Toxdvon, Vol . 25, No . 11, PD . 1249-1253, 1987 .

0041-0101/81 53 .00+ .00® 1987 PQpmon Journals Ltd .Printed in Great Britain .

ENZYMATIC ACTIVITIES OF MALAYAN COBRA(NAJA NAJA SPUTATRIX) VENOMS

NGET-HONG TAN and CHON-SENG TANDepartment of Biochemistry, University of Malaya, Kuala Lumpur, Malaysia

(Acceptedforpublication 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 Malayancobra venom were investigated . There was significant variation in the contents of L-amino acidoxidase, alkaline phosphomonoesterase, acetylcholinesterase, phospholipase A, 5'-nuclootidaseand hyaluronidase . The phosphodiesterase content was, however, constant . Storage of thelyophilized venom powder at 25*C for 1 month did not affect the enzymatic activities . The venomenzymatic activities were generally also stable at 4°C in 0.85% saline solution . After incubation at37°C for 39 days in 0.855 saline solution, the venom still retained considerable amounts ofenzymatic activities . SP-Sephadex C-25 ion-exchange chromatography of the venom showed thatthe phospholipase A, L-amino acid oxidase, 5'-nucleotidaw, phosphodiesterase and alkalinephosphomonoesterase exist in multiple forms .

SNAKE venoms contain a large number of enzymes (ZELLER, 1977 ; IWANAGA andSuzuKi, 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 venompoisoning is peripheral respiratory paralysis caused by neurotoxins (LEE, 1971). REiD(1964), however, observed that the major clinical feature of Malayan cobra (Ngia turjasputatrix) envenomation was local necrosis . It is thus of interest to investigate thebiochemical composition of Malayan cobra venom. Two toxic phospholipases A havebeen isolated from the Malayan cobra (TAN, 1982a), however, little is known about thecontents and characteristics of other enzymes in the venom. We report here the contentsand thermal stability of enzymes from four samples of Malayan cobra venom and thedistribution of these enzymes in the SP-Sephadex C-25 ion exchange chromatographicelution profile of the venom. The data will provide an information base for furtherinvestigations 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 SigmaChemical Company,St Louis, MO, U.S.A . The venoms were shiped airmail and arrived within two weeks atthe authors' laboratory . Venom sample 4 was obtained from the Snake and VenomInstitute, Penang, Malaysia . Venom samples 1, 2 and 4 were from individual cobras(suppliers' information) . Venom was dissolved (1 mg/ml) in physiological saline solutionand used for enzyme activity determinations and stability studies. Phospholipase Aactivity was determined according to the method of DEHAAS et al. (1968) .Phosphodiesterase and alkaline phosphomonoesterase activities were determined by the

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procedures described by Lo et al . (1966) using Ca-bisp-nitrophenylphosphate and p-nitrophenylphosphate, respectively, as substrates . One unit of enzyme activity wasdefined as the amount of enzyme that caused an increase of 0.001 absorbance units permin. 5'-Nucleotidase activity was determined using 5'-AMP as substrate (HEPPEL andHILMORE, 1955) . Hyaluronidase activity was determined turbidimetrically (Xu et al.,1982) and was expressed in NFU/mg (National Formulary Units/mg) . Protease activitywas measured by a method modified from that of KUNITZ (1947) as described by TAN etal . (1986), using casein as substrate . One unit of protease activity was defined as anincrease of 1 absorbance unit per hr at 280 nm. L-Amino acid oxidase activity was alsodetermined as described in TAN et al. (1986), using L-leucine as substrate. One unit ofenzyme activity was defined as the amount of enzyme that caused an increase of 0.001absorbance units at 436 nm per min . Acetylcholinesterase activity was determined usingacetylthiocholine 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 ofeluant was collected per tube . During the collection of tube 12 a linear, 0-0.8 M sodiumchloride gradient (250 ml to 250 ml) was started . The column effluent was analyzed forprotein 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 weresignificantly 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&) VENOMSEnzymatic activity

For definition of units see Text .

