Characterization of the biological and immunological properties of fractions of prairie rattlesnake (Crotalus viridis viridis) venom

oal-olova7 s3 .ao+ .oo ® 1987 Peraamon lournah l.td.

rosfrnn, voi . zs . Nn . Iz, pp . 1329-134z, 19x7. Printed io Great Britain .

CHARACTERIZATION OF THE BIOLOGICAL AND IMMUNOLOGICAL PROPERTIES OF FRACTIONS OF PRAIRIE RATTLESNAKE (CROTALUS VIRIDIS YIRIDIS) VENOM R.

CHARLOTTE L. OWNBY and TERRY COLBERG Departmrnt of Physiological Sciences, Oklahoma State University, Stillwater, Oklahoma 74078, U .S .A .

(Acceptedfor publication 22 July 1987) C . L . Owrlsx and T . R . Cot .BexG . Characterization of the biological and immunological properties of fractions of prairie rattlesnake (Crotales viridis viridis) venom. Toxieon 25, 1329-1342, 1987 . - Prairie rattlesnake (Crotales viridis viridLs) venom was separated using liquid column chromatography . The fractions were leafed for biological activity in mice and for irnmunological reactivity against polyvalent (Crotalidae) antivrnom and a monovalrnt antivrnom to the crude vrnom . Several of the basic fractions and most of the non-basic fractions had hemorrhagic activity. Six of eight basic fractions had direct myotoxic activity, two of the basic fractions produced edema 30 min after injection, and two were lethal . Polyvalrnt antivrnom contained few antibodies to the fractions of this vrnom, reacting with only two of the basic fractions . Monovalent antivenom formed mulitiple precipitin bands with almost all of the fractions . These results clearly demonstrate that most of the vrnom components are antigenic and immunogenic. Immunodiffusion using the monovalent antivrnom demonstrated that C. v. viridis venom contains many antigens common to 10 other crotaline vrnoms . One of the htmorrhagic components was presrnt in five of the other vrnoms tested, one of the direct myotoxic rnmponrnts was presrnt in three other venoms, and one of the lethal componrnts was common to two other vrnoms . Another highly active hemorrhagic componrnt was common to all of the vrnoms tested except that of Trirnereseres Jlovoviridis . INTRODUCTION

venoms are known to be complex mixtures, and rattlesnake venoms are especially complex (TU, 1982), consisting of numerous components having various chemical characteristics. These components possess a wide variety of biological activities, having systemic (HAWGOOD, 1982) as well as local (OWNBY, 1982) effects . Both the chemical characteristics and the biological activities of rattlesnake venoms have been studied extensively. The immunological properties of rattlesnake venoms have also been investigated . Flexner and Noguchi as early as 1904 produced antisera to rattlesnake venom and detected precipitin reactions with homologous venoms. Since then, most of the immunological studies of venoms have been directed at the analysis of the neutralizing ability of antivenoms, usually of commercial origin . The investigations of CRILEY (1956) and of GINGRICH and HOHENADEL (1956) are examples in which the polyvalent (Crotalidae) antivenom produced by Wyeth Laboratories Inc., Marietta, PA, was studied. There have been relatively few studies of the antigenic and immunogenic properties of rattlesnake venom components . MINTON (1957) used the Ouchterlony technique of immunodiffusion to study the reaction of 14 rattlesnake venoms against the polyvalent (Crotalidae) antivenom and against monovalent antivenom prepared against seven of the ALL SNAKE

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1330

C. L. OWNBY and T. R. COLBERG

venoms. He found that the venoms contained at least four to seven antigenic fractions, three being shared among the venoms . More recently MINTON et al. (1984) demonstrated, using an ELISA, extensive cross-reaction among the venoms of diffferent species of rattlesnakes and copperheads. Such investigations have allowed the enumeration of antigenic components of venoms and the collection of data on the existence of common antigens, but they have not led to the correlation of biological activities with antigenicity or immunogenicity of venom proteins . It is important to determine the biological activities of snake venom antigens, especially the antigens shared among a large number of species. If these antigens are biologically active, e.g. lethal, hemorrhagic, myotoxic, etc., and thus contribute markedly to the overall activity of the venom, it is important to assure that the antivenom used for treatment contains neutralizing antibodies to them. The purpose of the present investigation was to determine the relationship between biological activity and antigenicity of the components of one venom, that of Crotalus viridis viridis. Our approach was to separate the venom into fractions, determine the biological activity of each fraction, and determine the antigenicity of each fraction using two different antivenoms . Then, using the antigen-antibody reaction in immunodiffusion, to determine the presence of these antigens in 10 different crotaline venoms. MATERIALS AND METHODS Venons

C. v, viridis venom (Lot no . CV 184BHT, Montana) was purchased from Biotoxoins Inc., St . Cloud, Florida, U.S.A. Other venoms were purchased either from Biotoxina Inc. or from Miami Serpentarium Inc., Salt Lake City, Utah, U.S .A. Venom samples were either dissolved in buffer immediately before use if being ftacdonated or stored at 0°C until used in the immunadiffusion studies. Other materials

