pp.
361-365,
1991
)F SOME VENOMS OF 9PS (AMERICAN LANCI
;ICAL OF THE ~D VIPER)
ET-HONG TAN and GNANAJOTHYPONN Biochemistry, University of Malaya, Kual;
daysia
ATIVE STUDY OF THE
(Received 18 March 1991) ulant, phosphc Abstract--1. The hemorrhagic, procoagulant, anticoagular noesterase, 5'-nucleotidase, hyaluronidase, arginine ester hydrolase, oxidase and protease activities of 26 samples of)f venoms from 13 species the Sephadex G-75 gel filtration patterns for some of the venoms also vari 2. The results show that while there are considerable individual ind chara~ of many of the Bothrops venoms tested, there are some common corn levels. ven 3. The differences in the biological properties of the Bothrops B, differentiation of most Bothrops species examined.
INTRODUCTION l h ete genus Bothrops (American lance-headed viper), rich belongs to the Crotalinae subfamily (Tribe which 'otalini), is one of the largest of the genera of Crotalini nomous snakes, with more than 60 species (or over venomous 80 taxa) distributed throughout Central and South nerica and the West Indies (Hoge and Hoge, 1978). America te Bothrops contains both arboreal and terrestrial The rms; some have very limited known ranges and the forms relationshi ationships of the species within the genus are orly understood. Also, the genus is probably polypoorly phyletic. Some of the more re prominent members nakes B. atrox, B. asper, include the large terrestrial snakes
9. neuwiedi, B. alternatus B. jararaca, B. jararacussu, B. osed (also terrestrial) pit and B. bilineatus; the hog-nosed vipers B. cotiara, B. lansberg i and B. nasutus; and the arboreal B. nummifer and B. schlegeli. The biological properties, in particular the procoag ~rocoagulant activities of venoms from some of the more c o m m o n Bothrops have been investigated (Arag),on and Gubensek, 1981; Kamiguti and Hanada, 1985; 5; Kamiguti et al., 1987; Nahas et al., 1979; Tu, 1977 ). While some quantitatic activities of the Bothive data on certain enzymatic orted (Kocholaty et al., tops venoms have been reported rative study of the enzy1971; Mebs, 1970), a comparative ms is still lacking. matic activities of the venoms Several authors have reported )rted substantial individual variations in the biological ~,ical properties of some Bothrops venoms (Meier, 1986; )86; Taborska and Kornalik, 1985; Jimenez-Porras, , 1967). However, it was ing individual variation, reported that, notwithstandin venom biological properties could be used for differWe have also entiation o f B. nummifer and B. picadoi. 1 demonstrated that for venoms )ms of the genera Naja,
Trimeresurus, Agkistrodon, Bungarus and
Vipera,
venom biological propertiess can be used for the .., . ., differentiation of the species within (Tan and Tan, 1988a; Tan et al., Gnanajothy, 1990a, b, c). In this stuc
aline phosphomoA, L-amino acid re determined, and fiological activities genus and species n be used for the
from 13 species ul~.~lv~o,l v ~,lavlalo the biological b: rl J l v } p ~ l u * . , o v x venoms of Bothrops and examined exami the c o m m o n (26 samples) s~ characteristics of Bothrops venoms as well as the use chara biological properties for differentiation of of venom vel Bothrops species. MATERIALS AND METHODS METHq
Materials BioMerieux (France). All Cep3halite was obtained from BioMe grade and other reagents and substrates were of analytical al mrchased from Sigma Chemical mical Cc Co. (St. Louis, USA). were [c
Venoms A total of 26 samples of venoms from f 13 species of These Bothrops was used for the present investigation. ir aca (Jararaca) venom; three include four samples of B. jararaca (Jara ), B. atrox (Mapepire samples each of B. asper (Fer-de-lance), balsin), B. nummifer (Jumping viper) and anq B. schlegeli (Eyelash viper) venoms; two samples each eacl of B. alternatus (Urutu) and B. jararacussu (Jararacussu aracussu) venoms; and one sample each of B. bilineatus (Amazon Amazonian tree viper), B. cotiara(Cotiara), B. lansbergi (Lansberg' ,ansberg's hog-nosed viper), B. moojeni (Caissaca), B. nasuta (Horned (Hot hog-nosed pit The venoms