Venom 1

Venom 2

Venom 3

Venom 4(mean t S.D.; n=3)

Phospholipase A(umole/min/mg)

Alkaline phosphomonoesterase(units/mg)

Phosphodiesterase

112±6

3011

62±3

97±6

24±0.3

62±3

201±6

25:0.3

61±4

386±9

72±5

67±4(units/mg)

5'-Nucleotidase 214:10 334±23 429±25 621±19(umole phosphate/min/mg)

Protease 0.6±0.1 0.8:0.1 0 .5:0 .2 0.7±0.1(units/mg)

L-Amino acid oxidase 286:9 219±15 265±29 413±19(units/mg)

Hyaluronidase 10.5:1.8 5.6±0 .5 15 .3:0 .4 17.8±1 .3(NFU/mg)

Acetylcholinesterase 2 .7:0.1 4.1±0 .1 3.6±0.1 1 .2±0.1(umole/min/mg)

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 ofvenoms .The enzymatic activities of Malayan cobra venom were remarkably stable when the

venom was stored lyophilized at room temperature. The venom retained full enzymaticactivities after 1 month storage. This suggested that the enzymatic activities of cobravenom were not altered during the process of mail delivery of the venom samples. In0.85% saline solution all enzymatic activities investigated retained 100% activity afterincubation at 4°C for 10 days . 5'-Nucleotidase, alkaline phosphomonoesterase andacetylcholinesterase lost some activity upon prolonged storage (46 days) at 4°C in salinesolution, while the other enzymes retained their full activity . It is interesting to note thatthe venom solution retained significant enzymatic activities even after incubation at 37°Cfor prolonged periods. Phospholipase A and phosphodiesterase, for example, retained100% activity after incubation at 37°C for 39 days, while 5'-nucleotidase andacetylcholinesterase retained 55% and 15% activity, respectively, after the same period ofincubation . 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-FxcHANGECHROMATOGRAPHIC ELU7iON PROFILE of Naja Raja sputatrtx VENOM.

Two hundred milligrams of crude venom, dissolved in 10 ml of0.05 M sodium phosphate buffer,pH 6.0, was applied to a column (40 x 25 mm) equilibrated with the same buffer . Elution wascarried out with the same buffer with a linear (0-0 .8 M) sodium chloride gradient . Sevenmilliliters of effluent were collected per tube . SP-Sephadex C-25 elution profile of Naja Rajasputatrix venom: (A) absorbante at 280nm (. . . . . . ) (this curveis also shown inFig . 1 B-D) and thedistribution of phospholipase A activity () ; (B) the distribution of t.-amino acidoxidase ( -) and 5'-nucleoddaw (-O-O-O-) activities; (C) the distribution ofphosphodiesterasc (

) 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 atthis temperature. Alkaline phosphomonoesterase retained 47% and 35%, respectively, ofactivity after incubation at the same temperature for 6 days and 39 days . Hyaluronidaseactivity was very unstable : only a trace amount could be detected after incubating thevenom 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-exchangechromatography of the venom yielded 5 major protein peaks. The phospholipase A,L-amino acid oxidase, 5'-nucleotidase, phosphodiesterase and alkaline phosphomono-esterase exist in multiple forms . There were three peaks for phospholipase A, 5'-nucleotidase and alkaline phosphomonoesterase and two peaks each for L-amino acidoxidase and phosphodiesterase . The first two protein peaks represent a mixture of acidicproteins andwere very rich in enzymatic activities . Protein fraction 3 contains the bulk ofphosphodiesterase, as well as some L-amino acid oxidase and basic phospholipase Aactivities . Protein fractions 4 and 5 presumably contain mainly the polypeptideneurotoxins and cardiotoxins, together with hyaluronidase and the major forms of 5'-nucleotidase and alkaline phosphomonoesterase. Malayan cobra neurotoxins andcardiotoxins 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 byLO et al. (1966), who reported the presence of an acidic form of phosphodiesterase and abasic form of 5'-nucleotidase . On the other hand, the Malayan cobra venom examinedexhibited two basic forms of phospholipase A in addition to the acidic form, as well as abasic phosphomonoesterase, whereas in Nqja naja atra venom there was only one acidicform of phospholipase A and the phosphomonoesterase was present in the acidic proteinfraction . The two basic phospholipases A from Malayan cobra venom have been isolatedand characterized (TAN, 19ß2a) . Thus, even though Nqja naja sputatrix and Naja nqjaatra are both subspecies of Ngja ngja, the results of this study show some variations intheir venom enzyme profiles .

Acknowledgements - This work was supported by research grant Vote F 152/84A from the University ofMalaya and an IKM research award (1986) .

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ELLMAN, G. L., CouRTNEY, K . D., ANDRES, V. JR and FEATHERSTONE, R. M. (1961) A new and rapidcolourimetric 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.

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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 itscomponents . 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.

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