Toxiron. V°L 2(1. No . 2, pp. 509-512. 1982Printed m drcat Britdn.

0041-0101/B]ä020509-04 503.00/0Q 1982 Per~mam Ares Ltd.

ISOLATION OF A HEMORRHAGIC PRINCIPLE FROMBITIS ARIETANS (PUFF ADDER) SNAKE VENOM

D. MESS and F. PANHOLZERZentrum der Rechtsmedizin, University of Frankfurt, Frankfurt/Main, Germany

(Acceptedfor publication 3 August 1981)

D. MEBS and F. PANHOLZER. Isolation of a hemorrhagic printxpb from Bitis ~ictans (puff adder)snake venom. Toxicon 211, 509-512, 1982-By gel filtration on Sephacryl 5-200 and columnchromatography on CM-Sephadex C-25 one of the hemorrhagic principbs in the vmom of Bitfisarktans was isolated in a pure state. It was free ofcaseinolytic as well as arginine ester hydrolyzingactivity. Hemorrhagic activity was not ensconced with the lethal properties of the v~om (LUSo1.6 mg,/kg, sa injection), since the pure hemorrhagin possessed relatively low bthality (> 15 mg,/kg).

HEMORRHAGE is of great clinical importance in snakebite envenomations. This primarilylocal effect arotmd the bite involves cutaneous and subcutaneous tissues, but can spread overthe whole a®icted body part. In the case ofsevere complication, bleeding in organs such asintestines, lungs, kidneys and brain may occur. Many viperid and crotalid venoms possesshemorrhagc activity ; in particular, the venom ofthe puffadder, Bitis arietans, exhibits a highhemorrhagc activity when tested in mice or rabbits (OHSAICA et al., 1966 ; HoMMA and Tu,1971). In the present paper the attempts to isolate one of the venom factors producinghemorrhage are describedDesiccated venom from Bitis atzetans waspurchased from D. Muller, Johannesburg, SouthAfrica. The crude venom was fractionated by gel filtration on a Sephacryl S-200 column(130 x 2cm) which had been equilibrated with 0.1 M ammonium acetate, pH 5.8 . Furtherpurification ofthe fractions) producing hemorrhages was performed by chromatography onCM-Sephadex C-25 coltunns (15 x 1 cm) equilibrated with 0.05 M ammonium acetate, pH5.8, and eluted using a linear gradient up to 0.5 M ammonium acetate, pH 6.8 . Fractions of6ml were collected at a flow rate of 30 ml/hr. Protein concentration was determinedaccording to the method ofIAwRY etal. (1951), with bovine serum albumin as standard . Discelectrophoresis was carried out in polyacrylamide gel (7.5%) at pITs 8.5 and 4.3 for 120minat 8 V per tube. Hydrolysis of p-toluene-sulphonyl-L-arginine methyl ester (TAME) wasfollowed spectrophotometrically according to the method of HuMMEL (1959) ; one unit isdefined as the amount of enzyme which hydrolyzes 1 iemok TAME per min. Caseinhydrolysis wasassayed according to the procedure of Kuxrrz (1947). To 0.6 ml of 1~ caseinsolution (in 0.1 M Tris-HCI buffer, pH 8.5) 0.2 ml of an enzyme solution was added andincubated for 20 min at 37°C . The reaction was stopped by addition of 1 .2 ml of 5%trichloroacetic acid . The mixture was centrifuged and the absorbency of the supernatantmeasured at 280nm. One unit ofenzyme activity is defined as the amount ofenzyme whichcauses an increase ofabsorbency of0.001 per20min. In a semi~uantitative test hemorrhagic

sog

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activity was assayed by s.c . injection of varying doses into the back skin of mice, which werethen killed after 6 hr by chloroform inhalation . In a modification ofthe procedure describedby BIARNASON andTu (1978 ) thehighest amount ofvenom or itsfraction which did notcauseany hemorrhagc reaction was determined and called a unit. A defined or reproduciblehemorrhaggc spot was rarely observed, mostly there occurred diffuse bleeding on the surface ofthe back skin and muscles. The Lnso was determined by s.c. injection into mice, using fouranimals at each dose .By gel filtration on a Sephacryl 5-200 column the venom of Bitis arietans was separated

