- Email: [email protected]
Enzymatic activities of Calloselasma rhodostoma (Malayan pit viper) venom
Toxicon, Vol. 24, No. 6, pp. 626-630, 1986. Printed in Great Britain.
ENZYMATIC
004l -0101/86 $3.00+ .00 Pergamon Journals Ltd.
ACTIVITIES OF CALLOSELASMA RHODOSTOMA (MALAYAN PIT VIPER) VENOM
NGET-HONG TAN, M. S. KANTHIMATHI and CHON-SENG TAN Department of Biochemistry,Universityof Malaya, Kuala Lumpur, Malaysia (Accepted for publication 30 January 1986) N.-H. TAN, M. S. KANTHIMATHIarid C.-S. TAN.Enzymaticactivitiesof Calloselasmarhodostoma
(Malayan pit viper) venom. Toxicon 24, 626- 630, 1986. -- The enzymecontents of four venom samples of Caliogelasma rhodostoma were analyzed. The venoms contained phosphodiesterase, alkaline phosphomonoesterase, 5'-nucleotidase, protease, phospholipase A, L-amino acid oxidase, hyaluronidase, arginine ester hydrolase, arginine amidase, fibrinogenaseand coagulant enzymeactivities.There is significantvariation in the contents of coagulant enzyme,arginine ester hydrolase, hyaluronidase, protease, phosphodiesterase, alkaline phosphomonoesterase and Lamino acid oxidase. DEAE-Sephaceiion exchangechromatographyof the venom resolvedit into eight major protein fractions. The eight fractions wereheterogeneousand exhibitedmore than one type of enzymatic activity. The 5'-nucleotidase, alkaline phosphomonoesterase, protease, coagulant enzyme, arginine ester hydrolase, arginine amidase and fibrinogenaseexist in multiple forms.
ENZYMES are major components of snake venoms (JIMENEZ-PORRAS, 1970; ZELLER, 1977; TU, 1977). To fully understand the complex interaction between snake and prey, it is therefore necessary to study each enzyme in venoms. The coagulant enzyme of Calloselasma rhodostoma (Malayan pit viper, previously known as Agkistrodon rhodostoma), the commonest cause of snakebite in Malaysia (REID et al., 1963), has been extensively investigated (ESNOUF and TUNNAH, 1967; COLLINS and JONES, 1972; HATTON, 1973). TOOM et al. (1969) reported the presence o f hemorrhagins and proteases in the venom. Recently, OUYANO et al. (1983) isolated an a-fibrinogenase from Malayan pit viper venom. The occurrence of other enzymes in Malayan pit viper venom, however, has not been investigated. In this communication we report on the enzyme contents o f four venom samples o f Calloselasma rhodostoma and the distribution of these enzymes in the DEAE-Sephacel ion exchange chromatographic elution profile o f the venom. Venom samples 1,2 and 3 were from Caiioselasma rhodostoma from Northern Peninsular Malaysia and were supplied by the Institute o f Medical Research, Kuala Lumpur, Malaysia (sample 1 and 2), and Miami Serpentarium Laboratories, Miami, Florida, U.S.A. (sample 3). Venom sample 4 was from Cailoselasma rhodostoma from Southern Thailand and was supplied by Latoxan, Rosans, France. Ion exchange chromatography was performed on a DEAE-Sephacel (Pharmacia Fine Chemicals) ion exchange c o l u m n ( 4 0 x 2 5 ram) equilibrated with 0.05 M T r i s - H C 1 buffer, pH 8.7. Elution was carried out at a pH o f 8.7 and 5.1 ml o f eluant was collected per tube. During 626
Short Communications
627
collection of tube 15 a linear ( 0 - 0 . 2 M) sodium chloride gradient (250-250 ml) was started. After the collection of 80 tubes, 100 ml of 0.05 M T r i s - H C I buffer, pH 8.7, containing 0.4 M sodium chloride was used to elute the tightly bound proteins. Arginine ester hydrolase was assayed using a-benzoyl arginine ethyl ester as substrate (COLLINS and JONES, 1972). Arginine amidase activity was assayed with a-benzoylphenylalanyl-valyl-arginine p-nitroanilide (SVENDSEN et al., 1972). Phosphodiesterase and alkaline phosphomonoesterase activities were determined by the method described by Lo et al. (1966), using Ca-bis-p-nitrophenylphosphate and p-nitrophenylphosphate, respectively, as substrate. One unit of enzyme activity was defined as the amount of enzyme that caused an increase of 0.001 absorbance units per min. 5'-Nucleotidase activity was determined using 5'-AMP as substrate (HEPPEL and HILMORE, 1955). Phospholipase