Fractionation of midget faded rattlesnake (Crotalus viridis concolor) venom: Lethal fractions and enzymatic activities

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Tackna VoL 19, No. 4. pP. 517 527, 198l .

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FRACTIONATION OF MIDGET FADED RATTLESNAKE (CROTALUS VIRIDIS CONCOLOR) VENOM LETHAL FRACTIONS AND ENZYMATIC ACTIVITIES W. R. Poot and A. L. BIEBER Department of Chemistry, Arizona State University, Tempe, AZ 85281, U.S.A . (Acceptedfor publication

29

December

1980)

W. R. Poot and A. L. BIEeEx . Fracdoaation of midget faded rattlesnake (Crotales mridis concolor) venom: lethal fractions and enzymatic activities. Toxicon 19, 517-527, 1981 .-The venom of the midget faded rattlesnake has btxn resolved into tdght fractions using anion-exchange chromatography. Fractions 1, 6and 7 were lethal to mice at protein concentrations equivalent to 3 x Lnso of the crude venom. Fraction 7 (Lnso , 0~12pg/g) contained the major lethal components) of the vtmom. Using disc gel electrophoresis only one major and one minor protein component were present in this fraction. One major and one minor wmponent, both with pI values less than 5, were also observed after isoeltxtric focusing . One prott:in band with a pI > 9 was present in Fraction 1 after isoelectric focusing . Injection of this fraction resulted in paralysis of the hind limbs and respiratory distress immediately after injection. Crude venom and each fraction were leafed for phosphotüesteraae, phoapholipase, L-amino acid oxidase, arginine ester hydrolaxe and phosphomonoesterase activities. Phoaphodiesterase activity was detected only in Fraction 1. Phospholipase áctivities were present in Fractions 6 and 7. L-amino acid oxidase activity was present in Fractions 1 and 2. Arginine ester hydrolaxe activity was most prominent in Fractions 2-S . Phoxphomonoeaterase activity was not detectable in crude venom nor in any fraction derived there from. INTRODUCTION

family Crotalidae, are found only in the new world and as a group, their venoms have not received the detailed scrutiny that the venoms of the families Elapidae and Hydrophüdae have received (Tu, 1977). Crotalid venoms are complex mixtures, chiefly proteins, many of which have enzymatic activity. KOCHOLATY et al. (1971) reported the presence of t-amino acid oxidase, phosphodiesterase, protease, phospholipase A and synthetic ester hydrolyzing activities in a number of crotalid venoms. Few of these venoms have been fractionated and characterized with respect to the various enzymatic activities present . The midget faded rattlesnake, Crotalus viridis concolor, is a small rattlesnake indigenous only to the basins of the Colorado and Green rivers, including a small area of southwestern Wyoming, nearly all ofeastern Utah and extreme west-central Colorado (WoonsuRY,1929, 1958). Little information on C. v. concolor venom is available. GLENN and ST1tA1c1~IT (1977) determined the variation in lethal potency ofindividual venom specimens and compared the relative lethal potency with two other well-known, highly toxic crotalid venoms (Crotalus durissus terrificus and Crotalus scutulatus scutulatus, California variety) and with other C. viridis subspecies. FooTE and MncMAHON (1977) compared the electrophoretic patterns of 26 rattlesnake venoms, including venom from C. v. concolor, in an attempt to reconstruct or reaffirm their phylogeny. RATTLESNAICFS,

517

51 8

W. R. POOL and A. L. BIEBER

In this work, C. v. concolor venom was fractionated by ion exchange chromatography and the crude venom and isolated fractions were examined by disc gel electrophoresis and isoelectric focusing. Phosphodiesterase, L-amino acid oxidase, arginine ester hydrolase, phospholipase and phosphomonesterase enzyme activities were determined with the crude venom and the fractions obtained by ion exchange chromatography. The lethality of each fraction was assessed in white mice. MATERIALS AND METHODS