into five protein containing fractions (Fig. 1) . Three of them showed hemorrhagc as well ascasein and arginine ester hydrolyzing activity . Fraction II, being very active in this respect,was further resolved into three subfractions by chromatography on a CM-Sephadex C-25column . Whereas the first eluted peak possessed caseinolytic andTAME-hydrolytic as wellas hemorrhaggc activity, the following two peaks were entirely free ofthe first two activities, butwere still very active in producing hemorrhage. Fraction Ha-II-3 washomogeneous by discelectrophoresis. Although only two purification steps (even at 4°C) had been applied, therecovery of hemorrhagic activity was low (<20'/ after gel filtration and <8% after ion-exchange chromatography). An even lower yield (below 2%)wasobserved when fractions IIIand l:V were separated on CM-Sephadex C-25 . This might be partially due to the venom'shigh proteolytic, i.e ., caseinolytic, activity, which may be able to degrade other venomcomponents. The relatively high portion of insoluble material (20-30'x) observed when thecrude venom is dissolved in various buffer systems (0.1 M Tris-HCl buffer, pH 8.0 ; 0.1 M

OS

OAS M ammonium ocetat . pH 591QS~PH 6.8

hemorrhoyie octivity

10 20, 30 40 50 60 7 0 80r

-B°-jt-1

Bo-II-2

Bo-lI-3

t eoo

FIG. 1 . GEL F7LTRATION (ABOVE) OF 4OO m$ Blti3 (friCtalIS VENOM ON A SEPHACRYL S-ZiiO OOLUMN

(130 x l.3cm) ELtrrEn wtrtt 0.1 M AMMOrnuM ACErATE, pH 3 .8. Tr~ coemtrtm mtACnotvs ot=1~

sECOxn nEAtc (Ba-II) wEttE Art~ (natTOM) oNro A CM-SErItAnEtc C-23 coLUMN (13 x 1 ~)"Elution was performed usinga linear gadient of0.03M ammonium acetate, pH 3 .8, to0.3 M,pH 6.8 .

Cos .in-hydrdysis

l~ml i

7000

GO 800

101200

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511

TABLE 1 . PURIFICATION OF A HEMORRFIAGIC FACTOR FROM Bitis üriCtQltS VENOM

Data from a typical experiment are shown ; other experiments varied only slightly.One unit of hemorrhagic activity is defined as the highest amount of venom which does not producehemorrage .One unit of caseinolytic activity is defined as the amount of venom which causes an increase ofabsorbency of 0.001 per 20min at 280nm .

ammonium acetate, pH 7.0) may be the result of autodigestive processes. F. KORNALIK(personal communication) observed that the venom rapidly becomes turbid and cloudy aftermilking.

Lethality assay of the kactions from gel filtration indicated that fraction II, whichexhibited the highest hemorrhaggc activity, was also the most lethal, whereas fraction IV,which had low hemorrhagic, but high caseinolytic activities, possessed very low lethalproperties . However, when the subfractions ofBA-II were tested the pure hemorrhagin (Ba-II-3) was the least toxic (Table 1~ Although hemorrhaic principles alone seem to play aminor role in the venom's lethal action, they may contribute considerably to the clinicalpicture ofenvenomation in combination with other factors of still-unknown pharmacologi-cal properties.

Whether there is only one hemorrhagic factor, existing in several molecular forms,modified by the venom's protease, or whether various factors exist in the venom of BitisatYietans cannot be concluded from the present study. However, there is clear evidence that atleast the factor which has been isolated in a pure state is not identical with a proteasehydrolyzing casein. In some snake venoms hemorrhagic principles are associated withproteolytic activity, as in Agkistrodon halys blomlto~fti proteinase b (Os1nMn et al., 1971),hemorrhagic factor HFZ from Bothropsjararaca venom (MANDELHAUM et al ., 1976) and so-called hemorrhagc toxins from Crotalus atrox venom (BJA.RNASON and Tu, 1978). On theother hand, hemorrhagc factors entirely free of proteinase activity were isolated fromAgkistrodon halys blomho,~i (HR-I, OSHIMA et a1.,1972), Trimerestrrttsjlavotriridis(HR 2a andb, TAICAHASHI and OHSAKA, 1970) and Vipera palaestinae venoms (OVADIA, 1978). Thathemorrhage is at least partially an enzymatic process has been demonstrated by OHSAICA etal., (1973) on siolated basement membranes, which are disrupted with liberation of splitproducts in the presence ofproteinase-containing as well as ofproteinase-free hemorrhagins.

