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Enzymatic activities of Malayan cobra (Naja naja sputatrix) venoms
0041-0101/81 53 .00+ .00 ® 1987 PQpmon Journals Ltd .
Toxdvon, Vol . 25, No . 11, PD . 1249-1253, 1987 . Printed in Great Britain .
ENZYMATIC ACTIVITIES OF MALAYAN COBRA (NAJA NAJA SPUTA TRIX) VENOMS NGET-HONG TAN and CHON-SENG TAN Department of Biochemistry, University of Malaya, Kuala Lumpur, Malaysia (Accepted for publication 1 lune 1987) N .-H . TAN and C .-S . TAN . The enzymatic activities of Malayan cobra (Ngia ngia sputatrlx) venom . Toxicon 25, 1249-1253, 1987 . - The enzymatic activities of four samples of Malayan cobra venom were investigated . There was significant variation in the contents of L-amino acid oxidase, alkaline phosphomonoesterase, acetylcholinesterase, phospholipase A, 5'-nuclootidase and hyaluronidase . The phosphodiesterase content was, however, constant . Storage of the lyophilized venom powder at 25*C for 1 month did not affect the enzymatic activities . The venom enzymatic activities were generally also stable at 4°C in 0 .85% saline solution . After incubation at 37°C for 39 days in 0 .855 saline solution, the venom still retained considerable amounts of enzymatic activities . SP-Sephadex C-25 ion-exchange chromatography of the venom showed that the phospholipase A, L-amino acid oxidase, 5'-nucleotidaw, phosphodiesterase and alkaline phosphomonoesterase exist in multiple forms . SNAKE venoms contain a large number of enzymes (ZELLER, 1977; IWANAGA and SuzuKi, 1979). The occurrence of various enzymes in cobra venoms have been reported (KOCHOLATY et al., 1971 ; MEBS, 1970). The main cause of death due to cobra venom poisoning is peripheral respiratory paralysis caused by neurotoxins (LEE, 1971). REiD
(1964), however, observed that the major clinical feature of Malayan cobra (Ngia turja sputatrix) envenomation was local necrosis . It is thus of interest to investigate the biochemical composition of Malayan cobra venom. Two toxic phospholipases A have been isolated from the Malayan cobra (TAN, 1982a), however, little is known about the contents and characteristics of other enzymes in the venom. We report here the contents and thermal stability of enzymes from four samples of Malayan cobra venom and the distribution of these enzymes in the SP-Sephadex C-25 ion exchange chromatographic elution profile of the venom. The data will provide an information base for further investigations of the enzymes of Malayan cobra venom . Venom samples 1 and 2 were obtained from Miami Serpentarium Laboratories, Salt Lake City, UT, U.S.A ., whereas sample 3 was obtained from Sigma Chemical Company, St Louis, MO, U .S.A . The venoms were shiped airmail and arrived within two weeks at the authors' laboratory . Venom sample 4 was obtained from the Snake and Venom Institute, Penang, Malaysia . Venom samples 1, 2 and 4 were from individual cobras (suppliers' information) . Venom was dissolved (1 mg/ml) in physiological saline solution and used for enzyme activity determinations and stability studies. Phospholipase A activity was determined according to the method of DEHAAS et al. (1968) . Phosphodiesterase and alkaline phosphomonoesterase activities were determined by the 1249
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procedures described by Lo et al. (1966) using Ca-bisp-nitrophenylphosphate and pnitrophenylphosphate, respectively, as substrates . 