Comp. Biochem. Physiol. Vol. 93B, No. 4, pp. 757-762, 1989 0305-0491/89 $3.00 + 0.00 Printed in Great Britain © 1989 Pergamon Press pie

A COMPARATIVE STUDY OF THE ENZYMATIC AND TOXIC PROPERTIES OF VENOMS OF THE ASIAN

LANCE-HEADED PIT VIPER (GENUS TRIMERESURUS)

NGET-HONG TAN, ARUNMOZHIARASI ARMUGAM and CHON-SENG TAN

Department of Biochemistry, University of Malaya, Kuala Lumpur, Malaysia

(Received 28 November 1988)

Abstraet--l. The lethalities, anticoagulant effects, hermorrhagic, thrombin-like enzyme, hyaluronidase, protease, arginine ester hydrolase, 5'-nucleotidase, L-amino acid oxidase, alkaline phosphomonoesterase, phosphodiesterase and phospholipase A activities of twenty-three samples of venoms from twelve species of Asian lance-headed pit vipers (genus Trimeresurus) were examined.

2. The results indicate that notwithstanding individual variations in venom properties, the differences in biological properties of the Trimeresurus venoms can be used for the differentiation of venoms from different species of Trimeresurus.

3. The results also suggest that differences in the biological properties of snake venoms are useful parameters in the classification of snake species.

4. Our results indicate that venoms from the species T. okinavensis exhibited biological properties markedly different from other Trimeresurus venoms examined. This observation supports the recently proposed reclassification of T. okinavensis as a member of the genus Ovophis, rather than the genus Trimeresurus.

INTRODUCTION

The genus Trimeresurus (Asian lance-headed pit viper) are found in South-East Asia, China, India and Ryukyu Islands. Hoge and Hoge (1978) listed 41 species/subspecies of Trimeresurus in the oriental regions. The biological properties of venoms of some of the Trimeresurus species, including T. purpureo- maculatus, T. mucrosquamatus, T. flavoviridis, T. oki- navensis, T. gramineus, and T. elegans have been investigated (Tan and Tan, 1988a and references therein), and some of the toxic components of these venoms have also been isolated and characterized. The enzymatic properties of most of these venoms, however, have not been investigated. Also, little is known about the biological properties of other Trimeresurus venoms.

Certain authors (Goncalves and Deutsch, 1956; Jimenez-Porras, 1967) have suggested that venom properties might be used for the differentiation and classification of species, while others have argued that venom properties have limited value as taxonomic criteria due to intraspecific variations (Johnson, 1968). However, it is well established that there is correlation in the protease, acetylcholinesterase and arginine ester hydrolase activities of the venom and the taxonomic status of the snake at the familial level (Tu et al., 1965). Comparat ive study of the enzymatic activities of venoms from the genus Naja (common cobra) also revealed a correlation between the enzyme composit ion of venom and the taxonomic status of the snake at the species level (Tan and Tan, 1988b).

In the present study, we examined and compared the venom properties of twenty-three samples of venoms from twelve common species of Trimeresurus. The study also aims to investigate the taxonomic

information content of the Trimeresurus venom properties, as well as to examine the possibility of using the differences in venom biological activities to differentiate between venoms from different species of Trimeresurus. The data will also provide an infor- mation base for further investigations of properties of Trimeresurus venoms.

MATERIALS AND METHODS

Cephalite for kaolin-cephalin clotting time (kcct) deter- mination was obtained from bioMerieux (France). Mice were supplied by the Central Animal House, Faculty of Medicine, University of Malaya. All other reagents and substrates for enzyme assays were purchased from Sigma Chemical Company (St Louis, MO, USA).