HemorrhagicUnits

x 10s per mg

activity

Relative

CaseinolyticUniteper mg

activity

RelativeLDSo

mg/Icg a.c

Crude venom 2.5 1 .0 723 1 .0 1 .6Sephacryl S-200gel filtrationfraction

Ba-II 2 .9 1 .16 1108 1 .53 1 .0Ba-III 0.5 0.20 475 0.66 2 .5Ba-IV 0.6 Q24 2730 3.78 > 10

CM-Sephadex C-25chromatographyfraction

Ba-II-1 12 .5 5 .08 138 0.19 0.6Ba-II-2 3 .7 1 .48 0 - 1 .2Ba-II-3 7.5 3 .00 0 - > 15

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The present study aimed to isolate hemonhagins which could be used for immunization,thus improving the neutralizing capacity of antisera against hemorrhagic activity. However,the problem in obtaining these factors in a reasonable yield and the fact that the crudevenom's lethality does not oonespond to hemorrhagic activity prevents practical applicationof this aim.

REFERENCESBIARNASON, J. B. and Tu, A. T. (1978) Hemorrhagic toxins from western diamond rattksaake (Crotales atrox)venom :isolation and characterization of five toxins and the rok oftint in hemorrhagic toxin e. Biochemistry 17,3395.

Ho~a,, M.andTu, A.T. (1971) Morphology of local tissue damage in experimental snakeenvenomation . Br.J. rxp.Pathol. 52, 538.

HutYa~t., B. C. R+. (1959) A modified spectrophotometric determination of chymotrypsin, trypsin and thrombin .Can. J. Biochcm. Physiol. 37, 1393 .

Kuxrrz, M. (1947) Crystalline soybean trypsin inhibitor. J. gen. Physiol. 30, 291.Lowev, O. H., Rost:attovotr, N. J., Fenty A. L. and R~rro~t.t., R. J . (1951) Protein meaauretnwt with the Folin

phenol reagent. J. bioi. Chum. 193, 265.MNVOta,a~unt, F. R., Ratct~t, A. P. and Ass~tcutu, M. T. (1976) Some physical and biochemical characteristics ofHF=, one ofthe hemorrhagic factors in the venom ofBothrops Jaraca. In : Aninuri, Plant and Miuobial Toxins,Vol. I, p. 111 (Ot~tst,~, A., Hw~sw, K. and SAWAI, Y., Eda). New York : Pkaum Press.

Ottswtc~, A., OtNOw-Same,T., KotmqH, Kotano, S. and Mutt~rw, R. (1966) Bioc!>anical and pathological aspectsof hemorrhagic principks in snake veaoms with special reference to baba (Trimnesunu flaoooiridis) v~om.Meets. Inst. Butantan 33, 193.

OHSAKA, A., Jusr, M. and H~tteaeutvw, E. (1973) Action of evoke venom hemorrhagic principks on isolatedglomerular basetnmt membrane. Btochim biophys. Acts 323, 415.

Osetsu, G, IWANAGA, S. and Suzutct, T. (1971) Someproperties of proteinsee b in the venomofAgkistrodon halysblomhoffit Biochim. biophys. Acts 230, 416.

Ostttuu, G, OMORFSArOH, T, IWANAGA, S. and Suzutu, T. (1972) Studies on snake venom hemorrhagic factor I(HR-I)in the venom ofAgkistrodm halysblomhoffü. Itspurification and biological properties. J. Biochtm., Tokyo72, 1483 .

Ovxnu, M. (1978) Isolatie~n and charactervation of three hemorthagic factors from the venom of Yiperapalatstinae. Toxicon 16, 479.

Txtutt~set, T. and Ottswtu, A. (1970) Purification and some properties oftwo hemorrhagic principles (HR 2a and(HR-I) in thev~om ofAgkistrodonhaiys blomiwffü.Its purification and biological properties. J. Biochem., Tokyo72, 1483.