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). Hyaluronidase activity was determined turbidimetrically (Xu et al., 1982) and was expressed in NFU/mg (National Formulary Units/mg) . Protease activity was measured by a method modified from that of KUNITZ (1947) as described by TAN et al. (1986), using casein as substrate . One unit of protease activity was defined as an increase of 1 absorbance unit per hr at 280 nm. L-Amino acid oxidase activity was also determined as described in TAN et al. (1986), using L-leucine as substrate. One unit of enzyme activity was defined as the amount of enzyme that caused an increase of 0.001 absorbance units at 436 nm per min . Acetylcholinesterase activity was determined using acetylthiocholine as substrate (ELLMAN et al., 1961) . SP-Sephadex C-25 ion-exchange chromatography was performed on a column (40 x 25 mm) equilibrated with 0.05 M phosphate buffer, pH 6.0. Lyophilized venom (200 mg of venom sample 1) was dissolved in 10 ml of 0.05 M phosphate buffer (pH 6.0) and applied to the column . Elution was carried out at a constant pH of 6.0 and 7 ml of eluant was collected per tube. During the collection of tube 12 a linear, 0-0 .8 M sodium chloride gradient (250 ml to 250 ml) was started . The column effluent was analyzed for protein by absorbance measurements at 280 nm. The significance of difference in enzyme contents was determined by analysis of variance (ANOVA) using a statistical package Epistat (supplied by T. L. Gustafson, Round Rock, Texas, U.S .A.). The level of significance chosen was P=0 .05. The enzymatic activities of the four Malayan cobra venom samples are shown in Table 1 . The phosphodiesterase activities of the four venom samples were remarkably constant (61- 67 units/mg) . All four venom samples exhibited a comparable, low protease activity. On the other hand, the activities of other enzymes in the four venom samples were significantly different . The ranges of the enzymatic activities were: phospholipase A, 97 - 386 jAmole/min/mg ; alkaline phosphomonoesterase, 24 - 72 units/mg ; 5' nucleotidase, 214 - 621 ymole phosphate/min/mg ; L-amino acid oxidase, 219 -413 units/ TABLE 1 . ENZYMATIC ACTIVITIES OF FOUR SAMPLES OF MALAYAN COBRA (Ngja Rgja Sputatr&) VENOMS Venom 1 Venom 2 Venom 3 Venom 4 Enzymatic activity (mean t S.D.; n=3) Phospholipase A 112±6 97±6 201±6 386±9 (umole/min/mg) 3011 24±0.3 25:0.3 72±5 Alkaline phosphomonoesterase (units/mg) Phosphodiesterase 62±3 62±3 61±4 67±4 (units/mg) 214 :10 334±23 429±25 621±19 5'-Nucleotidase (umole phosphate/min/mg) 0.6±0.1 0.8:0.1 0.5:0.2 0.7±0.1 Protease (units/mg) L-Amino acid oxidase 286:9 219±15 265±29 413±19 (units/mg) 5.6±0.5 17.8±1 .3 Hyaluronidase 10.5:1 .8 15.3 :0.4 (NFU/mg) 2.7:0.1 4.1±0.1 3.6±0.1 1.2±0.1 Acetylcholinesterase (umole/min/mg) For definition of units see Text.
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mg ; hyaluronidase, 5.6 -17 .8 NFU/mg ; acetylcholinesterase, 1 .2 - 4.1 jAmole/min/mg. These data serve to emphasize the intraspecific variation in the enzyme contents of venoms . The enzymatic activities of Malayan cobra venom were remarkably stable when the venom was stored lyophilized at room temperature. The venom retained full enzymatic activities after 1 month storage. This suggested that the enzymatic activities of cobra venom were not altered during the process of mail delivery of the venom samples. In 0.85% saline solution all enzymatic activities investigated retained 100% activity after incubation at 4°C for 10 days . 5'-Nucleotidase, alkaline phosphomonoesterase and acetylcholinesterase lost some activity upon prolonged storage (46 days) at 4°C in saline solution, while the other enzymes retained their full activity . It is interesting to note that the venom solution retained significant enzymatic activities even after incubation at 37°C for prolonged periods. Phospholipase A and phosphodiesterase, for example, retained 100% activity after incubation at 37°C for 39 days, while 5'-nucleotidase and acetylcholinesterase retained 55% and 15% activity, respectively, after the same period of incubation . L-Amino acid oxidase was much less stable at 37°C : the venom lost 45% of
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THE DL4I'aBUT1oN OF ENZYMATic AcTIvrnEs IN THE SP-SEPHADEx C-25 ION-FxcHANGE CHROMATOGRAPHIC ELU7iON PROFILE of Naja Raja sputatrtx VENOM.
