7bxtcon, 1969, Vol. 7, pp. 185-188. Perl;amon Prou . Printed in t3reat Hdtrtn

SOME ENZYMATIC ACTIVITIES OF EGYPTIAN SNAKEVENOMS AND A SCORPION VENOM*

A. H. MOI-IAA4.D,A. KAntEI. and M. H. AYOBEDepartment of Physiology, Faculty of Medicine, Ain Shams University, Cairo, Egypt, U.A.R .

(Acceptedjor publication 20Jrrrte 1969)

Abstract-The enzymatic activities of proteinase, cholinesterase, tratlsaminases (glutamicoxalacetic transaminase and glutamic pyruvic transaminase) and amylase were estimatedin several Egyptian snake venoms and in a scorpion venom. All venoms showed proteinaseactivity in varying degrees, while cholinesterase was found only in Ngja hgje and Walterinesstaaegyptta venoms ; the glutamic pyruvic transaminase activity was recorded in all venoms exceptNgja rtigricollis and the scorpion venom, although no glutamic oxalacetic transaminase oramylase action was found in any of the venoms . All the enzymes were heat labile .

Tim AIM ofthis work was to determine some of the enzymatic activities such as proteinases,cholinesterases, amylases and transaminases in Egyptian snake venoms and a scorpionvenom.

MATERIALS AND METHODSVenoms

The venoms, obtained from the Physiology Department, Ain Shams University Serpen-tarium, were those of Naja haje, Walterinessia aegyptia, Cerastes cornutus, Echis carinatus,Cerastes vipera, Echis coloratus and Naja nigricollis . The snakes were allowed to injectvenom directly through a nylon diaphragm into an ice cold container. The venom wasimmediately dried in a desiccator in vacuo at 5° . For assay, the venom was dissolved inphysiological saline immediately before use. In some experiments fresh liquid venom wasused .

The Egyptian scorpion, Buthus quinquestriatus, venom was obtained by stimulating thegland with an induction shock.

Enzyme activitiesProteinase . The determination of proteinase activity was done according to ANSON

(1938), using urea denatured hemoglobin at pH7~4as a substrate, and tyrosine as a standard.The enzyme activity was measured photometrically at 660 m~ and expressed as ~M oftyrosine produced . 0~5 ml of a 0~2 per cent solution of sodium versengte was included inthe reaction system as an inhibitor for proteinase activity . (The heat stability of the enzymewas studied after boiling the venom for 3 min in neutral, alkaline and acid media. Also,incubation of the venom at 50° for 1 hr was tried.)

"This work was supported bya grant fromtheNational Institutes ofHealth, No . 322506, Hethesda, Maryland,U.S.A .

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A. H. MOHAMED, A. KAMEL and M. H. AYOBE

Cholinesterase . Activity was measured after the method of DE LA HUERGA et al. (1952),which involves the determination of choline ester by a hydroxymic reaction. The activitywas expressed in wM of acetyl at 540 mP. . The heat stability of the enzyme was studied, andthe effect ofeserine on the enzyme activity was investigated using 2 mg per reaction system .

Transaminases . The colorimetric method Of REITMAN and FRANKEL (1957) was used.Glutamic oxalacetic transaminase was determined using a substrate containing 200 mMnL-aspartic acid at pH 7~4, and incubated at 3T for 60 min . Glutamic pyruvic transaminasewas determined using a substrate containing 200 mM DL-alanine, pH 7~4 and incubated at37° for 30 min . The results of the enzyme activity were expressed in ~M of pyruvate permg venom. Heat stability of the enzyme was also examined .

Amylase . Activity was determined after KING (1964), using buffered starch, pH 7, as asubstrate, and measured at 650 m~.

RESULTSProteinase

This activity of the different venoms is shown in Table 1 . All venoms contained pro-teinase activity, which was highest in C. cornutus and E. coloratus venoms . Inhibition of theproteinase activity of N. nigricollis venom resulted after boiling for 3 min in neutral, acidicor alkaline media. Incubation of the venom at 56° for 1 hr resulted in a 65 per cent inhibitionof the enzyme activity, while EDTA was almost completely inhibitory (Table 2) .

TABLE 2. HEAT STABI[.IlY OF THE ENZYMES AND EFFECT OF EDTA AND ESERINE ON SOME ENZYME ACTIVICIFS

O=No activity ; -=not tested .

Venom used(1 mg)

Enzyme Control Boilingneutralmedium

Boilingacidic

medium

Boilingalkalinemedium

Incuba-tionat 50°

EDTA Eserine

Na1a Proteinase pMnigricollis tyrosine 0250 0 0 0 0130 0038Naja hgje Cholinesterase pM

acetylcholine 925 0 0 0 9~8 - 783Waltertnessia GPT ~Maegyptia pyruvate 0346 0 0 0 0340 - -

TABLE 1 . ENZYME CONTENT

Venom(1 mg)

OF THE DIFFERENTVENOMS

Proteinase(p.M

tyrosine)

EGYPTIAN SNAKE VENOM

Cholinesterase(pM

acetyle choline)

AND SCORPION

GPT(pM

pyruvate)

Naja ltgje 0261 925 0067Naja nigricollis 0246 0 0Walterlnessia aegyptia 0198 619 0346Echis carinatus 0244 0 0027Echis coloratus 0306 0 0319Cerastes cornutus 0468 0 0185Cerastes vtpera 0191 0 0310Buthus quinquestriatus 0146 0 0

O=No enzyme activity .