A activity was determined titrimetrically (DEHASS et ai., 1970). Hyaluronidase activity was determined turbidimetrically (XU et al., 1982). Acetylcholinesterase activity was determined using acetylthiocholine as substrate (ELLMANet ai., 1961). Coagulant activity was measured at 37°C by the clotting time assay (DENSON 1969) using human plasma. Proteolytic activity was measured by a modification of the method described by KUNITZ (1947). Two milliliters of 1%0 casein in 0.25 M sodium phosphate buffer, pH 7.75, and 0.1 ml of sample solution were incubated for 30 min at 37°C. The undigested casein was precipitated and the reaction terminated by adding 2 ml of 5°70 trichloroacetic acid. After centrifugation at 10000 1 for 10 min, the absorbance of the supernatant was measured at 280 nm. One unit of protease activity was defined as an increase of 0.001 absorbance units per hr at 280 nm. L-Amino acid oxidase activity was determined as described in the WORTHINGTON ENZYME MANUAL (1970) with some modification. Peroxidase 0.05 ml (0.007°?0,510 NIH units/mg) was added to 1 ml of 0.2 M triethanolamine buffer, pH 7.6, containing 0.1%0 L-leucine and 0.0065 % O-dianidisine and incubated for 3 rain at room temperature. Venom sample (0.1 ml) was then added and the increase in absorbance at 436 nm measured. One unit of enzyme activity was defined as the amount of enzyme that caused an increase of 0.001 absorbance units per rain. Fibrinogenase activity was determined using a method modified from SAPRU et al. (1983). Fibrinogen solution, 2 ml of 2% w/v in 0.05 M Tris - HC1 buffer, pH 7.75, was incubated with 0.1 ml of venom sample at room temperature for 15 rain and then 5 ml of 0.05 M imidazole-HCI buffer, pH 7.4, containing 0.9% sodium chloride was added. This was followed by addition of 0.1 ml of thrombin (50 NIH units) and the mixture was allowed to stand at room temperature for 30 rain and filtered. The absorbance at 280 ran of the filtrate was measured. One unit of enzyme activity was arbitrarily defined as the amount of enzyme that caused an increase of 0.001 absorbance units per rain. The LDsovalues of the venom were determined by i.v. injection into the caudal veins of mice (30~:3 g) and calculated according to the method of Spearman and Karber (WORLD HEALTH ORGANIZATION, 1981). The enzymatic activities of the four Calioselasma r h o d o s t o m a venom samples are shown in Table 1, and include phosphodiesterase, alkaline phosphomonoesterase, 5'nucleotidase, protease, phospholipase A, L-amino acid oxidase and hyaluronidase. In addition, like most crotalid venoms, it also contained arginine ester hydrolase, fibrinogenase and coagulant enzyme activities. The venom also exhibited arginine amidase activity, as assayed by a-benzoyl-phenylalanyl-valyl-argininep-nitroanilide, the substrate for thrombin-like enzymes (SVENDSENel al., 1972). The venom does not contain acetylcholinesterase activity, which is not surprising, since acetylcholinesterase is absent from Viperidae and Crotalidae venoms (ZELLER, 1977). There is little variation in the
628
Short Communications TABLE 1. ENZYMEACTIVITIESOF FOUR SAMPLESOF Calloselasma rhodostoma VENOM
Enzyme activity
Venom 1
Phosphodiesterase (units/mg) Alkaline phosphomonoesterase
237 ± 12
(units/mg) 5'-Nucleotidase (~molephosphate/min/mg) Protease (units/mg) Phospholipase A (tanole/min/mg) L-Amino acid oxidase (units/mg) Hyaluronidase (N.F.U./mg) Arginine ester hydrolase (/amole/min/mg) Arginin¢ amidase (nmole/min/mg) Coagulant activity (units/mg)
37 ± 1
Venom 2 Venom 3 (mean ± S.D.; n = 3 ) 87 ± 4 12.5 ±
206 ± 11 0.7
50 ± 3
Venom 4 156 ± 10 12.5 ±
0.7
142 ± 6
141 ± 8
136 ± 7
141 ± 7
4200 ± 100
5559 ± 150
3741 ± 120
5436 +_ 130
8.1 _+ 0.3
9.7 ± 0.5
9.6 ± 0.4
8.9 ± 0.4
6900 ± 150
3800 ± 110
3000 ± 105
6450 ± 160
0.16 ± 0.04
0.85 ± 0.05
0.74 ± 0.05
1.60 ± 0.06
8.3 ± 0.4
9.3 ± 0.4
7.3 ± 0.4
11.5 _+ 0.5
57 ± 3
60 + 2
60 ± 5
61 ± 3
54 ± 4
67 ± 5
42 ± 4
90 ± 6
LDso (i.v.) values of the four venom samples were 6.3, 5.5, 6.0 and 5.2 ~g/g, respectively, for the venom samples 1,2,3 and 4.