Two hundred milligrams of crude venom, dissolved in 10 ml of 0.05 M sodium phosphate buffer, pH 6 .0, was applied to a column (40 x 25 mm) equilibrated with the same buffer . Elution was carried out with the same buffer with a linear (0-0 .8 M) sodium chloride gradient . Seven milliliters of effluent were collected per tube . SP-Sephadex C-25 elution profile of Naja Raja sputatrix venom: (A) absorbante at 280 nm (. . . . . . ) (this curve is also shown in Fig . 1 B - D) and the ) distribution of phospholipase A activity ( ; (B) the distribution of t.-amino acid oxidase ( -) and 5'-nucleoddaw (-O-O-O-) activities; (C) the distribution of phosphodiesterasc ( ) and alkaline phosphomoooesterase (-O-O-O-) activities ; (D) the distribution of aaty1cholinesterase ( -) and hyaluronidase (-O-O-O-) activities. Enzymatic activities were means of duplicate analyses.
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L-amino acid oxidase activity after 6 days and retained only 4% of activity after 39 days at this temperature. Alkaline phosphomonoesterase retained 47% and 35%, respectively, of activity after incubation at the same temperature for 6 days and 39 days . Hyaluronidase activity was very unstable : only a trace amount could be detected after incubating the venom solution'at 37°C for two days. The distribution of Malayan cobra venom enzymatic activities on SP-Sephadex C-25 ion-exchange chromatography is shown in Fig. 1 . SP-Sephadex C-25 ion-exchange chromatography of the venom yielded 5 major protein peaks. The phospholipase A, L-amino acid oxidase, 5'-nucleotidase, phosphodiesterase and alkaline phosphomonoesterase exist in multiple forms . There were three peaks for phospholipase A, 5'nucleotidase and alkaline phosphomonoesterase and two peaks each for L-amino acid oxidase and phosphodiesterase . The first two protein peaks represent a mixture of acidic proteins and were very rich in enzymatic activities . Protein fraction 3 contains the bulk of phosphodiesterase, as well as some L-amino acid oxidase and basic phospholipase A activities . Protein fractions 4 and 5 presumably contain mainly the polypeptide neurotoxins and cardiotoxins, together with hyaluronidase and the major forms of 5'nucleotidase and alkaline phosphomonoesterase. Malayan cobra neurotoxins and cardiotoxins have been isolated (TAN, 1982b, 1983) . The elution pattern of Malayan cobra venom on SP-Sephadex C-25 ion-exchange chromatography shows some similarity to that of Ngja nqja atra venom as described by LO et al. (1966), who reported the presence of an acidic form of phosphodiesterase and a basic form of 5'-nucleotidase. On the other hand, the Malayan cobra venom examined exhibited two basic forms of phospholipase A in addition to the acidic form, as well as a basic phosphomonoesterase, whereas in Nqja naja atra venom there was only one acidic form of phospholipase A and the phosphomonoesterase was present in the acidic protein fraction. The two basic phospholipases A from Malayan cobra venom have been isolated and characterized (TAN, 19ß2a) . Thus, even though Nqja naja sputatrix and Naja nqja atra are both subspecies of Ngja ngja, the results of this study show some variations in their venom enzyme profiles . Acknowledgements - This work was supported by research grant Vote F 152/84A from the University of Malaya and an IKM research award (1986) . REFERENCES