Lyophilized midget faded rattlesnake venom (Crotalus viridis concolor) was a gift from Dr. R. Straight, Venom Research Laboratory, Veterans Administration Hospital, Salt Lake City, Utah. DEAE-Sepluidex A-50 was obtained from Pharmacia Fine Chemicals and pretreated sa described by thesupplier . All column chromatography was performed at 4°C . Servalyt ampholytea were obtained from Accurate Chemical and Scientific Corp. p-Tosyl-~arginine methyl ester ~ HCI (TAME) ; thymidine 5'-monophoxpho-p-nitrophenyl ester, ammonium salt ; odianisidine ~ 2HCI, horoeradish peroxidase, type II ; o-carboxyphenyl-phosphate ; and t,-a-phosphatidyl chlorine, egg yolk typé III E were purchased from Sigma Chemical Company. All other chemicals were reagent grade quality unless noted otherwise. All solutions were prepared using water distilled from an all-glass apparatus. Salt concentrations were estimated using a Radiometer Conductivity Meter, type CDM3 . DEAE-Sephadex A-50 was equilibrated with 0-05 MTris-HCI, ph 8~3, containing 0~1 M KCI. Lyophilized venom (240 mg) was dissolved in 2 ml of equilibration buffer, centrifuged for 10 min at 5000 p, and the clear supernatant solution was applied to a column (1 ~5 x 30cm). Afterwashing the venom solution into the column with 375ml of equilibration buffer, a linear gradient of KCI was started with 75 ml of equilibration buffer in the mixing chamber and 75 ml of 0-05 MTris, pH 8 ~3, containing 0~3 MKCl in the supply chamber. After depletion of the gradient, all remaining venom components were removed using an isocratic elution with 100m1 of 0-0SM Tris, pH 8-3, containing 2-0 M KCI. A flow rate of approximately 7 á ml/hr was used and samples of 125 ml were collected. The column e®uent was monitored at 280 nm with an ISCO UV-5 absorbance monitor equipped with an ISCO Model 1133 multiplexer-expander. Samples were pooled into fractions and lyophilized, after which the pooled fractions wen desalted by exhaustive dialysis in Spectrapor 6 membraae tubing (mol. wt. cutoff : approximately 2000 dallons) against multiple changesofglassdistilled water. The desalted fractions were lyophilized andstored in a dexiecator at -20°C. Allenzyme assays were performed with venom and venom fractions after theconcentrations hadbeen adjusted to producelinear razes of substratehydrolysis during the assay. Forassays of phosphodieaterase, c-aminoacid oxidase, arginine ester hydrolaxe and phosphomonoesterase, a Zeies M4 QIII monocluomator with a Gilford absorbance monitorand Honeywell xtrip chartrecorder wasaxed to measure thechange in abaorbance with time . A Radiometer TTTl automatic titrator equipped witó the SBR2/SBUI,/IZ'A31 titration assembly was used to measure proton release in the phosphoGpase assay. Phosphodiesterase activity was deurmined by the method Of RAZZELL and KHORANA (1959) using thymidine S'-monophospho-p-nitrophenyl ester as the substrau . Phospholipaseassays were performed by a pH-slat techniqueas dexaibed by CATS 8nd BIEBER (1976) using Só mM NaOH as titrant.Arginine ester hydrolaxe activity was measured as described by HUDí1~L (1959) using TAME as the subatrau . L-Amino acid oxidase activity was assayed by the apectrophotometric method Of NICHOLSON and KIM (1975) . Phosphomonoesterase activity was deurmined by the method of HoESree (1954). Disc polyacrylamide gel electrophonais of anionic and cationic prouins and isoelectric focusing in polyacryÍamide gels Were performed 8s dCxCribCd by CATS and BIEBER (1978). Toxicity assays wen performed with Swiss Webster white mice from a colony maintained at Arizona Stau University. Protein samples in 0~1 ml of 0~9 ~ saline were injected i.p. into 20 t 2 g mice. All Lu so determinations wen performed with 10 groups of mice with S individuals in each group. Lethal dose 50 ~ (Lnso ) values were estimated using the statistical method of Re© and MveNCH (1938). The mortality was recorded up to 48 hr following treatment.