contents of S'-nucleotidase, phospholipase and arginine amidase, however, the contents of other enzymes vary significantly. The variation in the activities of arginine ester hydrolase, hyaluronidase, protease, phosphodiesterase, L-amino acid oxidase and coagulant enzyme does not affect lethality of the venoms: the LDso(i.v.) values of the four venom samples varied only from 5.2 to 6.3 ~g/g. However, as phosphodiesterase, protease, arginine ester hydrolase, L-amino acid oxidase, hyaluronidase and coagulant enzymes have been reported to be involved in venom action (ZELLER, 1977; JIMENEZPORRAS, 1970), variation in enzymatic activities may lead to some differences in the pathophysiology of the venom action. DEAE-Sephacel ion exchange chromatography of the four venom samples yielded generally similar elution patterns. The venom proteins were resolved into eight fractions. Figure I shows the elution profile and enzyme activities of venom sample 4. Fraction I is a mixture of basic proteins. Fraction 2 contains a relatively small amount of protein, as assayed by the biuret method, but contains non-protein materials which exhibit a ~ of 310 nm in the absorption spectrum. Proteins in the other six fractions are acidic in nature. Phosphodiesterase activity occurs only in fraction l, while there are three distinct alkaline phosphomonoesterase peaks occurring in fractions 1,4 and 6. 5'-Nucleotidase activity occurs in all acidic protein fractions. Phospholipase A activity occurs only in fraction 5 while there are at least five distinct protease peaks (fractions 1,2,5 and 6 and the region between fractions 5 and 6). The basic protease occurring in fraction 1 is the major protease in the venom. The major coagulant enzyme occurs in fraction 5, with a minor coagulant component in fraction 3. Four arginine ester hydrolase peaks were observed, the two major peaks in fraction 3 and the region between fractions 5 and 6, and two minor peaks in fractions 2 and 8. There are four distinct fibrinogenase activity peaks occurring in fractions 1,3,6 and 7. Thus, all eight protein fractions resolved by DEAE-Sephacel ion
Short Communications Q
i
629 d ]" 120
600. h00'
,, o.~,
i t
~"
.x.~
..;~;x:
•
..
',...;,
tlO0 ~t
/ ~
111~
I t.,/
! ,ot!J
/'~
\
. I-z~-.t~
to Tum
6o
iil
t7
A lo
200'
!k
:0~
""
.tt'°°4lti!
~--
..
..i,.,....'l"~
r i ..... •....... llo
..-I. ~1
>., >
10 ]
~o ~.
20 ~! 20
t,0 A " 60 t,0
80" 80
ff I 00'~ . 3 ~T
~i
oz |
i
fr
20
60 60 TubeNu~l~
B0
FIG. 1. THE DISTRIBUTION OF ENZYMATIC ACTIVITIES IN THE DEAE-Sm,HACEL i o n EXCHANGE CHROMATOGRAPHIC ELUTION PROFILE OF Calloselasma rhodostoma VENOM.
One hundred milligrams of crude venom, dissolved in 10 nil of 0.05 M Tris - HCI buffer, pH 8.7, was applied to a column (40 × 25 ram) equilibrated with the same buffer. Elution was carried out with the lame buffer with a linear ( 0 - 0.2 M) sodium chloride gradient. Flow rate was 20 ml per hi" and 5.1 ml of effluent was collected per tube. (a) DEAE-Sephacel elution profile of Calloselasma rhodostoma venom, absorbance at 280 n m ( . . . . . . . . . . . . . . . . ) (this curve is also shown in Fig. 2 b - f ) ; Co) the distribution of phosphodiesterase ( ) and 5'-nucleotidase (-O-O-O-O-O-) activities; (c) the distribution of alkaline phosphomonoesterase ( ) and phospholipase A (-O-O-O-O-O-) activities; (d) the distribution of protease ( ) and L-amino acid oxidase (-0-0-0-0-0-) activities; (e) the distribution of arsinine ester hydrolase ( ) and arginine amidase (-0-0-0-(3-0-) activities; (f) the distribution of flbrinogenase ( ) and thrombin-like enzyme ( - 0 - 0 - 0 - 0 ~ ) activities.