L. L. M. (1968) Purification and properties of phospholipase A from porcine pancreas . Biochim. blophys. Acta 159, 103. ELLMAN, 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. HEPPEL, L . A. and HILMORE, R. J. (1955) 5'-Nucleotidase. In: Methods In Emymology, Vol. 11, p. 547 (CoLowtcx, S. P. and KAPLAN, N. O., Eds). New York : Academic Press. IwANAGA, S. and SUZUKI, T . (1979) Enzymes in snake venom. In : Handbook of Experimental Pharmacology, Vol. 52, p. 61 (LEE, C .-Y., Ed .) . Berlin : Springer-Verlag . KOCHOLATTY, W. F., LEDFoRD, E. B., DALY, J. G. and BILLINGS, T. A. (1971) Toxicity and some enzymatic properties and activities in the venoms of Crotalidae, Elapidae and Viperidae. Touricon 9, 131 . KuNITz, M. (1947) Crystalline soybean trypsin inhibitor II . General properties . J. gen. Physiol. 30, 291 . LEE, C. Y. (1971) Mode of action of cobra venom and its purified toxins . In : Neuropoisons. Their Pathophysiological Actions, Vol. 1, p. 21 (SltpsoN, L . L ., Ed .). New York : Plenum Press. Lo, T. B., CmN, Y. H. and LEE, C. Y. (1966) Chemical studies of Formosa cobra (Ngja nqja atra) venom (1). Chromatographic separation of crude venom on CM-Sephadex and preliminary characterization of its components . J. Chin . them. Soc., Taipei Ser. 11 13, 25 . MESS, D . (1970) A comparative study of enzyme activities in snake venoms. Int . J. Biochem. 1, 335 . REID, H. A. (1964) Cobra-bites . Br . med. J. 2, 540 . DEHAAs, G. H., POSTEMA, N. M., NIEUWENHUIZEN, W. and DEENEN,
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Tart, N. H. (1982a) Isolation and preliminary characterization of two toxic phospholipases A, from the venom
of the Malayan cobra (Naja ngia sputatrLY). Biochirn . biophys. Acta 719, 599.
TAN, N . H . (1982b) Cardiotoxins from the venom of Malayan cobra (Ngja ngja sputatrix). Archs Biochem.
Biophys . 218, 51 .
TAN, N . H . (1983) Isolation and characterization of two toxins from the venom of the Malayan cobra (Ngja nqja
sputatrix). Toxiton 21, 201 .
TAN, N . H . (1986) Enzymatic activities of Callosrlmma rhodostoma (Malayan pit viper) venom . Toxicon 24,
626. Xu, X ., WAm, X ., Xi, .X ., Ltu, J ., HUArtc, J . and Lu, Z. (1982) Purification and partial characterization of hyaluronidase from five pace snake (Agkistrodon acutus) venom . Toxieon 20, 973 . ZEuEte, E . A. (1977) Snake venom action: are enzymes involved in it? F.xpaientia 30, 121 .
Toxdvon, Vol . 25, No . 11, PD . 1249-1253, 1987 . Printed in Great Britain .
ENZYMATIC ACTIVITIES OF MALAYAN COBRA (NAJA NAJA SPUTA TRIX) VENOMS NGET-HONG TAN and CHON-SENG TAN Department of Biochemistry, University of Malaya, Kuala Lumpur, Malaysia (Accepted for publication 1 lune 1987) N .-H . TAN and C .-S . TAN . The enzymatic activities of Malayan cobra (Ngia ngia sputatrlx) venom . Toxicon 25, 1249-1253, 1987 . - The enzymatic activities of four samples of Malayan cobra venom were investigated . There was significant variation in the contents of L-amino acid oxidase, alkaline phosphomonoesterase, acetylcholinesterase, phospholipase A, 5'-nuclootidase and hyaluronidase . The phosphodiesterase content was, however, constant . Storage of the lyophilized venom powder at 25*C for 1 month did not affect the enzymatic activities . The venom enzymatic activities were generally also stable at 4°C in 0 .85% saline solution . After incubation at 37°C for 39 days in 0 .855 saline solution, the venom still retained considerable amounts of enzymatic activities . SP-Sephadex C-25 ion-exchange chromatography of the venom showed that the phospholipase A, L-amino acid oxidase, 5'-nucleotidaw, phosphodiesterase and alkaline phosphomonoesterase exist in multiple forms . SNAKE venoms contain a large number of enzymes (ZELLER, 1977; IWANAGA and SuzuKi, 1979). The occurrence of various enzymes in cobra venoms have been reported (KOCHOLATY et al., 1971 ; MEBS, 1970). The main cause of death due to cobra venom poisoning is peripheral respiratory paralysis caused by neurotoxins (LEE, 1971). REiD