Ion exchange celluloses were purchased from Whatman lnc., Clifton, NJ ; gel filtration medium was purchased from LKB Inc., Uppsala, Sweden ; and chromatofocusing gel was purchased from Pharmacia Fine Chemicals Inc., Piscataway, NJ . Elatrophoresis reagents were purchased from Hio-Rad Laboratories, Richman, CA . All other reagents were of analytical quality. Isbladon scheme

The fractionationprocedure was designed to fast separate the basic venom components from acidic or neutral components . This was done by using anion exchange chromatography so that the components having a net positive charge at a pH of 8.6 did not bind to a DEAE Cellulose column . At each step the fractions obtained were tested for biological activity in mice, and fractions having hemorrhagic, myotoxic or lethal activity were further separated. Active fractions were then further separated on the basis of molecular weight using Ultrogel AcA 54 . Fractions from the gel filtration column which were active were further separated using cation exchange chromatography with Whatman CM 52. If additional fractionation was desired, then either gel filtration, chromatofocusing or HPLC was used . All fractionationa were done is a cold room at 4°C . Betwxn steps, fractions were coacenuated in the cold using an Amicon Ultrafiltration cell anda YM-2 membrane . This system was also used to exchange buffers when necessary . The absorbante of the filtrate was monitored at 280 nm to determine whether protein was being lost during ultrafiltration Anion exchange

The DEAF Cellulose column (2.6 x 30 cm) was equilibrated with 0.01 M Tris, pH 8.6 . Two grams of C. v. viridis venom were dissolved in the same buffer and the solution centrifuged for 30 min m 40,000 x a, 4°C to remove particulate material . The supernatant was concentrated to about 10 ml, then applied to the column which was eluted with the same buffer at a flow rate of 20 ml/hr until the absorbante at 280 nm returned to baseline . Then an NaCI gradient (0-0 .5 M) in the 0.01 M Tris buffer, pH 8.6 wen used to elute the remaining neutral or acidic proteins . The column was washed with 0.5 M NaCI, but no additional protein was eluted .

Characterization of Rattlesnake Venom Fractions

1331

Gelfiltrntion Fractions from the DEAE column were father fractionated using molecular exclusion chromatography on Ultrogel AcA 54 . The column (3 .0 x 85 cm) was equilibrated with 0.01 M MOPS buffer, pH 7 .2 . Fractions were first exchanged into the MOPS buffer using ultrafiltration, and the concentrate was applied to the column . The column was eluted at a flow rate of 15 ml/hr with the 0.01 M MOPS buffer. Cation exchange Fractions from the AcA 54 column were further fractionated with a Whatman CM 52 column (2.0 x 25 cm) which had been equilibrated with 0.01 M MOPS, pH 7.2 . These fractions were concentrated by ultrafiltration, and sins they were already in the 0.01 M MOPS buffer were directly applied to the cation exchange column . The column was eluted with a linear gradient from 0 to 0.8 M NaCI. In some instances a step gradient of 1 .0 M NaCI was used at the end of the linear gradient to remove any additional protein from the column . ChromatoJocusing One of the fractions from the cation exchange column was further separated using chromatofowsing for separation of proteins based on differences in their isoelectric points . The column used was a Pharmacia PBE94 (3 .0 x 35 cm) which was eluted with Pharmacia's Polybuffer 74 over the pH range of 8.0-4 .0 or 7 .5-4.0 . HPLC One fraction from the cation exchange column with myotoxic activity appeared to be homognow and was thw not pooled . Each individual tube was tested for homogeneity by HPLC (Waters Associate Inc.) using a KBondapak C column . This column was eluted with a linear gradient of methanol-0 .2% formic acid . The effluent was monitored at both 254 nm and 280 nm. Electrophoresis (SDS-PAGE) Electrophoresis of the fractions was carried out in an SDS-PAGE (slab) system wing the Laemmli discontinuous buffo procedure (L~attitt,t, 1970). Gala containing 10ßb acrylamide were run at 30 mA/gel using a Tris-glycine buffer containing l~Jb SDS. They were allowed to run until the tracking dye or ion front was within 1 cm of the bottom of the gel. The gels were fixed with sulphosalicydic acid, TCA, stained with 0.125ßb Coomaaaie blue, SOßb NaOH, 10~ acetic acid and deatained until clear with 2Sß4 EtOH and Bale acetic acid . The gels were intentionally overloaded (0.1 mg/lane) to maximize our possibility of detecting con~n~+inants . Amino acid analysis Fractions from the HPLC column were collected and analyzed separately for amino acid rnmposition. First the solvent was evaporated under vacuum. This was followed by analysis of the residue after acid hydrolysis for 24 or 48 hr . The analyzer was modified for use with 2.8 mm columns and equipped with an sutolab system AA integrator . NOD -Phe was used as an internal standard . Antivenoms Polyvalent (Crotalidae) antivenin, subsequently called polyvalent antivenom, was purchased from Wyeth Laboratories Inc., Marietta, PA, U.S .A . The lot no . (18301) used for these studies had an expiration date of 5/88. The lyophilized antivenom was reconaituted with sterile distilled H,O as described in the pamphlet and was stored frozen at 0°C until needed for the immunodiffuaion studies. Monovalent antivenom to C. v. vlrldis venom was prepared in rabbits as previowly described by OwNaY et al. (1979) . Briefly, venom was dissolved in physiologic saline (0 .8Sß4 NaCI) to providea dose of 0.25 mg venom/kg rabbit . The venom solution was mixed with an equal volume of Freund's complete adjuvant for the initial igjection and with an equal volume of Freund's incomplete adjuvant for subsequent igjections. Injections were made aubacapularly each week for two weeks, then the rabbits were bled from the marginal ear vein every two weeks. Immr~nodjjJusion Ouchterlony double diffwion in 1 ßb agaroae was used to determiw the praena of antibodies to venom components in both the polyvalent antivenom and the monovalent antivenom, and to determine the amount of cross-reactivity between the fractions and other crude venom:. The procedure has been described previously by Owtvax tt al . (1979). Ten other crude venom: were tested to determine if any of the components of the C. v. viridis fractions were present in these heterologou : venom:. Each C. v. vlrldls fraction was placed in a peripheral well adjaaat to a peripheral well containing one of the heterologow crude venoma, andthe monovalent amivenom was plead In a central well . Precipitin bands were allowed to form, and a reaction of complete identity between a precipitin band against the fraction and s precipitin band against the heternlogous venom indicated a cross-reaction between the heterologow venom and the fraction of C. v. vfrldis venom.