exchange chromatography exhibited more than one enzymatic activity. For example, fraction 1 exhibited four enzymatic activities, viz. phosphodiesterase, alkaline phosphomonoesterase, protease and fibrinogenase. However, the fibrinogenase and protease activities may be due to the same enzyme, as Ouyang et al. (1983) have reported the isolation of a basic fibrinogenase that possessed protease activity. The region between fractions 5 and 6 is particularly heterogeneous. There are at least eight different types of enzymatic activity occurring in this region. It should be noted, however, that certain enzymes may possess more than one type of enzymatic activity. For example, the coagulant enzyme arvin also exhibits arginine ester hydrolase activity. In Calloselasma rhodostoma venom, the 5'-nucleotidase, alkaline phosphomonoesterase, protease, coagulant enzyme, arginlne ester hydrolase, arginine amidase and fibrinogenase exist in multiple forms. There were four distinct arginlne ester hydrolase peaks. This is in agreement with an observation made by TOOM et al. (1969). The same authors also reported the presence of three different proteolytic enzymes, whereas five protease peaks were observed in the present study. The shoulder on the arginine amidase peak indicates the presence of more than one form of amidase. ESNOUF and TUNNAH (1967) reported the presence of three coagulant enzymes. The DEAE-Sephacel ion exchanger employed in the present study, however, only resolved two coagulant enzyme peaks. There were at least four different forms of fibrinogenase. The fibrinogenase assay
630
Short Communications
method employed here, however, cannot detect the presence of fibrinogenases possess potent fibrinogen coagulant activity.
that
Acknowledgements--This work was supported by research grants Vote F 129/84 and 152/84 from the University of Malaya, Kuala Lumpur, Malaysia. REFERENCES COLLINS, J. P. and JONES, J. G. (1972) Studies on the active site of IRC-50 arvin, the purified coagulant enzyme from Agkistrodon rhodostoma venom. Eur. J. Biochem. 26, 510. DEHAss, G. H., SLOTaOOM, A. J., BONSEN, P. P. M., DEENEN, L. L. M., MAROUX, S., PUIGSERVER,A. Y. and DESNUELLE, P. (1970) Studies on phospholipase A and it zymogen from porcine pancreas. I. The complete amino acid sequence. Biochim. biophys. Acta 221, 31. DENSON, K. W. E. 0969) Coagulant and anticoagulant action of snake venoms. Toxicon 7, 5. ELLMAI~, G. L., COURTNEY, K. D., ANDRES, V. JR and FEATHERSTONE, R. M. (1961) A new and rapid colourimetric determination of acetylcholinesterase activity. Biochem. Pharmac. 7, 88. ESNOUF, M. P. and TUNNAH, G. W. 0967) The isolation and properties of the thrombin-like activity from Ancistrodon rhodostoma venom. Br. J. Haemat. 13, 581. HATTON, M. W. C. (1973) Studies on the coagulant enzyme from Agkistrodon rhodostoma venom. Biochem. J. 131,799. HEPPEL, L. A. and HILMORE, R. J. (1955) 5'-Nucleotidase. In: Methods In Enzymology (CoLowICK, S. P. and KAPLAN, N. O., Eds), Vol. II, p. 547. New York: Academic Press. JIMENEz-PORRAS, J. M. (1970) Biochemistry of snake venoms. Clin. Toxic. 3 389. KUNITZ, M. (1947) Crystalline soybean trypsin inhibitor II. General properties. J. gen. Physiol. 30, 291. Lo, T. B., CHEN, Y. H. and LEE, C. Y. (1966) Chemical studies of Formosan cobra (Najo naja atra) venom (I). Chromatogral~hic separation of crude venom on CM-Sephadex and preliminary characterization of its components~rJ. Chin. chem. Soc., Taipei, Scr I I . 13, 25. OUYANG, C., HWANO, L. J. and HUANG, T. F. (1983) a-Fibrinogenase from Agkistrodon rhodostoma (Malayan pit viper) snake venom. Toxicon 21, 25. REID, H. A., THEAN, P. C. and MARTIN, W. J. (1963) Epidemiology of snake bite in Northern Malaya. Br. med. J. 1,992. SAPRU, Z. Z., Tu, A. T. and BAILEY, G. S. (1983) Purification and characterization of a fibrinogenase from the venom of western diamondback rattlesnake (Crotalus atrox). Biochim. biophys. Acta 747, 225. SVENDSEN, L., BLOMBACK,M. and OLSSON, P. I. (1972) Substrates for determination of trypsin, thrombin and thrnmbin-like enzymes. Folio haemat., Lpz. 98, 446. TOOM, P. M., SQUIRE, P. G. and Tu, A. T. (1969) Characterization of the enzymatic and biological activities of snake venoms by isoelectric focussing. Biochim. biophys. Acta 181, 339. Tu, A. T. (1977) Venoms: Chemistry And Molecular Biology. New York: John Wiley & Sons. WORTHINGTONENZYMEMANUAL (1977) L-Amino acid oxidase, p. 49. Worthington Biochemical Corporation, U.S.A. WORLD HEALTH ORGANISATION (1981) Progress in the characterization of venoms and standardization of antivenoms. WHO Offset Publication No. 58, p. 23. Xu, X., W~¢3, X., XI, X., LIU, J., HUANG, J. and Lu, Z. 0982) Purification and partial characterization of hyaluronidase from five pace snake (Agkistrodon acutus) venom. Toxicon 20, 973. ZELLER, E. A. (1977) Snake venom action: are enzymes involved in it? Experientia 30, 121.