(1964), however, observed that the major clinical feature of Malayan cobra (Ngia turja sputatrix) envenomation was local necrosis . It is thus of interest to investigate the biochemical composition of Malayan cobra venom. Two toxic phospholipases A have been isolated from the Malayan cobra (TAN, 1982a), however, little is known about the contents and characteristics of other enzymes in the venom. We report here the contents and thermal stability of enzymes from four samples of Malayan cobra venom and the distribution of these enzymes in the SP-Sephadex C-25 ion exchange chromatographic elution profile of the venom. The data will provide an information base for further investigations of the enzymes of Malayan cobra venom . Venom samples 1 and 2 were obtained from Miami Serpentarium Laboratories, Salt Lake City, UT, U.S.A ., whereas sample 3 was obtained from Sigma Chemical Company, St Louis, MO, U .S.A . The venoms were shiped airmail and arrived within two weeks at the authors' laboratory . Venom sample 4 was obtained from the Snake and Venom Institute, Penang, Malaysia . Venom samples 1, 2 and 4 were from individual cobras (suppliers' information) . Venom was dissolved (1 mg/ml) in physiological saline solution and used for enzyme activity determinations and stability studies. Phospholipase A activity was determined according to the method of DEHAAS et al. (1968) . Phosphodiesterase and alkaline phosphomonoesterase activities were determined by the 1249
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procedures described by Lo et al. (1966) using Ca-bisp-nitrophenylphosphate and pnitrophenylphosphate, respectively, as substrates . 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). Hyaluronidase activity was determined turbidimetrically (Xu et al., 1982) and was expressed in NFU/mg (National Formulary Units/mg) . Protease activity was measured by a method modified from that of KUNITZ (1947) as described by TAN et al. (1986), using casein as substrate . One unit of protease activity was defined as an increase of 1 absorbance unit per hr at 280 nm. L-Amino acid oxidase activity was also determined as described in TAN et al. (1986), using L-leucine as substrate. One unit of enzyme activity was defined as the amount of enzyme that caused an increase of 0.001 absorbance units at 436 nm per min . Acetylcholinesterase activity was determined using acetylthiocholine as substrate (ELLMAN et al., 1961) . SP-Sephadex C-25 ion-exchange chromatography was performed on a column (40 x 25 mm) equilibrated with 0.05 M phosphate buffer, pH 6.0. Lyophilized venom (200 mg of venom sample 1) was dissolved in 10 ml of 0.05 M phosphate buffer (pH 6.0) and applied to the column . Elution was carried out at a constant pH of 6.0 and 7 ml of eluant was collected per tube. During the collection of tube 12 a linear, 0-0 .8 M sodium chloride gradient (250 ml to 250 ml) was started . The column effluent was analyzed for protein by absorbance measurements at 280 nm. The significance of difference in enzyme contents was determined by analysis of variance (ANOVA) using a statistical package Epistat (supplied by T. L. Gustafson, Round Rock, Texas, U.S .A.). The level of significance chosen was P=0 .05. The enzymatic activities of the four Malayan cobra venom samples are shown in Table 1 . The phosphodiesterase activities of the four venom samples were remarkably constant (61- 67 units/mg) . All four venom samples exhibited a comparable, low protease activity. On the other hand, the activities of other enzymes in the four venom samples were significantly different . The ranges of the enzymatic activities were: phospholipase A, 97 - 386 jAmole/min/mg ; alkaline phosphomonoesterase, 24 - 72 units/mg ; 5' nucleotidase, 214 - 621 ymole phosphate/min/mg ; L-amino acid oxidase, 219 -413 units/ TABLE 1 . ENZYMATIC ACTIVITIES OF FOUR SAMPLES OF MALAYAN COBRA (Ngja Rgja Sputatr&) VENOMS Venom 1 Venom 2 Venom 3 Venom 4 Enzymatic activity (mean t S.D.; n=3) Phospholipase A 112±6 97±6 201±6 386±9 (umole/min/mg) 3011 24±0.3 25:0.3 72±5 Alkaline phosphomonoesterase (units/mg) Phosphodiesterase 62±3 62±3 61±4 67±4 (units/mg) 214 :10 334±23 429±25 621±19 5'-Nucleotidase (umole phosphate/min/mg) 0.6±0.1 0.8:0.1 0.5:0.2 0.7±0.1 Protease (units/mg) L-Amino acid oxidase 286:9 219±15 265±29 413±19 (units/mg) 5.6±0.5 17.8±1 .3 Hyaluronidase 10.5:1 .8 15.3 :0.4 (NFU/mg) 2.7:0.1 4.1±0.1 3.6±0.1 1.2±0.1 Acetylcholinesterase (umole/min/mg) For definition of units see Text.