Egyptian Snake and Scorpion Venoms

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CholinesteraseActivity was found in N. haje and W. aegyptia venoms . Its activity was lost afterboiling

for 3 min in neutral, acidic or alkaline media . The incubation of the venom at 50° for 1 hrinhibited 85 per cent of the enzyme activity, while more than 90 per cent was destroyed bythe addition of eserine (Table 2).

TransaminasesNo glutamic oxalacetic transaminase activity was recorded . Glutamic pyruvic trans-

aminase activity was found in all venoms except that ofN. nigricollis and scorpion venom .The activity was highest in W. aegyptia and E. coloratus venoms. The enzyme activity in W.aegyptia venom was inhibited by boiling, but the activity was not affected by incubating thevenom for 1 hr at 56° .

AmylaseNo amylase activity could be detected .

Fresh venomThe experiments carried out on dried venoms in which no enzyme activity could be

recorded were repeated using fresh liquid venoms . The findings were also negative.

DISCUSSIONThe observed marked proteinase activity of N. haje and N. nigricollis venoms is con-

sistent with the rich trypsin-like action of the venom from Naja naja, as stated by Gxosx(1936), RAO and RAO (1956) . REro (1964 ; 1968) has reported the clinical necrotizing pro-perties ofAsian cobra venom. However, Elapidae venoms possess very little or no proteinaseactivity (ZELLIIt, 1951 ; BoQuET, 1966 ; Tu et al., 1966 and RussELl., 1967) .

The absence of cholinesterase activity in the venom of N. nigricollis supports the resultsobtained by DE BOER (1956) arid SCHWICK 8IId DICKGIESSER (1963), who reported barelydetectable activity in 2 N. nigricollis and Dendroaspis polylepis venoms and excluded faultydrying techniques as a cause ; despite the rule by Gxosx et al. (1939), ZEI.><.Ex (1949),MELDRUM (1965), RUSSELL (1967) and C~R1sTENSEx (1968) that elapid venoms arecharacteristically rich in cholinesterase activity.

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Venoms, Vol . I, p . 437, (BUCHERL, W., Bucxr.Er, E. and DEULOFEU, V., Eds.). New York : Academic Press .DE BOER, J. (1956) Toxicity of some South African snake venoms . Their relation to the acetylcholinesterase

content . Med. Proe. 2, 545.DE LA HUERGA, J., YESIMCK, C. änd POPPER, H. (1952) Colorimetric method for determination of serum

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ofdifferent pH . J. lndfan drem. Soc . 13, 451 .Gxosx, B. N., DuT-r, P. and CY~owDxuRY, D. K. (1939) Enzymes in snake venoms-V . Detection of

dipeptidase, polypeptidase, carboxypolypeptidase and esterase in different snake venoms . J. Indtan them .Soc. 16, 75 .

KnvG,E. (1964) Amylases. In : Microanalysis in Medical Biochemtstry, 4th ed., p. 106. London : Churchill .MELDRUM, B. S. (1965) The actions ofsnakevenoms on nerve and muscle .The pharmacology ofphospholipaseAand of polypeptide toxins . Pharmac . Rev . 17, 393.

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Rno, S. S. and Rno, S. S. (1956) Proteases of cobra (Naja raja) venoms . In : Venoms, p. 467, (BUCKLEY, E. E.and PORGE4, N., Eds.) . Washington : American Association for the Advancement ofScience .

REro, H. A. (1964) Cobra bites. Br . Med. J. 2, 540.Rrm, H. A. (1968) Snake bite in the tropics. Br. Med. J. 3, 359.RErrMarr, S. and FRANKEL, S. (1957) Colorimetric methods for estimation of transaminases. Am . J. clan.

Path . 28, 56.RUS4ELL, F. E. (1967) Pharmacology ofanimal venoms . Clin . Pharnrac . Ther. S, 849.Scxwicx, G. and IiICxGIES4ER, F. (1963) Probleme der Antigen-und Ferment-analyse in Zusammenhangmit der Herstellung polyvalenter Schlangengiftseren . In : Die Cij1 schlangen der Erde, p. 35 . Behring werke-Mitteilungen Marburg.

Tv, A. T., Passet,R. B. and Tu,T. (1966) Proteolytic enzyme activities of snake venoms . Toxicon 4, 59 .Zett.eR, E. A. (1949) Uber die Cholinesterase der Schlangen gifte . Helv . Chim . Acta 32, 94.Zer.~R, E. A. (1951) Enzymes as essential components of bacterial and animal toxins . In : 7ïee Enrymes,Vol. 1, part 2, p. 986. NewYork : Academic Press.