ENZYMATIC
004l -0101/86 $3.00+ .00 Pergamon Journals Ltd.
ACTIVITIES OF CALLOSELASMA RHODOSTOMA (MALAYAN PIT VIPER) VENOM
NGET-HONG TAN, M. S. KANTHIMATHI and CHON-SENG TAN Department of Biochemistry,Universityof Malaya, Kuala Lumpur, Malaysia (Accepted for publication 30 January 1986) N.-H. TAN, M. S. KANTHIMATHIarid C.-S. TAN.Enzymaticactivitiesof Calloselasmarhodostoma
(Malayan pit viper) venom. Toxicon 24, 626- 630, 1986. -- The enzymecontents of four venom samples of Caliogelasma rhodostoma were analyzed. The venoms contained phosphodiesterase, alkaline phosphomonoesterase, 5'-nucleotidase, protease, phospholipase A, L-amino acid oxidase, hyaluronidase, arginine ester hydrolase, arginine amidase, fibrinogenaseand coagulant enzymeactivities.There is significantvariation in the contents of coagulant enzyme,arginine ester hydrolase, hyaluronidase, protease, phosphodiesterase, alkaline phosphomonoesterase and Lamino acid oxidase. DEAE-Sephaceiion exchangechromatographyof the venom resolvedit into eight major protein fractions. The eight fractions wereheterogeneousand exhibitedmore than one type of enzymatic activity. The 5'-nucleotidase, alkaline phosphomonoesterase, protease, coagulant enzyme, arginine ester hydrolase, arginine amidase and fibrinogenaseexist in multiple forms.
ENZYMES are major components of snake venoms (JIMENEZ-PORRAS, 1970; ZELLER, 1977; TU, 1977). To fully understand the complex interaction between snake and prey, it is therefore necessary to study each enzyme in venoms. The coagulant enzyme of Calloselasma rhodostoma (Malayan pit viper, previously known as Agkistrodon rhodostoma), the commonest cause of snakebite in Malaysia (REID et al., 1963), has been extensively investigated (ESNOUF and TUNNAH, 1967; COLLINS and JONES, 1972; HATTON, 1973). TOOM et al. (1969) reported the presence o f hemorrhagins and proteases in the venom. Recently, OUYANO et al. (1983) isolated an a-fibrinogenase from Malayan pit viper venom. The occurrence of other enzymes in Malayan pit viper venom, however, has not been investigated. In this communication we report on the enzyme contents o f four venom samples o f Calloselasma rhodostoma and the distribution of these enzymes in the DEAE-Sephacel ion exchange chromatographic elution profile o f the venom. Venom samples 1,2 and 3 were from Caiioselasma rhodostoma from Northern Peninsular Malaysia and were supplied by the Institute o f Medical Research, Kuala Lumpur, Malaysia (sample 1 and 2), and Miami Serpentarium Laboratories, Miami, Florida, U.S.A. (sample 3). Venom sample 4 was from Cailoselasma rhodostoma from Southern Thailand and was supplied by Latoxan, Rosans, France. Ion exchange chromatography was performed on a DEAE-Sephacel (Pharmacia Fine Chemicals) ion exchange c o l u m n ( 4 0 x 2 5 ram) equilibrated with 0.05 M T r i s - H C 1 buffer, pH 8.7. Elution was carried out at a pH o f 8.7 and 5.1 ml o f eluant was collected per tube. During 626
Short Communications
627
collection of tube 15 a linear ( 0 - 0 . 2 M) sodium chloride gradient (250-250 ml) was started. After the collection of 80 tubes, 100 ml of 0.05 M T r i s - H C I buffer, pH 8.7, containing 0.4 M sodium chloride was used to elute the tightly bound proteins. Arginine ester hydrolase was assayed using a-benzoyl arginine ethyl ester as substrate (COLLINS and JONES, 1972). Arginine amidase activity was assayed with a-benzoylphenylalanyl-valyl-arginine p-nitroanilide (SVENDSEN et al., 1972). Phosphodiesterase and alkaline phosphomonoesterase activities were determined by the method described by Lo et al. (1966), using Ca-bis-p-nitrophenylphosphate and p-nitrophenylphosphate, respectively, as substrate. One unit of enzyme activity was defined as the amount of enzyme that caused an increase of 0.001 absorbance units per min. 5'-Nucleotidase activity was determined using 5'-AMP as substrate (HEPPEL and HILMORE, 1955). Phospholipase A activity was determined titrimetrically (DEHASS et ai., 1970). Hyaluronidase