Venoms

A total of twenty-three venom samples were used in this study. These include four venom samples from T. flavoviridis, two venom samples from 1". elegans, two venom samples from T. okinavensis, three venom samples from T. mucrosquamatus, four venom samples from T. albolabris, two venom samples from T. purpureomacula- tus and one sample each from T. macrops, 1". tokarensis, T. stejnegeri, T. stejnegeri formosensis, T. popeorium and T. sumatranus. T. stejnegeri formosensis venom and one sample of T. mucrosquamatus venom were generous gifts from Liau M.Y. of National Institute of Preventive Medicine, Taiwan. (T. stejnegeri formosensis venom, the venom from Formosan green habu, was often misidentified as T. gramineus venom, Hoge and Hoge (1978) listed it as T. stejnegeri formosensis.) Other samples were purchased from Miami Serpentarium Laboratories (Salt Lake City, USA); Latoxan (Rosans, France); Dr R. D. G. Theakston (Liverpool, UK); Sigma Chemical Company (St Louis, USA) and Snake and Venom Institute, Malaysia (see Table I for source for each venom sample). All of the venom samples are pooled samples and except for the

757

758 NGET-HONG TAN et al.

T. albolabris venom sample and the T. purpureomaculatus venom which were obtained from local sources, were shipped airmail and arrived within two weeks at the authors' laboratory.

Determination o f enzymatic activities

Venom (1 mg/ml) in physiological saline was used for enzymatic activity determination. Protease, 5'-nucleotidase, hyaluronidase and L-amino acid oxidase activities were determined as described previously (Tan and Tan, 1988b). Phosphodiesterase and alkaline phosphomonoesterase activities were determined by a method modified from Tan and Tan (1988b): the hydrolysis of the substrate in both cases was followed by measuring the rate of increase of absorbance at 400 nm instead of 440 nm, and one unit of enzyme activity was defined as the amount of enzyme that caused the increase of 0.001 absorbance unit per min. Arginine ester hydrolase activity was determined according to Collins and Jones (1972). Phospholipase A activity was determined acidimetrically using egg yolk suspension as substrate (Tan and Tan, 1988c).

Determination of lethality and hemorrhagic activi O,

LDs0 (i.v.) values of the venom sample was determined by intravenous injection of the sample (in physiological saline) into the caudal vein of mice (25 + 3 gm) and calculated according to the method of Spearman and Karber (World Health Organization, 1981).

Hemorrhagic activity was determined using method modified from Kondo et al. (1960). Fifty #1 of venom (1 mg/ml) was injected intradermally into the dorsal area of unanesthetized mice (25 _+ 3 gm, 2 mice were used for each venom sample). The animals were sacrificed after 1 hr. Two diameters were obtained for the spot of hemorrhage by measuring the longest diameter of the spot and the diameter perpendicular to the first measurement. Hemorrhage effect was quantitated as the product of the two diameters.

Determination o f thrombin -like activity and kaolin -cephalin clotting time

Thrombin-like activity was determined as described by Denson (1969). The clotting mixture contained 0.4ml of 0.5% bovine fibrinogen and the clotting time was recorded upon the addition of 0.I ml venom (1 mg/mt).

Kaolin--cephalin clotting time was determined according to Bell and Alton (1954). Citrated platelet poor plasma (100/zl) was incubated with 100/~1 of Cephalite at 37'C for 3 min. Fifty/zl of venom (1 mg/ml) was added followed by 50 #1 of 50 mM calcium chloride and clotting time was then recorded.

RESULTS

The lethali ty, thrombin- l i ke enzyme , at t t icoagulant and hemorrhagic activi t ies ~ / T r i m e r e s u r u s t~enoms

The lethality, th rombin- l ike enzyme, an t i coagu lan t and h e m o r r h a g i c activities o f the Tr imeresurus venoms are s h o w n in Table 1. E x a m i n a t i o n o f the LDs0 (i.v.) values, th rombin- l ike activities and anti- coagu lan t effects (as measu red by kcct assay) o f the different v e n o m samples f rom T. f lavoir idis , T. elegans, T. mucrosquamatus , T. purpureomacu la tus , T. albolabris and T. okinat,ensis indicates tha t there is no m a r k e d individual var ia t ion in these proper t ies .