viper) and B. pradoi (Prado's pit viper) venoms. v( ntarium Laboratories were obtained from Miami Serpenta Rosans, France), Quality (Salt Lake City, USA), Latoxan (Rosan Florida, USA), Ophidia Venoms for Medical Research (Florid Venin (Tavannes, Switzerland), Ventoxin Ventoxil (Maryland, USA) and Sigma Chemical Co. The source ( ~urce of each venom sample is indicated in Table 1. Most of the venom samples are pooled samples and were shipped by airmail a and arrived within 2 weeks at the authors' laborat0r]y. The venoms were dissolved in 0.85% saline at a concentrat pncentration of 1 mg/ml and then used for all biological activity determinations. det(
Determination of hemorrhagic activity Hemorrhagic activity was determined in mice (Kondo et al., 1960). Fifty #1 of the venom sa sample was injected intradermally into the dorsal area of mice mic (25 + 3 g) and the animals were sacrificed 1 hr later. The size of the hemorm the visceral side of the meter of the spot and the e first measurement were
qGET-HONG TAN and GNANAJOTHYPONNU! ~¢as quantitated as the
ting time (KCCT) d platelet-poor rabbit of the venom solution containing the rabbit ) at 37°C followed by chloride. The clotting h (in sec). was determined using f 0.5% bovine fibrinoa and the clotting time was recorded (in sec) using a ~pwatch upon addition of 0.1 ml of the venom sample enson, 1969). Other enzymatic activities. Protease, phosphodiesterase, :aline phosphomonoesterase, L-amino acid oxidase, 5'-nuotidase, acetylcholinesterase and hyaluronidase activities re determined as described previously (Tan and Tan, ]8a; Tan and Gnanajothy, 1990a) using casein, bis-4rophenyl phosphate, 4-nitrophenyl phosphate, L-leucine, AMP, acetylhiocholine and human umbilical cord aluronic acid, respectively, as substrates. Arginine ester :lrolase and phospholipase A activity were determined ng ~-benzoyl arginine ethyl ester (Collins and Jones, 1972) :1 egg yolk suspension (Tan and Tan, 1988b), respectively, substrates. One unit of protease activity was arbitrarily ~ned as an increase of I absorbance unit per hr at 280 nm. ,aluronidase activity was expressed as NFU (national mulary unit)/mg. Activities for the other enzymes are exl~ressed either in #mol (for arginine ester hydrolase, acet~ ;tylcholinesterase and phospholipase A) or nmol (for nucleotidase, alkaline phosphomonoesterase, phosphodi5'-nucleotidase. esterase erase and L-amino acid oxidase) substrate transformed or product released/min/mg venom. LU~ILA~
XXAIALUI~
~ll~tlllla~b
~.~
llU
~L
~.J
/U
tI~Vlll~
UOtUlO"
Se~~hadex G-75 gel filtration chromatography o f the venoms Venom Venom solution (2 mg/ml 0.85% saline) was fractionated by a Sephadex G-75 gel filtration column (12 x 250 mm) equilibrated filibrated with physiological saline. The flow rate was 25 ml/hr L~ and I ml of effluent was collected per tube. Protein t absorbance concentration was monitored by absorban bance measurement at 280 nm. A calibration curve wass constructed using bovine ic anhydrase and soybean serum albumin, pepsin, carbonic trypsin inhibitor as standard proteins.
Statistical analysis The significance of difference in enzyme activities was determined by t-test. The level of significance chosen was P < 0.05. RESULTS
Hemorrhagic, procoagulant and nticoa m d antic anticoagulant activities o f the venoms Table 1 shows the h e m o r r hagic ha and thrombin-like enzyme activities o f the Bothro ~rops venoms as well as the effect o f the venoms on thee K C C T o f platelet-poor rabbit plasma. All Bothrops venoms examined amined elicited hemorrhages in mice• The h e m o r r h a~gic effect o f the venoms, however, ranged from trace to to 6 cm 2 with substantial individual variation• M o s t Bothrops venoms tested ;ted exhibited procoagulant activity as measured by K~,CCT C C T an and thrombin-like enzyme assays• B. bilineatus and B. s c h l e g e l i v e n o m s both did not shorten the K C CZT T but eexhibited moderate thrombin-like activity• A m o n g th oms tested, only two venoms (B. h nasuta) were anticoagulant: they ( .
.
.
.
*L--
D--~I-
.
.
.
.
.
.
.
.
.