activity was determined turbidimetrically (XU et al., 1982). Acetylcholinesterase activity was determined using acetylthiocholine as substrate (ELLMANet ai., 1961). Coagulant activity was measured at 37°C by the clotting time assay (DENSON 1969) using human plasma. Proteolytic activity was measured by a modification of the method described by KUNITZ (1947). Two milliliters of 1%0 casein in 0.25 M sodium phosphate buffer, pH 7.75, and 0.1 ml of sample solution were incubated for 30 min at 37°C. The undigested casein was precipitated and the reaction terminated by adding 2 ml of 5°70 trichloroacetic acid. After centrifugation at 10000 1 for 10 min, the absorbance of the supernatant was measured at 280 nm. One unit of protease activity was defined as an increase of 0.001 absorbance units per hr at 280 nm. L-Amino acid oxidase activity was determined as described in the WORTHINGTON ENZYME MANUAL (1970) with some modification. Peroxidase 0.05 ml (0.007°?0,510 NIH units/mg) was added to 1 ml of 0.2 M triethanolamine buffer, pH 7.6, containing 0.1%0 L-leucine and 0.0065 % O-dianidisine and incubated for 3 rain at room temperature. Venom sample (0.1 ml) was then added and the increase in absorbance at 436 nm measured. One unit of enzyme activity was defined as the amount of enzyme that caused an increase of 0.001 absorbance units per rain. Fibrinogenase activity was determined using a method modified from SAPRU et al. (1983). Fibrinogen solution, 2 ml of 2% w/v in 0.05 M Tris - HC1 buffer, pH 7.75, was incubated with 0.1 ml of venom sample at room temperature for 15 rain and then 5 ml of 0.05 M imidazole-HCI buffer, pH 7.4, containing 0.9% sodium chloride was added. This was followed by addition of 0.1 ml of thrombin (50 NIH units) and the mixture was allowed to stand at room temperature for 30 rain and filtered. The absorbance at 280 ran of the filtrate was measured. One unit of enzyme activity was arbitrarily defined as the amount of enzyme that caused an increase of 0.001 absorbance units per rain. The LDsovalues of the venom were determined by i.v. injection into the caudal veins of mice (30~:3 g) and calculated according to the method of Spearman and Karber (WORLD HEALTH ORGANIZATION, 1981). The enzymatic activities of the four Calioselasma r h o d o s t o m a venom samples are shown in Table 1, and include phosphodiesterase, alkaline phosphomonoesterase, 5'nucleotidase, protease, phospholipase A, L-amino acid oxidase and hyaluronidase. In addition, like most crotalid venoms, it also contained arginine ester hydrolase, fibrinogenase and coagulant enzyme activities. The venom also exhibited arginine amidase activity, as assayed by a-benzoyl-phenylalanyl-valyl-argininep-nitroanilide, the substrate for thrombin-like enzymes (SVENDSENel al., 1972). The venom does not contain acetylcholinesterase activity, which is not surprising, since acetylcholinesterase is absent from Viperidae and Crotalidae venoms (ZELLER, 1977). There is little variation in the
628
Short Communications TABLE 1. ENZYMEACTIVITIESOF FOUR SAMPLESOF Calloselasma rhodostoma VENOM
Enzyme activity
Venom 1
Phosphodiesterase (units/mg) Alkaline phosphomonoesterase
237 ± 12
(units/mg) 5'-Nucleotidase (~molephosphate/min/mg) Protease (units/mg) Phospholipase A (tanole/min/mg) L-Amino acid oxidase (units/mg) Hyaluronidase (N.F.U./mg) Arginine ester hydrolase (/amole/min/mg) Arginin¢ amidase (nmole/min/mg) Coagulant activity (units/mg)
37 ± 1
Venom 2 Venom 3 (mean ± S.D.; n = 3 ) 87 ± 4 12.5 ±
206 ± 11 0.7
50 ± 3
Venom 4 156 ± 10 12.5 ±
0.7
142 ± 6
141 ± 8
136 ± 7
141 ± 7
4200 ± 100
5559 ± 150
3741 ± 120
5436 +_ 130
8.1 _+ 0.3
9.7 ± 0.5
9.6 ± 0.4
8.9 ± 0.4
6900 ± 150
3800 ± 110
3000 ± 105
6450 ± 160
0.16 ± 0.04
0.85 ± 0.05
0.74 ± 0.05
1.60 ± 0.06
8.3 ± 0.4
9.3 ± 0.4
7.3 ± 0.4
11.5 _+ 0.5
57 ± 3
60 + 2
60 ± 5
61 ± 3
54 ± 4
67 ± 5
42 ± 4
90 ± 6
LDso (i.v.) values of the four venom samples were 6.3, 5.5, 6.0 and 5.2 ~g/g, respectively, for the venom samples 1,2,3 and 4.