The LDs0 (i.v.) values o f the Tr imeresurus v e n o m s range f rom 0 . 5 # g / g to 10l~g/g. V e n o m s f rom T. albolabris, T. sumatranus , T. s te /negeri f o r m o s e n s i s and T. purpureomacu la tus are highly lethal with LDs0 (i.v.) values o f <1 l~g/g: while v e n o m s f rom T. )qavot~iridis, T. clegans, T. mucrosquamatus , T. popeorum, T. s te /negeri and T. okinavensis are modera te ly lethal. T. tokarensis and T. macrops venoms , on the o ther hand, exhibited low lethality with LDs0 (i.v.) values of 7.5/~g/g and 1 0 # g / g , respectively.

O f the Trimeresurus ve noms examined , v e n o m s f rom six species, including T. albolabris, T. popeorum, T. macrops , T. s tejnegeri , T. s te /negeri f o r m o s e n s i s and T. purpureomacu la tus exhibited s t rong to mode r a t e th rombin- l ike activity, while ve noms f rom the o ther six species (T. ,)qat:o~,iridis, T. elegans, T. mucrosquamatus , T. tokarensis , 1". suma t ranus and T. ok inavens is ) did no t exhibit a detectable level of

Table 1. Lethality, thrombin-like, anticoagulant and hemorrhagic activities of 7)'imeresurus venoms*

Sample no. Venoms LDs~ ~ (i.v.) TLE KCCT Hemorrhage (Source)t

1 T..flavoriridis 3.8 > 30 m > 10 m 0.25 (Miami) 2 T. [lavoviridis 4.8 > 30 m > 10 m 0.25 (LIV) 3 T. flavoviridis 3.8 > 30 m > 10 m 1 (Sigma) 4 72 flavoviridis 3.8 > 30 m > 10 m 1.50 (LTX) 5 T. tokarensis 7.5 > 30 m > 10 m 0.25 (LTX) 6 T. mucrosquamatus 1.9 > 30 m > 10 m 2.25 (LTX) 7 72 mucrosquamatus 1.7 > 30 m > 10 m 1.50 (Miami) 8 T. mucrosquamatus 1.7 > 30 m > 10 m 1.50 (Taiwan~ 9 T. albolabris 0.5 80 ± 5 s > 10 m 1.50 (Sigma)

10 T. albolabris 0.5 120 ± 13 s > 10 m 1.50 (LTX) I I T. albolabris 0.5 65 + 5 s > I 0 m 4 ( LI V) 12 T. albolabris 0.5 70 + 10s > 10m 4 (SEA) 13 7". sumatranus 0.6 >30m > 10m 9 (LIV) 14 72 purpureomaculatus 0.9 60 + 10 s 220 +_ 29 s 6 (Miami) 15 T. purpureomaculatus 0.5 50 + 5 s 200 _+ 30 s 9 (SEA) 16 7". stejnegeri 1.9 50 ± 5 s 380 + 45 s 4 (Sigma) 17 72 s. ~rmosensis 0.8 25 ± 3 s 175 + 25 s 2.25 (Taiwan) 18 T. popeorum 1.9 53 + 7 s 175 _+ 35 s 2.25 (LIV) 19 T. elegans 3.8 > 30 m 70 ± 10 s 2.25 (Miami) 20 T. elegans 3.8 > 30 m 90 + 15 s I (Sigma) 21 ?2 macrops 10.0 285 ± 19 s 100 ± 20 s 2 (LIVI 22 72 okinavensis 5.0 > 30 m 20 + 3 s 1.50 (Miami) 23 T. okinavensis 5.0 >30 m 18 + 2 s I (Sigma~

*Abbreviations and units: thrombin-like enzyme (TLE) activity in clotting time (s, second or m, min): kaolin~:ephalin clotting time (kcct) in sec (s) or min (m), control was 60 ± 10 s; hemorrhage activity in cm ~.

tSources of venoms: LIV: Dr R. D. G. Theakston, Liverpool, UK; Miami: Miami Serpentarium Laboratories, USA: Sigma: Sigma Chemical Company, USA; LTX: Latoxan, France; Taiwan: MY Liau, Taiwan: SEA: South East Asia Venom Institute, Malaysia.

Pit viper

thrombin-like activity when bovine fibrinogen was used as the substrate.