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C. L. OWNBY and T. R. COLBERG

Assayjor hemorrhagic and myotazic activity Fractions were tested for their ability to induce hemorrhage and myonecrosis by intramuscular injection into female white mice (CD-1, Charles River). Mice were injected with a dose of 2 mg/kg of each fraction in avolume equivalent to 0.05 ml/25 g mouse. The injection was made into the dorso-lateral aspect of the right thigh, and tissue was taken from theventro-medial aspect of the same thigh to avoidareas damaged by the needle . The mice were killed by cervical dislocation at either 30 min or 24 hr after the injection. Samples of muscle tissue were taken, processed as previously described (Owtv>iv et al., 1976), and examined with the light microscope for presence of necrotic muscle cells. The amount of hemorrhage present in the thigh at these time periods was estimated by a semi-quantitative method . if gross examination revealed no hemorrhage, the fraction was assigned a negative (-); if there was a small spot of hemorrhage which did not cover the entire thigh, the fraction was assigned one plus (+); if there was moderate hemorrhage wvering part of the thigh, two pluses (+ +) were assigned; if the hemorrhagic area covered the entire thigh, the fraction was assigned three pluses (+ + +) . Alternative assay for hemorrhagic activity For some fractions only hemorrhagic activity was determined . This was done by examining the mice grossly 1-1 .5 hr after the injection (i .m.) of the fraction . The results were recorded using the semi-quantitative method described above. RESULTS Fractionation of crude venom

For clarification, fractions are labelled A - K beginning with A for fractions from the DEAF column and a new letter designation for each subsequent column ; all fractions from the same column have the same letter designation. Figure 1 shows the elution profile from the fractionation of crude venom on the DEAF Cellulose column . This column yielded five fractions, two (Al and A2) eluted before the salt gradient and were wellresolved from the last throe which were not well-resolved from each other. Both of the basic fractions (A1 and A2) had hemorrhagic activity at both 30 min and 24 hr. The results of the biological assays are summarized in Table l . Of these two fractions only A1 caused myonecrosis at 30 min after injection, indicating that there might be a

n~ Nuu~t (~.aq FYa. 1 . FawcnOtv~rtor~ of cause Crotahis vlridit vlridls vtiraM ON DEAF cet.t.uwse. Eluted with 0.01 M Tris, pH 8.6 at a flow rate of 20 ml/hr, NaCI gradient (0-O .S M) in 0.01 M Tris, pH 8.6 started at tube 90, 0.5 M NaCI in 0.01 M Tris, pH 8.6 added at tube 160.

1333

Characterization of Rattlesnake Venom Fractions T AAi C

1.

BIOLOOICAI. ACrIVIiTES OF THt3

DEAE FRwcrtotvs of Crotadrs viridis viridis verroh Myonecrosist

Other

Fraction

Hemorrhage'

30 min

24 hr

Al A2 A3 A4 AS

++ ++ +++ + +

+ -

+ + + + +

30 min

24 hr

E,T*

E,T

E E

E

" Hemorrhage determined by gross obaavation after i,m . injection of fraction into mice: indicatea no visible hemorrhage, + indicates slight hemorrhage covering small part of the thigh, + + indicates moderate hemorrhage covering moat of the thigh, + + + indicates severe hemorrhage covering entire thigh . tMyoneaosia determined by examining sections of muscle with the light microscope: indicatea no damaged muscle cells present, + indicates pr~eaena of damaged muscle cep :. *E = edema : T ~ thrombi in vessels .