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mg ; hyaluronidase, 5.6 -17 .8 NFU/mg ; acetylcholinesterase, 1 .2 - 4.1 jAmole/min/mg. These data serve to emphasize the intraspecific variation in the enzyme contents of venoms . The enzymatic activities of Malayan cobra venom were remarkably stable when the venom was stored lyophilized at room temperature. The venom retained full enzymatic activities after 1 month storage. This suggested that the enzymatic activities of cobra venom were not altered during the process of mail delivery of the venom samples. In 0.85% saline solution all enzymatic activities investigated retained 100% activity after incubation at 4°C for 10 days . 5'-Nucleotidase, alkaline phosphomonoesterase and acetylcholinesterase lost some activity upon prolonged storage (46 days) at 4°C in saline solution, while the other enzymes retained their full activity . It is interesting to note that the venom solution retained significant enzymatic activities even after incubation at 37°C for prolonged periods. Phospholipase A and phosphodiesterase, for example, retained 100% activity after incubation at 37°C for 39 days, while 5'-nucleotidase and acetylcholinesterase retained 55% and 15% activity, respectively, after the same period of incubation . L-Amino acid oxidase was much less stable at 37°C : the venom lost 45% of
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Fia . 1 .
THE DL4I'aBUT1oN OF ENZYMATic AcTIvrnEs IN THE SP-SEPHADEx C-25 ION-FxcHANGE CHROMATOGRAPHIC ELU7iON PROFILE of Naja Raja sputatrtx VENOM.
Two hundred milligrams of crude venom, dissolved in 10 ml of 0.05 M sodium phosphate buffer, pH 6 .0, was applied to a column (40 x 25 mm) equilibrated with the same buffer . Elution was carried out with the same buffer with a linear (0-0 .8 M) sodium chloride gradient . Seven milliliters of effluent were collected per tube . SP-Sephadex C-25 elution profile of Naja Raja sputatrix venom: (A) absorbante at 280 nm (. . . . . . ) (this curve is also shown in Fig . 1 B - D) and the ) distribution of phospholipase A activity ( ; (B) the distribution of t.-amino acid oxidase ( -) and 5'-nucleoddaw (-O-O-O-) activities; (C) the distribution of phosphodiesterasc ( ) and alkaline phosphomoooesterase (-O-O-O-) activities ; (D) the distribution of aaty1cholinesterase ( -) and hyaluronidase (-O-O-O-) activities. Enzymatic activities were means of duplicate analyses.