Venoms from T. flavoviridis, T. mucrosquamatus, T. tokarensis, T. albolabris and T. sumatranus exhibited strong anticoagulant activity: 50 #g of the venom prolonged the kaolin-cephalin clotting time to more than 10 min. Venoms from T. stejnegeri, T. s. formosensis, T. purpureomaculatus and T. popeorum exhibited moderate anticoagulant activity, while venoms from T. macrops and T. elegans exhibited weak anticoagulant activity, as assayed by the kaolin-cephalin clotting time method. The only Trimeresurus venom examined that did not prolong kaolin-cephalin clotting time was that of T. okinavensis. In fact, both T. okinavensis venom samples shortened kaolin--cephalin clotting time, indicating the presence of non-thrombin-like procoagulant in the venom.

All Trimeresurus venom samples examined exhib- ited hemorrhagic effect in mice. T. purpureomaculatus venom samples exhibited exceptionally strong hemorrhagic activity. The venom samples of T. flavoviridis, T. elegans and T. albolabris exhibited marked individual variation in hemorrhagic activity.

The enzymatic activities of Trimeresurus venoms

Table 2 shows the enzymatic activities of the Trimeresurus venoms.

Hyaluronidase activity. Venoms of T. flavoviridis, T. albolabris, T. popeorum, T. macrops, T. stejnegeri, T. stejnegeri formosensis and T. okinavensis exhibited high hyaluronidase activity, while venom of T. tokarensis exhibited moderate hyaluronidase activ- ity. Venoms from T. elegans, T. mucrosquamatus, T. purpureomaculatus and T. sumatranus, exhibited low hyaluronidase activity. Comparison of hyaluronidase contents in venom samples of T. flavoviridis, T. elegans, T. mucrosquamatus, T. purpureomaculatus, T. albolabris and T. oki- navensis suggests that there is no marked individual variation in the content of hyaluronidase.

Protease activity. Both venom samples from T. okinavensis exhibited very high protease activity (7.1 and 8.2 unit/mg, respectively). The other Trimeresurus venoms examined exhibited compar- able, moderate protease activity of between 1.2 to 3.2 unit/mg. There is some individual variation in protease contents of T. albolabris venoms.

5'-Nucleotidase activity. Trimeresurus venoms generally exhibited high 5'-nucleotidase activity, even though marked individual variation is also evident in venoms from certain species, particularly in T. albolabris venoms. However, both T. okinavensis venom samples exhibited a low 5'-nucleotidase activity.

Arginine ester hydrolase activity. The arginine ester hydrolase activities of Trimeresurus venoms range widely, from a relative low of 0.5/~mol/min/mg in T. tokarensis venom to 16.6/zmol/min/mg in T. mucrosquamatus venoms. Although there is some individual variation in the arginine ester hydrolase content of T. mucrosquamatus and T. albolabris venoms, it is also evident that T. mucrosquamatus and T. okinavensis venoms exhibited exceptionally high arginine ester hydrolase activity. Also, T. popeorum venom and both samples of T. elegans venoms exhib-

C.B.P, 9 3 / 4 ~

venoms 759

ited moderately high arginine ester hydrolase activity while T. flavoviridis, T. macrops and T. tokarensis venoms exhibited low arginine ester hydrolase activity.

L-Amino acid oxidase. Most Trimeresurus venoms examined exhibited moderately high L-amino acid oxidase activity. There is marked individual vari- ation in the L-amino acid oxidase contents of T. mucrosquamatus and T. okinavensis venoms.

Alkaline phosphomonoesterase activity. The alka- line phosphomonoesterase activities of Trimeresurus venoms examined range widely (from 13 to 190 units/mg). Individual variation is evident among the venoms samples from T. flavoviridis, T. mucrosquamatus and T. albolabris.

Phosphodiesterase activity. The Trimeresurus venoms examined exhibited high phosphodiesterase activity, except for T. okinavensis venoms, which exhibited very low phosphodiesterase activity. Examination of the phosphodiesterase contents of various samples of venoms of T. flavoviridis, T. elegans, T. mucrosquamatus, T. purpureomaculatus, T. albolabris and T. okinavensis indicates that there is no marked individual variation in the content of phosphodiesterase.

