ) with high hemoly tic power (35-100% ), showing high statistical signifi cance. This indicates that the hemolytic power is more concentrated in crystallized venoms (Table 4 and Figure 1).
W hen hemolysis rates were studied as a funct ion of the manner in which blood was obtained, red blood cells from defibrinated blood were hemolysed at a lower rate than red blood cells from blood collected with heparin at all intervals (Table 5 and Figure 2). When the hemolytic power of whole venom fractions was tested (Table 6), the concentrated fractions sho wed individual hemolytic power varying from fraction to fraction. Human red blood cells were found to be more susceptible to the action of whole venom and venom fractions than sheep red blood cells.
We conclude that, under the conditions em ployed in the present study, rattlesnake venom has hemolytic power which increases when the venom is crystallized. The red blood cells should be properly prepared for the lysis reactions. W e suggest that the
L i m a E G , C o s ta P I, L a u r e C J . S o u t h A m e r i c a n r a t t l e s n a k e v e n o m : it s h e m o l y t i c p o w e r . R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c i n a T r o p i c a l 2 2 : 1 7 1 - 1 7 5 , O u t- D e z , 1 9 8 9 .
power of the venom is related to venom concentration and to the purity of its fractions.
RESUM O
Foi estudado o poder hemolitico do veneno da cas cavel ( C r o t a l u s d u r i s s u s te r r i fic u s ). Encontrou-se grande número de suas frações sem capacidade de hemolisar eritró- citos de carneiro. O veneno “ in natura” , recentemente extraído, e em estado líquido tem pouca atividade litica. A cristalização do veneno aumenta sua concentração e poder lítico. Os resultados de hemólise do sangue de carneiro obtidos em placas e tubos foram comparados evidenciando um grande número de animais com venenos com alto poder hemolítico. Os valores não foram proporcionais quando os mesmos venenos foram examinados com hemáceas de ho mem. Neste caso os percentuais de hemólise foram mais baixos. Pode-se verificar que o poder hemolítico do veneno se relaciona com a concentração e pureza de suas frações.
P a l a v r a s - c h a v e s : Veneno de cascavel. Poder hemolítico.
A C K N O W L E D G M E N T S
We are grateful to M aria Aparecida Nonato Fernandes, W ander Cosine Ribeiro da Silva, Clemen tina Pereira de Oliveira Antonini and Rosângela Catarina Peral M esquita for technical assistance.
R E FE R E N C E S
1. Conti MAB. Estrutura primária da cadeia B da Crotapo- tina. Tese de Doutorado. Faculdade Medicina de Ribei rão Preto, Universidade de São Paulo, 1986.
2. Gabilan N H. Estrutura primária da cadeia C da Crota- potina. Tese de Doutourado. Faculdade Medicina de Ribeirão Preto, Universidade de São Paulo, 1984. 3. Garcia-Lima E, Laure CJA. Study of bacterial contami
nation of rattlesnake venom. Revista da Sociedade
Brasi-leira de M edicina Tropical 20: 19-21, 1987.
4. Goucher CR, Flowers HH. The chemical modification of neurogenic and proteolytic activities of venom and the use of E D T A to produc e A g k i s t r o d u m p i s c i v o r u s venom toxoid. Toxicon 2: 139-147, 1964.
5. Grotto L, M oroz C, DeVries A, Goldblum N . Isolation of vipera palestinae hemorrhagin and distinction between its hemorrhagic and proteolytic activities. Biochimica et Biophysica A cta 133: 356-362, 1967.
ö.H aberm ann E. Bee and wasp venoms. Science 177: 314-322, 1972.
7. Houssay BA. Classification des actions de venins de serpents sur l’organismes animal. Comptes Rendus des Séances de la Société de Biologie 105: 308-310, 1930. 8. Huang SY, Perez JC. Comparative study on hemorrha gic and proteolytic activities of snake venoms. Toxicon 18: 421-426, 1980.
9. Kelen EM A , Rosenfeld G , Nudel F. Hemolytic activity of animal venoms. II. Variation in relation to erythrocyte species. Memórias do Institute Butantan 30: 133-142, 1960-1961-1962.
10. Laure CJ. Estrutura primária da Crotamina. Tese de Livre-Docência. Faculdade Medicina de Ribeirão Preto, Universidade de São Paulo, 1975.
11. O’Connor R, Peck ML. Venoms of apidae. I n : Bettini S. (ed.) Arthropod Venoms. Springer Verlag p. 613-618,
1978.
12 Omori-Satoh T. Antihemorrhagic factor as a proteinase inhibitor isolated from the serum of T r i m e r e s u r u s f l a v o -v ir id is . Biochimica et Byophysica A cta 495: 93-98, 1977.
13. Ownby CL, Geren CR. Pathogenesis of hemorrhage induced by hemorrhagic proteinase IV from timber rattlesnake ( C r o t a l u s h o r r i d u s h o r r i d u s ) venom. Toxi con 25: 517-526, 1987.
14. Rosenfeld G, Kelen EM A , Nudel F. Hemolytic activity of animal venoms. I. Classification in different types and activities. Memórias do Instituto Butantan 30:117-132, 1960-1961-1962.