contents of S'-nucleotidase, phospholipase and arginine amidase, however, the contents of other enzymes vary significantly. The variation in the activities of arginine ester hydrolase, hyaluronidase, protease, phosphodiesterase, L-amino acid oxidase and coagulant enzyme does not affect lethality of the venoms: the LDso(i.v.) values of the four venom samples varied only from 5.2 to 6.3 ~g/g. However, as phosphodiesterase, protease, arginine ester hydrolase, L-amino acid oxidase, hyaluronidase and coagulant enzymes have been reported to be involved in venom action (ZELLER, 1977; JIMENEZPORRAS, 1970), variation in enzymatic activities may lead to some differences in the pathophysiology of the venom action. DEAE-Sephacel ion exchange chromatography of the four venom samples yielded generally similar elution patterns. The venom proteins were resolved into eight fractions. Figure I shows the elution profile and enzyme activities of venom sample 4. Fraction I is a mixture of basic proteins. Fraction 2 contains a relatively small amount of protein, as assayed by the biuret method, but contains non-protein materials which exhibit a ~ of 310 nm in the absorption spectrum. Proteins in the other six fractions are acidic in nature. Phosphodiesterase activity occurs only in fraction l, while there are three distinct alkaline phosphomonoesterase peaks occurring in fractions 1,4 and 6. 5'-Nucleotidase activity occurs in all acidic protein fractions. Phospholipase A activity occurs only in fraction 5 while there are at least five distinct protease peaks (fractions 1,2,5 and 6 and the region between fractions 5 and 6). The basic protease occurring in fraction 1 is the major protease in the venom. The major coagulant enzyme occurs in fraction 5, with a minor coagulant component in fraction 3. Four arginine ester hydrolase peaks were observed, the two major peaks in fraction 3 and the region between fractions 5 and 6, and two minor peaks in fractions 2 and 8. There are four distinct fibrinogenase activity peaks occurring in fractions 1,3,6 and 7. Thus, all eight protein fractions resolved by DEAE-Sephacel ion
Short Communications Q
i
629 d ]" 120
600. h00'
,, o.~,
i t
~"
.x.~
..;~;x:
•
..
',...;,
tlO0 ~t
/ ~
111~
I t.,/
! ,ot!J
/'~
\
. I-z~-.t~
to Tum
6o
iil
t7
A lo
200'
!k
:0~
""
.tt'°°4lti!
~--
..
..i,.,....'l"~
r i ..... •....... llo
..-I. ~1
>., >
10 ]
~o ~.
20 ~! 20
t,0 A " 60 t,0
80" 80
ff I 00'~ . 3 ~T
~i
oz |
i
fr
20
60 60 TubeNu~l~
B0
FIG. 1. THE DISTRIBUTION OF ENZYMATIC ACTIVITIES IN THE DEAE-Sm,HACEL i o n EXCHANGE CHROMATOGRAPHIC ELUTION PROFILE OF Calloselasma rhodostoma VENOM.
One hundred milligrams of crude venom, dissolved in 10 nil of 0.05 M Tris - HCI buffer, pH 8.7, was applied to a column (40 × 25 ram) equilibrated with the same buffer. Elution was carried out with the lame buffer with a linear ( 0 - 0.2 M) sodium chloride gradient. Flow rate was 20 ml per hi" and 5.1 ml of effluent was collected per tube. (a) DEAE-Sephacel elution profile of Calloselasma rhodostoma venom, absorbance at 280 n m ( . . . . . . . . . . . . . . . . ) (this curve is also shown in Fig. 2 b - f ) ; Co) the distribution of phosphodiesterase ( ) and 5'-nucleotidase (-O-O-O-O-O-) activities; (c) the distribution of alkaline phosphomonoesterase ( ) and phospholipase A (-O-O-O-O-O-) activities; (d) the distribution of protease ( ) and L-amino acid oxidase (-0-0-0-0-0-) activities; (e) the distribution of arsinine ester hydrolase ( ) and arginine amidase (-0-0-0-(3-0-) activities; (f) the distribution of flbrinogenase ( ) and thrombin-like enzyme ( - 0 - 0 - 0 - 0 ~ ) activities.
exchange chromatography exhibited more than one enzymatic activity. For example, fraction 1 exhibited four enzymatic activities, viz. phosphodiesterase, alkaline phosphomonoesterase, protease and fibrinogenase. However, the fibrinogenase and protease activities may be due to the same enzyme, as Ouyang et al. (1983) have reported the isolation of a basic fibrinogenase that possessed protease activity. The region between fractions 5 and 6 is particularly heterogeneous. There are at least eight different types of enzymatic activity occurring in this region. It should be noted, however, that certain enzymes may possess more than one type of enzymatic activity. For example, the coagulant enzyme arvin also exhibits arginine ester hydrolase activity. In Calloselasma rhodostoma venom, the 5'-nucleotidase, alkaline phosphomonoesterase, protease, coagulant enzyme, arginlne ester hydrolase, arginine amidase and fibrinogenase exist in multiple forms. There were four distinct arginlne ester hydrolase peaks. This is in agreement with an observation made by TOOM et al. (1969). The same authors also reported the presence of three different proteolytic enzymes, whereas five protease peaks were observed in the present study. The shoulder on the arginine amidase peak indicates the presence of more than one form of amidase. ESNOUF and TUNNAH (1967) reported the presence of three coagulant enzymes. The DEAE-Sephacel ion exchanger employed in the present study, however, only resolved two coagulant enzyme peaks. There were at least four different forms of fibrinogenase. The fibrinogenase assay
630
Short Communications
method employed here, however, cannot detect the presence of fibrinogenases possess potent fibrinogen coagulant activity.