Phospholipase A activity. Trimeresurus venoms exhibited moderate level of phospholipase A activity, which ranges from 78gmol/min/mg to 279/~mol/min/mg. Marked individual variation was observed in the phospholipase A content of T. albolabris venoms.

DISCUSSION

Individual variations in the biological properties o f Trimeresurus venoms

Willemse (1978) demonstrated a wide range of individual variation in the electrophoretic patterns of some snake venoms. Several authors also reported that biological activities of venoms may be subjected to great variations depending on the condition of the snake (e.g. age, nutrition etc.) and the handling of the venom and its storage (see Iwanaga and Suzuki, 1979). In a comparative study of cobra venom enzymatic activities, Tan and Tan (1988b), however, demonstrated that while there are individual variations in the venom enzyme contents, interspecific differences in the venom contents of phospholipase A, acetylcholinesterase, hyaluronidase and phosphodiesterase are significantly more marked and these differences can be used for the differ- entiation of venoms from different species of the genus Naja. Thus, information on venom properties can be used to differentiate species from the same genus if the magnitude of interspecific variation is greater than that of intraspecific (individual) vari- ation. In this connection, in the Trimeresurus venoms examined, the lethality, thrombin-like enzyme, anti- coagulant, hyaluronidase, arginine ester hydrolase, and phosphodiesterase activities are potentially useful parameters in the differentiation of venoms from different species of Trimeresurus, and that data on relative contents of protease, 5'-nucleotidase, L-amino acid oxidase, alkaline phosphomonoesterase and phospholipase A are of limited use in this respect.

Tab

le 2

. E

nzym

atic

act

ivit

ies

of T

rim

eres

urus

ven

oms*

Enz

yme

acti

viti

es (n

= 3

) Sa

mpl

e no

. V

enom

H

YA

P

RO

N

UC

A

EH

L

AA

O

PM

E

PD

E

PL

A

(Sou

rce)

1 72

flav

ovir

idis

32

_+2

2.6_

+0.1

20

75_+

152

1.

1 _

+0

.1

2390

_+45

19

_+2

1010

± 1

5 22

1 _+

13

(Mia

mi)

2

T. fl

avov

irid

is

32_+

2 2.

3_+0

.2

1550

_+ 1

04

1.24

40.1

21

99_+

40

34_+

2 93

9_+

12

2154

4 13

(L

IV)

3 T.

flav

ovir

idis

34

_+1

2.6_

+0.2

17

52_+

65

1.0

-+ 0

. I

2033

4425

17

441

925+

_18

227_

+21

(Sig

ma)

4

T. fl

avov

irid

is

36_+

2 2.

6_+0

.2

2459

_+ 1

09

1.04

40.1

18

94_+

17

23 ±

1

9144

4 12

24

1 ±

13

(LT

X)

5 T.

tok

aren

sis

23

±1

3.

1_+0

.2

2306

_+16

7 0.

5_+1

.0

2216

4427

75

_+2

946_

+23

228-

+13

(L

TX

) 6

T. m

ucro

squa

mat

us

5+

I

2.5-

+0.

3 21

0844

211

16.6

_+ 1

.2

1764

_+22

t2

0-+

8 57

1 _+

11

1694

4 13

(L

TX

) 7

I2 m

ucro

squa

mat

us

344

1 3.

2_+0

.3

1584

4498

9.

1 _+

0.7

1599

44 1

9 46

_+2

464_

+ 16

18

944

13

(Mia

mi)

8

T. m

ucro

squa

mat

us

10_+

1 2.

6_+

0.2

2154

4413

4 14

.7-+

1.1

747+

11

100_

+4

5004

419

2264

47

(Tai

wan

) 9

72 a

lbol

abri

s 36

443

1.2_

+0.2

14

20_+

55

2.34

40.1

21

20_+

39

65_+

3 10

89_+

23

1434

47

(Sig

ma)

10

T.

alb

olab

ris

3544

2 2.

1+_0

.1

786_

+36

1.74

40.1

17

98-+

19

190_

+9

9604

423

1104

47

(LT

X)

II

T. a

lbol

abri

s 34

_+3

2.1

_+0.

2 16

90_+

112

3.