direct-acting myotoxin in this fraction . The necrotic muscle cells had the pathologic appearance of cells damaged by phospholipase myotoxins, i.e. delta lesions and clumped myofibrils . Fraction A1 also caused myonecrosis at 24 hr, but the necrotic cells were either vaculolated or had an amorphous hyaline appearance . The other fractions produced myonecrosis at 24 hr but not at 30 min, suggesting that this myotoxicity could be an indirect result of hemorrhagic and ischemic conditions rather than a direct action of a toxin. Thus, three different types of muscle cell damage were observed with the fractions. One was observed 30 min after injection and consisted of cells with delta lesions and clumped myofibrils . The other two types were observed only 24 hr after injection and consisted of cells with large vacuoles or cells with an amorphous hyaline appearance . To investigate the myotoxic activity of the venom, A1 was further fractionated . Fraction A2 was also further fractionated because it contained hemorrhagic activity, edema-forming activity and caused thrombosis at both 30 min and 24 hr. Fractionation of basic components Frnctionation ofDEAEfraction A1. The result of fractionating A1 with gel filtration is shown in Fig. 2H. This step resulted in eight fractions of which three contained hemorrhagic activity (see Table 2), six contained myotoxic activity at 30 min, and svl caused myonecrosis at 24 hr. Fractions BS and B6 did not cause hemorrhage, but induced myonecrosis at 30 min and death by 24 hr after injection. Fractions Bl and BS did not cause hemorrhage, but induced slight myonecrosis, primarily vacuolation, at 24 hr. Fraction B7 did not cause hemorrhage, but caused vacuolation of muscle cells identical to that caused by myotoxin a. This fraction was further separated. Figure 2C shows the elution profile from the fractionation of B7 on the cation exchange column . The material from this column eluted essentially in one fraction (Cl), having myotoxin a-like activity in the light microscopic assay (Table 2). This fraction was not pooled, but the contents of individual tubes were tested by HPLC. The HPLC column resulted in two poorly resolved peaks (not shown) . When the contents of these two peaks were assayed for amino acid composition, they wen shown to be identical to each other and to myotoxin a previously isolated from C. v. viridis venom (OwlvsY et al., 1979). Figure 2D shows the elution profile from the separation of fraction B3 on the cation exchange column . Elution of this column resulted in five fractions. Of these, fractions D1 and D2 were low in protein and produced no hemorrhage . D3, D4 and DS all induced

1334

C. L. OWNBY and T. R. COLHERG .~i

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Characterization of Rattlesnake Venom Fractions

1335

TABLE I . BIOLOGICAL ACTIV171FS OF THE BASIC FRACTIONS DERIVED FROM FRACTION viridis viridis vENGM

Fraction

Hemorrhage"

Bl B2

++

Myonecrosist 30 min 24 hr +

+ + .

A1

30 min

OF

CrotOIus

Other

24 hr

E*

+ + + + L* + L + + + + nd nd nd nd ++ nd nd +++ nd nd + nd nd E +++ nd nd _ _ _ L FS L F6 + + G1 + nd nd . G2 nd nd G3 nd nd G4 nd nd "Hemorrhage determined by gross observation after i.m . injecxion of fraction into mix: indicates no visible hemorrhage, + indicates slight hemorrhage covering small part of the thigh, + + indicates moderate hemorrhage covering most of the thigh, + + + indicates severe hemorrhage covering the entire thigh. tMyonecrosis determined by examining sections of muscle with the fight microscope: indicatea no damaged muscle cells presort, + indicates presence of damaged muscle cells. *E = edema; C ~ congested blood vessels ; L = lethal, all mice igjected with fraction died; nd ~ not determined. B4 BS H6 B7 B8 C1 D1 D2 D3 D4 DS E1

hemorrhage, with D4 having the greatest activity . DS induced a slight amount of hemorrhage, but it also caused some edema. These results are summarized in Table 2. SDS-PAGE showed that fraction D4 contained one major protein of high molecular weight p 40,000) and more than 10 minor components of lower molecular weights . Figure 2E shows the elution profile of the fractionation of fraction D4 with gel filtration . This step resulted in one major peak (E1) containing high hemorrhagic activity (Table 2). The major component appeared to be a basic, high molecular weight protein. Figure 2F is the elution profile from the cation exchange separation of fraction BS. This step resulted in seven fractions of which only three contained enough protein for testing biological activity (Table 2). Fraction F3 did not cause hemorrhage or myonecrosis but had an i.m . LDP of about 0.05 mg/kg. Fraction FS also did not cause hemorrhage or myonecrosis but had an i.m . LDP of about 0.09 mg/kg. Death from these fractions was very rapid, occurring within 10 min after their injection, and could have been due to respiratory paralysis. Fraction F6 did not cause hemorrhage, but did induce myonecrosis, and had an LDP of greater than 1 .64 mg/kg. SDS-PAGE showed fractions FS and F6 to contain one major proton and one or two minor contaminants . Fractionation ofDEAEfraction A2. Figure 3 is the elution profile of the separation of DEAE fraction A2 on the Ultrogel AcAS4 column which resulted in four fractions (G1-G4) . Only fraction G1 caused hemorrhage (Table 2), and since the amount of

1336

C. L. OWNBY and T. R. COLBERG

FIG. 3. FRACTIONATION OF DEAF CELLULOSE FRACTION A2 (FIG . 1) WITH ULTROGEL ACA 54. Eluted with 0.01 M MOPS, pH 7 .2, flow rate of 1S ml/hr.