Table 1. Procoagul,'
tt and hemorrhagicactivities of 5 venoms
Venom of species
KCCT (see)
TLE (sec)
Venom sources
B. alternatus 1
361-365,
1991
)F SOME VENOMS OF 9PS (AMERICAN LANCI
;ICAL OF THE ~D VIPER)
ET-HONG TAN and GNANAJOTHYPONN Biochemistry, University of Malaya, Kual;
daysia
ATIVE STUDY OF THE
(Received 18 March 1991) ulant, phosphc Abstract--1. The hemorrhagic, procoagulant, anticoagular noesterase, 5'-nucleotidase, hyaluronidase, arginine ester hydrolase, oxidase and protease activities of 26 samples of)f venoms from 13 species the Sephadex G-75 gel filtration patterns for some of the venoms also vari 2. The results show that while there are considerable individual ind chara~ of many of the Bothrops venoms tested, there are some common corn levels. ven 3. The differences in the biological properties of the Bothrops B, differentiation of most Bothrops species examined.
INTRODUCTION l h ete genus Bothrops (American lance-headed viper), rich belongs to the Crotalinae subfamily (Tribe which 'otalini), is one of the largest of the genera of Crotalini nomous snakes, with more than 60 species (or over venomous 80 taxa) distributed throughout Central and South nerica and the West Indies (Hoge and Hoge, 1978). America te Bothrops contains both arboreal and terrestrial The rms; some have very limited known ranges and the forms relationshi ationships of the species within the genus are orly understood. Also, the genus is probably polypoorly phyletic. Some of the more re prominent members nakes B. atrox, B. asper, include the large terrestrial snakes
9. neuwiedi, B. alternatus B. jararaca, B. jararacussu, B. osed (also terrestrial) pit and B. bilineatus; the hog-nosed vipers B. cotiara, B. lansberg i and B. nasutus; and the arboreal B. nummifer and B. schlegeli. The biological properties, in particular the procoag ~rocoagulant activities of venoms from some of the more c o m m o n Bothrops have been investigated (Arag),on and Gubensek, 1981; Kamiguti and Hanada, 1985; 5; Kamiguti et al., 1987; Nahas et al., 1979; Tu, 1977 ). While some quantitatic activities of the Bothive data on certain enzymatic orted (Kocholaty et al., tops venoms have been reported rative study of the enzy1971; Mebs, 1970), a comparative ms is still lacking. matic activities of the venoms Several authors have reported )rted substantial individual variations in the biological ~,ical properties of some Bothrops venoms (Meier, 1986; )86; Taborska and Kornalik, 1985; Jimenez-Porras, , 1967). However, it was ing individual variation, reported that, notwithstandin venom biological properties could be used for differWe have also entiation o f B. nummifer and B. picadoi. 1 demonstrated that for venoms )ms of the genera Naja,
Trimeresurus, Agkistrodon, Bungarus and
Vipera,
venom biological propertiess can be used for the .., . ., differentiation of the species within (Tan and Tan, 1988a; Tan et al., Gnanajothy, 1990a, b, c). In this stuc
aline phosphomoA, L-amino acid re determined, and fiological activities genus and species n be used for the
from 13 species ul~.~lv~o,l v ~,lavlalo the biological b: rl J l v } p ~ l u * . , o v x venoms of Bothrops and examined exami the c o m m o n (26 samples) s~ characteristics of Bothrops venoms as well as the use chara biological properties for differentiation of of venom vel Bothrops species. MATERIALS AND METHODS METHq
Materials BioMerieux (France). All Cep3halite was obtained from BioMe grade and other reagents and substrates were of analytical al mrchased from Sigma Chemical mical Cc Co. (St. Louis, USA). were [c
Venoms A total of 26 samples of venoms from f 13 species of These Bothrops was used for the present investigation. ir aca (Jararaca) venom; three include four samples of B. jararaca (Jara ), B. atrox (Mapepire samples each of B. asper (Fer-de-lance), balsin), B. nummifer (Jumping viper) and anq B. schlegeli (Eyelash viper) venoms; two samples each eacl of B. alternatus (Urutu) and B. jararacussu (Jararacussu aracussu) venoms; and one sample each of B. bilineatus (Amazon Amazonian tree viper), B. cotiara(Cotiara), B. lansbergi (Lansberg' ,ansberg's hog-nosed viper), B. moojeni (Caissaca), B. nasuta (Horned (Hot hog-nosed pit The venoms