that
Acknowledgements--This work was supported by research grants Vote F 129/84 and 152/84 from the University of Malaya, Kuala Lumpur, Malaysia. REFERENCES COLLINS, J. P. and JONES, J. G. (1972) Studies on the active site of IRC-50 arvin, the purified coagulant enzyme from Agkistrodon rhodostoma venom. Eur. J. Biochem. 26, 510. DEHAss, G. H., SLOTaOOM, A. J., BONSEN, P. P. M., DEENEN, L. L. M., MAROUX, S., PUIGSERVER,A. Y. and DESNUELLE, P. (1970) Studies on phospholipase A and it zymogen from porcine pancreas. I. The complete amino acid sequence. Biochim. biophys. Acta 221, 31. DENSON, K. W. E. 0969) Coagulant and anticoagulant action of snake venoms. Toxicon 7, 5. ELLMAI~, G. L., COURTNEY, K. D., ANDRES, V. JR and FEATHERSTONE, R. M. (1961) A new and rapid colourimetric determination of acetylcholinesterase activity. Biochem. Pharmac. 7, 88. ESNOUF, M. P. and TUNNAH, G. W. 0967) The isolation and properties of the thrombin-like activity from Ancistrodon rhodostoma venom. Br. J. Haemat. 13, 581. HATTON, M. W. C. (1973) Studies on the coagulant enzyme from Agkistrodon rhodostoma venom. Biochem. J. 131,799. HEPPEL, L. A. and HILMORE, R. J. (1955) 5'-Nucleotidase. In: Methods In Enzymology (CoLowICK, S. P. and KAPLAN, N. O., Eds), Vol. II, p. 547. New York: Academic Press. JIMENEz-PORRAS, J. M. (1970) Biochemistry of snake venoms. Clin. Toxic. 3 389. KUNITZ, M. (1947) Crystalline soybean trypsin inhibitor II. General properties. J. gen. Physiol. 30, 291. Lo, T. B., CHEN, Y. H. and LEE, C. Y. (1966) Chemical studies of Formosan cobra (Najo naja atra) venom (I). Chromatogral~hic separation of crude venom on CM-Sephadex and preliminary characterization of its components~rJ. Chin. chem. Soc., Taipei, Scr I I . 13, 25. OUYANG, C., HWANO, L. J. and HUANG, T. F. (1983) a-Fibrinogenase from Agkistrodon rhodostoma (Malayan pit viper) snake venom. Toxicon 21, 25. REID, H. A., THEAN, P. C. and MARTIN, W. J. (1963) Epidemiology of snake bite in Northern Malaya. Br. med. J. 1,992. SAPRU, Z. Z., Tu, A. T. and BAILEY, G. S. (1983) Purification and characterization of a fibrinogenase from the venom of western diamondback rattlesnake (Crotalus atrox). Biochim. biophys. Acta 747, 225. SVENDSEN, L., BLOMBACK,M. and OLSSON, P. I. (1972) Substrates for determination of trypsin, thrombin and thrnmbin-like enzymes. Folio haemat., Lpz. 98, 446. TOOM, P. M., SQUIRE, P. G. and Tu, A. T. (1969) Characterization of the enzymatic and biological activities of snake venoms by isoelectric focussing. Biochim. biophys. Acta 181, 339. Tu, A. T. (1977) Venoms: Chemistry And Molecular Biology. New York: John Wiley & Sons. WORTHINGTONENZYMEMANUAL (1977) L-Amino acid oxidase, p. 49. Worthington Biochemical Corporation, U.S.A. WORLD HEALTH ORGANISATION (1981) Progress in the characterization of venoms and standardization of antivenoms. WHO Offset Publication No. 58, p. 23. Xu, X., W~¢3, X., XI, X., LIU, J., HUANG, J. and Lu, Z. 0982) Purification and partial characterization of hyaluronidase from five pace snake (Agkistrodon acutus) venom. Toxicon 20, 973. ZELLER, E. A. (1977) Snake venom action: are enzymes involved in it? Experientia 30, 121.