protein in these fractions was very low, they were not assayed for myonecrosis. Electrophoresis indicated that G1 contained one major component and one minor component, both in the high molecular weight range (80,000-100,000) . Fractionation of non-basic components Fractionation of DEAF fraction A3. Fraction A3 contained the highest hemorrhagic activity of all of the DEAF fractions. Separation of this fraction by gel filtration resulted in four fractions as shown in Fig. 4H. The first three fractions were hemorrhagic (Table 3), but fraction H3 had the highest hemorrhagic activity and was thus further fractionated . Figure 4I is the elution profile from the separation of fraction H3 on the cation exchange column . This step resulted in one very large peak (I1) and one much smaller peak (I2) . Fraction I1 had very high hemorrhagic activity (Table 3) and SDS-PAGE showed that it contained two major components close together in molecular weight and several minor components of both higher and lower molecular weight . Since fraction I1 had such high hemorrhagic activity, it was further separated using chromatofocusing . Figure 4J is the elution profile from the separation of fraction I1 on the Pharmacia PBE94 column after elution with a pH range of 8 - 4. This fractionation resulted in five pabrly resolved peaks (Jl -JS). Biological testing of these fractions showed that fraction J2 had the highest hemorrhagic activity (Table 3). Thus this fraction was further separated using chromatofocusing over a different pH range. Figure 4K shows the result of separation of fraction J2 with chromatofocusing over the pH range of 7 .5 - 4 .0. Elution under these conditions resulted in one peak, K1 . This fraction had high hemorrhagic activity (Table 3) and upon SDS-PAGE was shown to contain two major bands close together at about 36,000 and 40,000 mol. wt . The fraction also contained six to seven minor proteins of lower molecular weight . The electrophoretic pattern of this fraction by SDS-PAGE was not very different from that of fraction I1 from the anion exchange column ; only three proteins of higher molecular weights were removed in two fractionation steps.

Characterization of Rattlesnake Venom Fractions Y

1337

I,

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Y. w.

Y"

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Y"

tun

rra (u.+/,u~

w

w

w

w

w

w

tIR "w0 (L~wl/Tln)

7r - w-,w

FIG . 4. FRACTIONATION OF NON-BASIC COMPONENTS FROM DEAF FRACTION A3 (FIG . 1). Elution profiles for : (H) separation of fraction A3 with Ultrogel AcA 54 ; (I) separation of fraction H3 with Whatmaa CM52 ; (J) separation of Fraction I1 with Pharmacia PBE94 chromatofocusing medium, pH range 8 .0-4 .0; (K) separation of fraction J2 with Pharmacia PBE94 chromatofocusing medium, pH range 7 .3-4 .0 . Elution conditions are described under Materials and Methods .

TABLE 3 . HEMORRHAOIC ACTIVITY OF THE NON-BASIC FRACTIONS DERIVED FROM FRACTION A3 OF CrorQhrs YlIldLS VllldlS VENOM

Fraction H1 H2 H3 H4 II J1 J2 J3 J4 KI

Hemorrhaggc activity' ++ ++ +++ +++ +++ +++

'Hemorrhage determined by observation after i .m. injection of fraction into mice: - indintea no hemorrhage visible, + indiates alight hemorrhage covering small part of the thigh, + +indicates moderate hemorrhage covering most of the thigh, + + + indicates severe hemorrhage covering the entire thigh .

C . L . OWNBY and

133 8 TABLE

4.

T.

R : COLBERG

RESULTS OF IMMUNODIFFUSION ASSAYS OF FRACTIONS OF Crotahcs viridis viridis vENOM

Fraction" Vcnom AI A2 A3 A4 AS B2 B3 B4 BS B6 B7 C1 Dl D3 F3 F4 FS F6* F7~ Kl';

Polyvalrnt antivrnomt

Monovalrnt antiveaomt

2 1 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0

5 6 6 4 5 3 4 3 6 2 2 1 1 3 1 1 1 1 0 0 2

" Concentration was 1 .0 mg/ml unless otherwise indicated . tTiumbers indicate the number of precipitin bends obtained . *Concentration was 0 .25 mg/ml . 'Concentration was 0 .07 mg/ml . "Concentration wea 0 .34 mg/ml .

Immunodiffusion Table 4 shows the results of immunodiffusion tests between the fractions described above and two antivenoms. When reacted against the polyvalent antivenom, few precipitin bands were formed . In fact the only fractions which appeared to react with this antivenom were fractions A1 and its component fractions B2 and B3. In contrast to these results, the monovalent antivenom appeared to contain many antibodies to the venom fractions. All but two of the fractions elicited precipitin reactions with the monovalent antivenom, and most of the fractions resulted in two or more bands, indicating the TABLE S . RESULTS OF IMMUNODIFFUSiON ASSAYS TO DETERMINE THE CROSS-REACTIVITY BErweEN FRwcnoNS of Crotalus viridis viridis vENOM AND TEN OTHER cROrALINE vENOMs

Crotaline venom' C. atrox C. v . bdleri C. adamanteus C. v. rnberus C. rober C. d. tenj/"uus C. h . horridus S. m. barbouri B. atrox T. Jlavoviridis

Cross-reacting fractions

B2,B3,B4,DI,D3,H1,H2,H3,K1 B2,B3,B4,D1,D3,H1,H2,H3,K1 B3,H4,B7,C1,D1,D3,H1,H2,H3,K1 B3,B4,B6,B7,C1,D1,D3,H1,H2,H3,Kl B2,B3,B4,F3,HI,H2,H3,K1 B6,DI,D3,H1,H2,K1 B2,B3,B4,F3,H1,H2,H3,K1 B3,B4,B6,H1,H2,H3,K1 B2,B3,B4,D1,D3,H1,H2,K1 B3,B4,DI,D3,H1,H2

" C. = Croralus; S . = Sistrurus; B . = Botkropa; T. = 7ilnrerrsurus; d . = durissus; h . = horridus; rn . ~ miliarus .