viper) and B. pradoi (Prado's pit viper) venoms. v( ntarium Laboratories were obtained from Miami Serpenta Rosans, France), Quality (Salt Lake City, USA), Latoxan (Rosan Florida, USA), Ophidia Venoms for Medical Research (Florid Venin (Tavannes, Switzerland), Ventoxin Ventoxil (Maryland, USA) and Sigma Chemical Co. The source ( ~urce of each venom sample is indicated in Table 1. Most of the venom samples are pooled samples and were shipped by airmail a and arrived within 2 weeks at the authors' laborat0r]y. The venoms were dissolved in 0.85% saline at a concentrat pncentration of 1 mg/ml and then used for all biological activity determinations. det(
Determination of hemorrhagic activity Hemorrhagic activity was determined in mice (Kondo et al., 1960). Fifty #1 of the venom sa sample was injected intradermally into the dorsal area of mice mic (25 + 3 g) and the animals were sacrificed 1 hr later. The size of the hemorm the visceral side of the meter of the spot and the e first measurement were
qGET-HONG TAN and GNANAJOTHYPONNU! ~¢as quantitated as the
ting time (KCCT) d platelet-poor rabbit of the venom solution containing the rabbit ) at 37°C followed by chloride. The clotting h (in sec). was determined using f 0.5% bovine fibrinoa and the clotting time was recorded (in sec) using a ~pwatch upon addition of 0.1 ml of the venom sample enson, 1969). Other enzymatic activities. Protease, phosphodiesterase, :aline phosphomonoesterase, L-amino acid oxidase, 5'-nuotidase, acetylcholinesterase and hyaluronidase activities re determined as described previously (Tan and Tan, ]8a; Tan and Gnanajothy, 1990a) using casein, bis-4rophenyl phosphate, 4-nitrophenyl phosphate, L-leucine, AMP, acetylhiocholine and human umbilical cord aluronic acid, respectively, as substrates. Arginine ester :lrolase and phospholipase A activity were determined ng ~-benzoyl arginine ethyl ester (Collins and Jones, 1972) :1 egg yolk suspension (Tan and Tan, 1988b), respectively, substrates. One unit of protease activity was arbitrarily ~ned as an increase of I absorbance unit per hr at 280 nm. ,aluronidase activity was expressed as NFU (national mulary unit)/mg. Activities for the other enzymes are exl~ressed either in #mol (for arginine ester hydrolase, acet~ ;tylcholinesterase and phospholipase A) or nmol (for nucleotidase, alkaline phosphomonoesterase, phosphodi5'-nucleotidase. esterase erase and L-amino acid oxidase) substrate transformed or product released/min/mg venom. LU~ILA~
XXAIALUI~
~ll~tlllla~b
~.~
llU
~L
~.J
/U
tI~Vlll~
UOtUlO"
Se~~hadex G-75 gel filtration chromatography o f the venoms Venom Venom solution (2 mg/ml 0.85% saline) was fractionated by a Sephadex G-75 gel filtration column (12 x 250 mm) equilibrated filibrated with physiological saline. The flow rate was 25 ml/hr L~ and I ml of effluent was collected per tube. Protein t absorbance concentration was monitored by absorban bance measurement at 280 nm. A calibration curve wass constructed using bovine ic anhydrase and soybean serum albumin, pepsin, carbonic trypsin inhibitor as standard proteins.
Statistical analysis The significance of difference in enzyme activities was determined by t-test. The level of significance chosen was P < 0.05. RESULTS
Hemorrhagic, procoagulant and nticoa m d antic anticoagulant activities o f the venoms Table 1 shows the h e m o r r hagic ha and thrombin-like enzyme activities o f the Bothro ~rops venoms as well as the effect o f the venoms on thee K C C T o f platelet-poor rabbit plasma. All Bothrops venoms examined amined elicited hemorrhages in mice• The h e m o r r h a~gic effect o f the venoms, however, ranged from trace to to 6 cm 2 with substantial individual variation• M o s t Bothrops venoms tested ;ted exhibited procoagulant activity as measured by K~,CCT C C T an and thrombin-like enzyme assays• B. bilineatus and B. s c h l e g e l i v e n o m s both did not shorten the K C CZT T but eexhibited moderate thrombin-like activity• A m o n g th oms tested, only two venoms (B. h nasuta) were anticoagulant: they ( .
.
.
.
*L--
D--~I-
.
.
.
.
.
.
.
.
.
Table 1. Procoagul,'
tt and hemorrhagicactivities of 5 venoms
Venom of species
KCCT (see)
TLE (sec)
Venom sources
B. alternatus 1