1 _+

0.2

1929

_+32

12

0_+6

84

2_+

12

91 _

+7

(LIV

) 12

T.

alb

olab

ris

37_+

3 1.

8_+0

.2

2101

4411

9 2.

7440

.1

1799

_+21

30

_+2

1010

4423

78

_+7

(SE

A)

13

T. s

umat

ranu

s 74

41

1.9_

+0.2

23

36_+

118

2.44

40.1

23

37_+

34

54_+

3 14

46_+

26

260_

+13

(LIV

) 14

T.

pur

pure

omac

ulat

us

14 _

+ 2

2.8

_+ 0

.2

1204

44 6

7 3.

4 _+

0.2

23

46 4

4 28

23 _

+ 1

1024

_+

23

247

_+ 21

(M

iam

i)

15

T. p

urpu

reom

acul

atus

15

± 1

2.

2 +

0.2

1152

+ 5

6 2.

7 ±

0.2

2250

+ 3

9 13

± 2

98

7 44

28

185

± 13

(S

EA

) 16

T.

ste

jneg

eri

32_+

2 1.

4_+0

.1

2545

_+20

4 3.

4_+

+0.

2 12

94±

10

95_+

4 73

1 _+

11

2284

-21

(Sig

ma)

17

T.

sfo

rmos

ensi

s 48

± 4

1.

2 ±

0.1

3180

-!-_

245

2.9

± 0.

2 13

17 -+

7

100

± 4

857

± 23

27

9 -+

13

(Tai

wan

) 18

T.

pop

eoru

m

3744

2 2.

3±0.

2 10

22-+

78

5.14

40.3

21

55_+

28

22_+

1 34

6441

2 24

7442

1 (L

IV)

19

72 e

lega

ns

4±

1

2.1.

_+ 0

.1

2192

_+ 1

19

5.2_

+0.3

13

12+_

12

44

+2

66

4_+

10

91 _

+7

(Mia

mi)

20

72

ele

gans

6_

+ 1

2.8±

0.2

2767

_+21

0 5.

6 ±

0.3

1460

± 1

3 57

444

75

0±

16

13

0±

7

(Sig

ma)

21

72

mac

rops

34

_+4

2.5_

+0.2

19

56±

167

1.

4_.+

0.1

3389

± 4

1 47

_+2

621

_+ 1

2 12

4_+7

(L

IV)

22

72 o

kina

vens

is

40

+3

7.

1 ±

0.5

380_

+27

11.4

±0.

8 8

78

±7

13

± 1

18

_+ 1

91

+7

(M

iam

i)

23

T. o

kina

vens

is

39 4

4 4

8.2

± 0.

6 43

7 _+

34

11.2

44

1.2

2393

4- 3

2 16

44

1 25

44

1 13

0 44

7

(Sig

ma)

Z

O

,q

*Abb

revi

atio

ns a

nd u

nits

: hya

luro

nida

se (H

YA

) ac

tivi

ty in

Nat

iona

l F

orm

ular

y U

nit/

mg.

Pro

teas

e (P

RO

) ac

tivi

ty in

uni

t/m

g, 5

'-nu

cleo

tida

se (

NU

C)

acti

vity

in n

mol

pho

spha

te/m

in/m

g.

Arg

inin

e es

ter

hydr

olas

e (A

EH

) ac

tivi

ty i

n um

ol/m

in/m

g,

L-a

min

o ac

id

oxid

ase

(LA

AO

) ac

tivi

ty i

n un

it/r

ag,

alka

line

pho

spho

mon

oest

eras

e (P

ME

) ac

tivi

ty i

n un

it/m

g.

Phos

phod

iest

eras

e (P

DE

) ac

tivi

ty i

n un

it/m

g, p

hosp

hoti

pase

A (

PL

A)

acti

vity

in

pmol

/min

/mg.

Pit viper venoms 761

However, even in cases where substantial interspecific differences exist the relative contents of these enzymes may still be useful for the differentiation of venoms or may even have taxonomic value (vide infra).