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L-amino acid oxidase activity after 6 days and retained only 4% of activity after 39 days at this temperature. Alkaline phosphomonoesterase retained 47% and 35%, respectively, of activity after incubation at the same temperature for 6 days and 39 days . Hyaluronidase activity was very unstable : only a trace amount could be detected after incubating the venom solution'at 37°C for two days. The distribution of Malayan cobra venom enzymatic activities on SP-Sephadex C-25 ion-exchange chromatography is shown in Fig. 1 . SP-Sephadex C-25 ion-exchange chromatography of the venom yielded 5 major protein peaks. The phospholipase A, L-amino acid oxidase, 5'-nucleotidase, phosphodiesterase and alkaline phosphomonoesterase exist in multiple forms . There were three peaks for phospholipase A, 5'nucleotidase and alkaline phosphomonoesterase and two peaks each for L-amino acid oxidase and phosphodiesterase . The first two protein peaks represent a mixture of acidic proteins and were very rich in enzymatic activities . Protein fraction 3 contains the bulk of phosphodiesterase, as well as some L-amino acid oxidase and basic phospholipase A activities . Protein fractions 4 and 5 presumably contain mainly the polypeptide neurotoxins and cardiotoxins, together with hyaluronidase and the major forms of 5'nucleotidase and alkaline phosphomonoesterase. Malayan cobra neurotoxins and cardiotoxins have been isolated (TAN, 1982b, 1983) . The elution pattern of Malayan cobra venom on SP-Sephadex C-25 ion-exchange chromatography shows some similarity to that of Ngja nqja atra venom as described by LO et al. (1966), who reported the presence of an acidic form of phosphodiesterase and a basic form of 5'-nucleotidase. On the other hand, the Malayan cobra venom examined exhibited two basic forms of phospholipase A in addition to the acidic form, as well as a basic phosphomonoesterase, whereas in Nqja naja atra venom there was only one acidic form of phospholipase A and the phosphomonoesterase was present in the acidic protein fraction. The two basic phospholipases A from Malayan cobra venom have been isolated and characterized (TAN, 19ß2a) . Thus, even though Nqja naja sputatrix and Naja nqja atra are both subspecies of Ngja ngja, the results of this study show some variations in their venom enzyme profiles . Acknowledgements - This work was supported by research grant Vote F 152/84A from the University of Malaya and an IKM research award (1986) . REFERENCES
L. L. M. (1968) Purification and properties of phospholipase A from porcine pancreas . Biochim. blophys. Acta 159, 103. ELLMAN, 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. HEPPEL, L . A. and HILMORE, R. J. (1955) 5'-Nucleotidase. In: Methods In Emymology, Vol. 11, p. 547 (CoLowtcx, S. P. and KAPLAN, N. O., Eds). New York : Academic Press. IwANAGA, S. and SUZUKI, T . (1979) Enzymes in snake venom. In : Handbook of Experimental Pharmacology, Vol. 52, p. 61 (LEE, C .-Y., Ed .) . Berlin : Springer-Verlag . KOCHOLATTY, W. F., LEDFoRD, E. B., DALY, J. G. and BILLINGS, T. A. (1971) Toxicity and some enzymatic properties and activities in the venoms of Crotalidae, Elapidae and Viperidae. Touricon 9, 131 . KuNITz, M. (1947) Crystalline soybean trypsin inhibitor II . General properties . J. gen. Physiol. 30, 291 . LEE, C. Y. (1971) Mode of action of cobra venom and its purified toxins . In : Neuropoisons. Their Pathophysiological Actions, Vol. 1, p. 21 (SltpsoN, L . L ., Ed .). New York : Plenum Press. Lo, T. B., CmN, Y. H. and LEE, C. Y. (1966) Chemical studies of Formosa cobra (Ngja nqja atra) venom (1). Chromatographic separation of crude venom on CM-Sephadex and preliminary characterization of its components . J. Chin . them. Soc., Taipei Ser. 11 13, 25 . MESS, D . (1970) A comparative study of enzyme activities in snake venoms. Int . J. Biochem. 1, 335 . REID, H. A. (1964) Cobra-bites . Br . med. J. 2, 540 . DEHAAs, G. H., POSTEMA, N. M., NIEUWENHUIZEN, W. and DEENEN,
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Tart, N. H. (1982a) Isolation and preliminary characterization of two toxic phospholipases A, from the venom
of the Malayan cobra (Naja ngia sputatrLY). Biochirn . biophys. Acta 719, 599.
TAN, N . H . (1982b) Cardiotoxins from the venom of Malayan cobra (Ngja ngja sputatrix). Archs Biochem.
Biophys . 218, 51 .
TAN, N . H . (1983) Isolation and characterization of two toxins from the venom of the Malayan cobra (Ngja nqja
sputatrix). Toxiton 21, 201 .
TAN, N . H . (1986) Enzymatic activities of Callosrlmma rhodostoma (Malayan pit viper) venom . Toxicon 24,
626. Xu, X ., WAm, X ., Xi, .X ., Ltu, J ., HUArtc, J . and Lu, Z. (1982) Purification and partial characterization of hyaluronidase from five pace snake (Agkistrodon acutus) venom . Toxieon 20, 973 . ZEuEte, E . A. (1977) Snake venom action: are enzymes involved in it? F.xpaientia 30, 121 .