Charactaiution of Rattlanake Veaom Fractiona

1339

complexity of the fractions as well as the ability of these components to be immunogenic and antigenic. Fraction C1, which has myotoxic activity, and fractions F3 and F5, which are lethal, do not react with the polyvalent antivenom, but form one precipitin band against the monovalent antivenom. Fractions Kl and B4, which are hemorrhagc, reacted with the monovalent antivenom forming six and two precipitin bands, respectively, but failed to react with the polyvalent antivenom. Friction K1 had strong hemorrhagc activity (sce Table 3). Table S shows the results of immunodiffusion tests which were used to determine the amount of cross-reactivity between these venom fractions and other snake venoms. This was done by reacting the fractions from C. v. viridis venom and 10 heterologous crude venoms (1 mg/ml) against the monovalent antivenom prepared to C. v. -viridis venom. All of the heterologous venoms formed at least one precipitin band with this antivenom. Fraction B2 which causes hemorrhage at 30 min, slight myonecrosis at 30 min and definite myonecrosis at 24 hr appeared to be present in five other venoms. Fraction B6 which does not cause hemorrhage, but does cause myonecrosis in 30 min appeared to be present in two other venoms. Fraction F3 which is one of the lethal fractions appeared to be present in two other venoms. Fraction H3 cross-reacted with seven of the venoms and one of its components, fraction K1 reacted with all of the venoms except that of T. jlavoviridis . DISCUSSION

These results show that C. v. viridis venom contains numerous hemorrhagc and nyotoxic components . The venom also contains components which induce edema, thrombosis, and congestion of blood vessels as well as death of mice. The hemorrhagc activity of the venom resides in both basic and non-basic fractions. Similar results were reported by BJARNASON and Tu (1978) who isolated five hemorrhaagc toxins from the venom of Crotalus atrox. They found that four of the toxins were acidic and one was basic. NIICAI et al. (1984, 1985) subsequently isolated two more hemorrhaggc toxins from C. atrox venom, both having neutral isoelectric points . Thus it appears that both C. v. viridis and C. atrox venoms contain numerous hemorrhaagc toxins having basic, acidic and neutral isoelectric points . Our results are different from those of KuxECxl and KRFSS (1985) who reported the presence of only one hemorrhaic toxin in C. adamanteus venom. Some venoms may be more complex than others in their content of hemorrhagc toxins ; both C. viridis viridis and C. atrox venoms are complex. The results of this study indicate that the myotoxic activity of C. v. viridis venom consists of at least three types . In the first type, which occurs within 15 - 30 min after injection, the damaged muscle cells contain delta lesions and dense clumps of myofibrils . This type of myonecrosis appears identical to the myonecrosis induced by the phospholipase Ai toxins such as notexin (HARRIS et al., 1975), taipoxin (HARRIS and MALTIN, 1982) and crotoxin (GOPALAKRISHNAKONE et al., 1984). Fractions B2 - B7 and fraction F6 contained this type of activity. In the second type of myonecrosis, which is present only 24 hr after injection, the pathologic state of the muscle cells in identical to that caused by myotoxin a, i.e. vacuolation. Fractions B1, B7 and B8 contained this type of activity . Fraction B7 also caused myonecrosis of the first type at 30 min, but this could be due to the presence of a phospholipase A2-like component since fraction B6 also contained this kind of activity . Myotoxin a was subsequently isolated from B7. However, fraction B1 contained myotoxin a-like activity, but consisted of much higher molecular

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C. L. OWNBY and T. R. COLBERG