The biological properties of Trimeresurus venoms and the taxonomic status of Trimeresurus okinavensis

It is evident from Tables 1 and 2 that the two venom samples of Trimeresurus okinavensis exhibited biological properties that are markedly different from other Trimeresurus venoms. While all other Trimeresurus venoms exhibited weak to strong anti- coagulant action as demonstrated in their abilities to prolong kaolin--cephalin clotting time, Trimeresurus okinavensis venoms shortened kaolin-cephalin clot- ting time. This is presumably due to the presence of a Factor X activating component, which has been reported to be present in T. okinavensis venom fractions (Andersson, 1972). Also both samples of T. okinavensis venom exhibited an extremely low phosphodiesterase and 5'-nucleotidase activities as well as an exceptionally high protease activity. In addition, the venom samples also exhibited a very high arginine esterase activity, although similarly high arginine esterase activity was also found in T. mucrosquamatus venom samples. In this con- nection, it is interesting to note that the species T. okinavensis has recently been removed from the genus Trimeresurus and reclassified as member of the genus Ovophis (Hoge and Hoge, 1978). Our above results on the comparative study of biological activi- ties of T. okinavensis venoms and other Trimeresurus venoms further support that this species does not belong to the genus Trimeresurus. Earlier study (Tan and Tan, 1989) has demonstrated that venoms of Tropidolaemus wagleri, a species formerly also con- sidered as member of Trimeresurus, also exhibited biological activities distinctly different from other Trimeresurus venoms. These observations together suggest that biochemical differences in venoms can be a useful aid in the classification of snakes.

Thus, excluding venoms of T. okinavensis (or more accurately, Ovophis okinavensis), the Trimeresurus venoms examined exhibit the following characteristic biological properties: moderate levels of protease and phospholipase A activities; high levels of 5'-nucleotidase and phosphodiesterase activities and moderately high level of L-amino acid oxidase activ- ity. The contents of hyaluronidase and alkaline phos- phomonoesterase vary widely. All Trimeresurus venoms also exhibited anticoagulant activity, even though the potency varies widely, and like all crotalid venoms, all Trimeresurus venoms examined exhibited hemorrhagic activity. The LDs0 (i.v.) values of the venoms in mice range from a highly toxic 0.5/~g/g for T. albolabris venoms to 10 #g/g for T. macrops venom. Of the eleven species of 'true' Trimeresurus examined, thrombin-like activity could be detected from the venoms of six species. While venoms of T.flavoviridis, T. mucrosquamatus, T. elegans, T. tokarensis and T. sumatranus appeared to be devoid of thrombin-like activity, fractionation studies, however, have demon- strated the presence of thrombin-like enzymes in T. flavoviridis and T. sumatranus venoms (Kosugi et al., 1986; Tan and Tan, unpublished). The thrombin- like activity in these venoms are presumably too

weak to be detected by the method described here. Also, a thrombin-like enzyme has been purified from T. okinavensis venom (Andersson, 1972), which also exhibited no thrombin-like activity as measured by the method described here.

Differentiation between Trimeresurus species by the differences in the biological properties o f the venoms

Close examination of data in Tables 1 and 2 indicates that most of the venoms fron the eleven species of 'true' Trimeresurus examined here can be differentiated from each other based on the differ- ences in their biological properties. The venoms can be divided into three groups according to their anti- coagulant activities. Group 1, with potent anti- coagulant activity (kect > 10 min), includes venoms from T. flavoviridis, T. mucrosquamatus, T. tokaren- sis, T. albolabris and T. sumatranus. Group 2 consists of venoms exhibiting moderate anticoagulant activity and includes venoms from T. purpureomaculatus, T. popeorum, T. stejnegeri and its subspecies T. stejnegeriformosensis. Group 3 consists of venoms with very weak anticoagulant activity and includes venoms from T. elegans and T. macrops.