weight components . It may be that other toxins can induce the same type of myonecrosis as myotoxin a. The third type of myonecrosis, which was present at 24 hr but not at 30 min after injection, was always associated with the presence of hemorrhage at 30 min. It was characterized by muscle cells which had a homogeneous, hyaline appearance at the light microscopic level. It is probably due to ischemic conditions resulting from severe hemorrhage as suggested by GLEASON et al. (1983), who called these toxins myotoxichemorrhagins . Thus, C. v. viridis venom contains toxins which cause myonecrosis directly (phospholipase-like and myotoxin a) and indirectly (myotoxic-hemorrhagins) . From our studies presented here it appears that all of the direct myotoxic activity of C. v, viridis venom is contained in the most basic fraction, i.e. fraction A1 (see Table 1). This is the first report indicating presence of discrete `lethal' toxins in the venom of C. v. viridis. Although the purpose of this study was not to purify toxins, the results show that two of the relatively pure fractions, F3 and FS, contain lethal components . With i.m. LDP values of O.OS mg/kg and 0.09 mg/kg, respectively, F3 and FS are 20 - 60 times more lethal than the crude venom which has an i.m . LDP of 6.0 mg/kg. Both fractions resulted in death of mice within 10 min of i.m. injection, whereas lower doses resulted in longer survival times. Neither fraction caused hemorrhage or myonecrosis. The lethal fractions of C. v, viridis are not hemorrhagic, whereas those of the timber rattlesnake, C. horridus horridus (SULLIVAN et al., 1979) and of the eastern cottonmouth, A . piscivorespiscivores (MoRnty and GEREN, 1979) possess hemorrhagic activity . Results from the immunodiffusion assay of the various C. v. viridis venom fractions against the polyvalent (Crotalidae) antivenom show that this antivenom contains few precipitating antibodies to C. v. viridis components . Only two bands formed against the crude venom. MINTON (1957) using immunodiffusion reported that most rattlesnake venoms he tested formed four to seven precipitin bands with polyvalent antivenom. The differences between our results and his could be due to the use of different lots of polyvalent antivenom or to tl_e different concentrations of venoms tested, i .e. he used S mg/ml and we used 1 mg/ml. However, it is important that we obtained at least five precipitin bands when the crude venom was reacted against the monovalent antivenom prepared against C. v. viridis venom. This demonstrates that the venom components are indeed immunogenic, and that the titer of precipitating antibodies to them is very low in the polyvalent antivenom. These results could help explain why the polyvalent antivenom may be poor in neutralizing capability for the local tissue damaging activities of some rattlesnake venoms (OWNBY et al., 1983). However, the polyvalent antivenom does contain precipitating antibodies to fraction A1 and two of its subfractions, fractions B2 and B3 (see Table 4). Both of these fractions contain high hemorrhagic activity, and the presence of antibodies in the polyvalent antivenom to these components might explain the results of OWNBY et al. (1984), that the polyvalent antivenom neutralizes some of the hemorrhagic activity of this venom. Our results show that the polyvalent antivenom does not contain detectable amounts of precipitating antibodies to other components, such as many of the ones which cause myonecrosis either directly or indirectly . These results could help explain the lack of neutralization of myonecrosis by the polyvalent antivenom (OwrrsY et al., 1983). No precipitating antibodies were detected to any of the lethal fractions of C. v. viridis venom (BS, B6, F3, FS, see Table 4). This lack of antibodies might suggest that the polyvalent antivenom would have poor neutralizing ability for the lethal effects of C. v. viridis venom. However, OWNBY et al. (1983) reported that polyvalent antivenom neutralized a dose 10 times the LD,~ (i.v.). Thus, it could be that the lethal activity of

Characterization of Rattlesnake Vrnom Fractions

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fractions F3 and FS is only a small part of the total lethal activity of the venom. On the other hand, the polyvalent antivenom could contain non-precipitating antibodies which neutralize these and other toxins responsible for lethality. Caution must be exercised when interpreting immunodiffusion data and extrapolating it to neutralizing capability of antivenom. The presence of neutralizing antibodies can be demonstrated in the absence of precipitating antibodies (COHEN et al., 1971). However, it is clear from our results that the components of C. v. viridis venom are immunogenic, stimulating the production of antibodies at least in the rabbit, and that they are antigenic. Only two of the fractions tested failed to form precipitating bands with the monovalent antivenom, and these were tested at very low concentrations . The results presented here show that C. v. viridis venom contains antigens which produce cross-reacting precipitating antibodies. When the monovalent antivenom was reacted with 10 other venoms, precipitin bands were formed against all of them, even with venoms from two less related snakes, B. atrox and T. flavoviridis. Although crossreaction between C. v. viridis venom and B. atrox venom has not been previously reported, MINTON et al. (1984) reported cross-reaction between C. atrox venom and the venoms of B. riper and T. flavoviridis using an ELISA. Our immunodiffusion results also indicate that several components are common to all of the venoms tested . Table S shows that two of the basic fractions, B3 and B4, cross-react with all venoms tested except that of C. d. terrificus. Two of the non-basic fractions, H1 and H2, cross-react with all of the venoms tested . Thus, as previously shown (MINTON, 1967; MINTON et al., 1984) crotaline venoms do contain several common components . We have shown that some of these common components are hemorrhagic and myotoxic, and thus it might be possible to use these components as immunogens to produce an antivenom which would neutralize the hemorrhagic and myotoxic activity of a wide variety of crotaline venoms . Acknowledgements - The authors thank Dr BRUCe LESSLEY, Department of Physiological Sciences for technical advice and ED JOHNSON for critically reading the manuscript . Thin study was supported by Public Health Service Grant 3 ROl AI16623-07 from the National Institute of Allergy and Infectious Diseases . C. L. OwNaY is the recipient of a Research Career Developmrnt Award (K04 A100474) from this Institute. REFERENCES BJARNASON, J. B. and Tu, A. T. (1978) Hemorrhagic toxins from western diamondback rattlesnake (Crotales atrox) venom: isolation and characterization of five toxins and the role of zinc in hemorrhagic toxin e. Biochemistry 17, 3393 . COHEN, P., BERxeLEY, W. H. and $ELIOMANN, E. B. (1971) Coral snake vrnoms . In vitro relation of neutralizing and pricipitating antibodies . Am . J. trop. Med. Flyg . 20, 646. CRtLev, 8. R. (1956) Developmrnt of a multivalent antivenin for the family Crotalidae . In : Venoms, p. 373 (BUCKLEY, E. E. and PoaaE,s, N., Eds) . Washington, D.C.: American Association for the Advancernrnt of Science. GttvoRtcH, W. C. and HOHENADEL, J. C. (1956) Standardization of polyvalrnt antivenin. In : Vtnoms, p. 381 (BUQCLEY, E. E. and POROES, N., Eds). Washington, D.C.: American Association for the Advancemrnt of Scirnce. GLEASON, M. L., ODELL, G. V. and OWNaY, C. L. (1983) Isolation and biological activity of viriditoxin and a viriditoxin variant from Crotales viridis viridis vrnoms . J. Taxk. -Toxin Rev. 2, 235. GOPALAKRISFINAKONE, P., DEMPSI'ER, D. W., HAWOOOD, B. J. Snd ELDER, H. Y. (1984) CCllular and mitochondria) changes induced in the structure of marine skeletal muscle by crotoxin, a neurotoxin phoapholipaae A, complex . Toxirnn 22, .85. HARR15, J. H. and MALTtN, C. A. (1982) Myotoxic activity of the crude vrnom and the principle neurotoxin, taipoxin, of the Australian taipan, Oxyuranus srrrtellatrcs. Br. J. Pharmac. 7f, 67 . HARRts, J. B., JOHNSON, M. A. and KARLSSON, E. (1975) Pathological responses of rat skeletal muscle to a single subcutaneous injection of a toxin isolated from the vrnom of the Australian tiger snake, Notechls scvtatws saetates. Clip . exp. PJrarmar. Physiol. 2, 383 .