Among the venoms from the five species of Trimeresurus in group 1, venoms from T. albolabris are characterized by high hyaluronidase activity, moderate level of arginine ester hydrolase activity and a high lethality. T. albolabris is also the only Trimeresurus species examined the venom of which exhibited both strong anticoagulant and thrombin- like activities. Venoms from T. mucrosquamatus exhibited a low hyaluronidase activity and an excep- tional high arginine ester hydrolase activity and moderate lethality. Venom from T. sumatranus exhibited low hyaluronidase activity, moderate level of arginine ester hydrolase activity and high lethality. Venoms from T.flavoviridis and T. tokaren- sis possess similar biological properties and cannot be differentiated from each other based on the biological properties alone. They both exhibited medium to high hyaluronidase activity, relatively low arginine ester hydrolase activity and moderate to low lethality. There are some minor differences between biological properties of venoms of T. flavoviridis and T. tokarensis, however, the differences (contents of hyaluronidase, protease, arginine ester hydrolase and alkaline phosphomonoesterase activities and LDs0 value) are too small and therefore of doubtful significance.

Among the venoms from the four Trimeresurus species in Group 2, venoms from T. stejnegeri and its subspecies T. stejnegeriformosensis possess similar biological properties and cannot be differentiated from each other based on venom biological proper- ties. An earlier comparative study of cobra enzyme activities (Tan and Tan, 1988b) also indicated rela- tively little subspecific variation in venom properties. Venoms from T. purpureomaculatus exhibited moder- ately low hyaluronidase activity and strong hemor- rhagic activity, while venoms from 7". stejnegeri, T. stejnegeri formosensis and T. popeorum exhibited high hyaluronidase and moderate hemorrhagic activities. T. purpureomaculatus venoms also differ markedly from T. stejnegeri venoms in protease, 5'-nucleotidase, L-amino acid oxidase and alkaline

762 NGET-HONG TAN et al.

phosphomonoes te rase activities, and from T. popeorum venom in arginine ester hydrolase and phosphodies terase activities. On the other hand, the two T. stejnegeri venoms differ markedly f rom T. popeorum venom in protease, 5 '-nucleotidase, arginine ester hydrolase, L-amino acid oxidase, alka- line phosphomonoes te rase and phosphodies terase activities.

Venoms of the two species of Trimeresurus in G r o u p 3, T. elegans and T. macrops, exhibited markedly different biological activities, in part icular , T. macrops has a low lethality, exhibi ted low arginine ester hydrolase, high L-amino acid oxidase, hya luronidase and modera te thrombin- l ike activities, while T. elegans has a modera te lethality, exhibited high arginine ester hydrolase, low hyaluronidase and lower t~-amino acid oxidase activities and is devoid of thrombin- l ike enzyme activity. The two venoms can thus be readily differentiated based on these biological differences.

In conclusion, our results show that in format ion on venom biological activities alone is sufficient to differentiate venoms of most of the species of Trimeresurus examined here (except between venoms of T. flavoviridis and T. tokarensis). Unfor tuna te ly , venoms from the other twenty-nine species/ subspecies of Trimeresurus are not available for investigations. While we do not expect to be able to differentiate venoms of every species of Trimeresurus based on the differences in venom biological proper- ties alone, the results presented here and our earlier studies on cobra venom enzymes (Tan and Tan, 1988b) suggest tha t no twi ths tand ing individual vari- ations, venom biological propert ies conta in useful t axonomic informat ion and also can be a useful aid in the differentiat ion of snake venoms from the same genus, part icular ly venoms from morphological ly similar snakes tha t inhabi t the same geographical area. Our findings, for example, allow the differ- en t ia t ion of venoms from the three morphological ly similar green tree pit vipers inhabi t ing South East Asia, i . e .T , macrops, T. popeorum and T. albolabris. Misidentif icat ion of these snakes has virtually rendered all work on Trimeresurus venoms in Tha i l and a lmost unin terpre tab le (Warrell , 1986). Our results show that the venoms of these three species of Trimeresurus possess markedly different biological properties; T. albolabris venom exhibited bo th s t rong ant icoagulant and strong thrombin- l ike activities and is highly lethal. 7'. macrops venom, on the o ther hand, has a low lethality, exhibited weak an t icoagulan t and modera te thrombin- l ike activities while 7". popeorum venom is moderate ly lethal, exhibited modera te anti- coagulant effect and s t rong thrombin- l ike activity.

Acknowledgement--This work was supported by a research grant, Vote F 129/84A, from the University of Malaya, Malaysia.

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