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Hwwcoon, B . J . (1982) Physiological and pharmacological effects of rattlesnake venoms. In : Rattlesnake Venoms: Their Actions and Treatment, p . 121 (Tu, A . T ., Ed .) . New York : Marvel Dekker . Kutet=,cxt, T . and Kte>;ss, L . F . (1985) Purification and partial characterization of the hemorrhaic factor from the venom of Crotales adamantees (eastern diamondback rattlesnake) . Toxicon 23, 657. LAEMMLI, U . K . (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature 227, 680 . Mtt1r'orv, S . A . (1957) An immunological investigation of rattlesnake venoms by the agar diffusion method . Am . J. trop. Med. Hyg . 6, 1097 . Mtnrrav, S . A ., Wetrtsrtart, S . A. and Wtt .ne, C . E . (1984) An enzyme-linked immunoassay for detection of North American pit viper venoms. Clin. Taxic . 22, 303. Moeww, J . B . and GereErt, C . R . (1979) A comparison of biological and chmûcal properties of three North American (CrotaGdae) snake venoms . Toxicon 17, 237 . Nttw, T ., Motet, N ., KtSHIDA, M ., SuottiAxA, H . and Tu, A . T . (1984) Isolation and biochemical characterization of hemorrhaic toxin j from the venom of Crotales atrox (western diamondback rattlesnake) . Amps Biochem . Biophys . 231, 309 . Ntxwt, T ., Moat, N ., KISHIDA, M ., Tsueot, M . and SUGIHARA, H . (1985) Isolation and characterization of hemorrhagic toxin g from the venom of Crotales atrox (western diamondback rattlesnake). Am . J. trop. Med. Hyg. 34, 1167. OWNBY, C . L . (1982) Pathology of rattlesnake envenomation . In : Rattlesnake Venoms: Their Actions and Treatment, p . 163 (Tu, A . T ., Ed .) . New York : Marvel Dekker . OwtvaY, C . L., CAMERON, D . and Tu, A . T . (1976) Isolation of myotoxic component from rattlesnake venom : electron microscopic analysis of muscle damage . Am . J. Path . 85, 149 . OwrtaY, C . L ., WooDS, W . M . SIId ODELL, G . V . (1979) Antiserum to myotozin from prairie rattlesnake (Crotales viridis viridis) venom . Toxirnn 17, 373 . Owtvav, C . L., OnFJ..t., G . V ., Woons, W . M . and Cot .uenc, T . R. (1983) Ability of antiserum to myotoxin a from prairie rattlesnake (Crotolus viridis virldis) venom to neutralize local myotoxicity and lethal effects of myotoxin a and homologous crude venom . Toxicon 21, 35 . OwtveY, C . L ., COLHERG, T . R . and OUEI.t., G . V . (1984) A new method for quantitating hemorrhage induced by rattlesnake venoms: ability of polyvalent antivmom to neutralize hemorrhagic activity . Taxiton 22, 227 . SULLIVAN, J . A ., FAxa, E . and Gt:AEN, C . R. (1979) Fractionation and partial characterization of toxic components of timber rattlesnake venom. Toxirnn 17, 269 . Tu, A . T . (1982) Chemistry of rattlesnake venoms. In : Rattlesnake Venoms: Their Actions and Treatment, p. 251 (Tu, A . T ., Ed .) . New York : Marvel Dekker .