RESULTS

Figure 1 represents a typical reproducible separation of240 mg ofmidget fadedrattlesnake venom by DEAE Sephadex column chromatography. The eluate was pooled as indicated by the arrows . To assess their lethality, each ofthe venom fractions was tested at 075 ~g/g, three times the ~so ofthe crude venom. Groups ofSve mice were used for each test. Fractions FDlb, FD-6 and FD-7 elicited physiological responses when injected i.p. into white mice. Test individuals injected with 075 pg/g of FD-6 or FD-7 died within 24 hr of treatment. Injection of fraction FD-lb at the same concentration resulted in an almost immediate extreme tonic hyperextension ofthe hind limbs. This effect is similar to that observed by BONILLA and FIERO

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FIG. 1. FRACTIONATION OF MIDGET FADED RATTLESNAKE VENOM HY DEAE SEPHADEX A-SO COLUMN CHROMATOGRAPHY .

A 13 x 30cm DEAF Sephadex A-SO column wan equilibrated with SOmM Tris-HCI, pH 8~3, containing 0~1 M KCl and 240mg of lyophilized venom, was applied in 2m1 of the equilibration buffer. Nonretained protein was eluted with 373 ml of the same buffer. An increasing salt gradient consisting of 7S ml of equilibration buffer and 7S ml of 50 mM Trie-HCI, pH 8 ~3, containing Oß M KG was used to elute bound venom components . A final wash of SOmM Tris-HCI, pH 8~3, ooataining 2 ~0 M KCl was used to elute the remainder of the bound venom components The column e®uent was monitored at 280nm (-)with an ISCO UV-S1 absorbaace monitor. The conductivity (---) in mS of each fraction was measured.

(1971) following injection of basic proteins isolated from several other North American rattlesnake venoms. Few deaths were observed after administration of fraction FD-lb even at dosages of 6 ~gfg. Higher dosages increased the duration of the physiological response and, at the highest concentration tested, hindlimb paralysis was still present 48 hr after injection. Of the 240 mg of crude venom applied to the column only 164 mg (67 ~) was recovered afitter lyophilization ofthe dialyzed eluate pools (Table 1). Of the recovered material, 77'6 mg (47 ~) was present in FD-lb, 13 ~9 mg (8~S /) in FD-6 and 43 ~8 mg (27 /) in FD-7. The remaining 175 % of the recovered material was distributed throughout the other pools. It should be noted that those fractions which elicited physiological responses represent the

W. R. POOL and A. L. BEIBER

520

TABLE 1 . ENZYMATIC ACTIVITIES IN POOLED ELUATE FROM THE FRACI70NATION OF C . U . COIICOiOI VENOM

Crude venom FD-la FD-lb FD-2 FD-3 FD-4 FD-S FD-6 FD-7 FD-8 FD_g

Weight (mg) "

Phosphodiesteraset

Arginine ester hydrolaset

c-Amino acid oxidaset

Phospholipaset

240 32 77~6 9~2 1~4 1~2 4-0 13~9 43~8 8~7 0~7

872-0 919-0 203-0 0~73 0~14 0 0 0 4~89 0 0

645-0 1~40 133-0 190-0 125-0 117-0 125-0 29-0 41 ~5 20~2 0

0~31 1-05 47~3 2~72 0~10 0~10 0-07 0-08 0 0 0

7~75 0 0 0 0 6-09 15~1 23~9 146.0 0 0

"Weight of nondialy7able material recovered from pooled column fractions. tTotal activity in the pooled fractions in pmoles/min of product formed (International Units).

major portion of the recovered material . Fraction FD-7 is the primary lethal fraction of C. v. concolor venom. Its LD so was assessed in white mice and found to be 012 f 0~02~g/g . Polyacrylamide gel electrophoresis at pH 9~6 (Tris-glycine system) and pH 3~8 (ß-alanine system) were used for characterization of the crude venom and pooled eluates (Fig. 2) . Iscelectric focusing was likewise employed for the assessment of the venom and its fractions (Figs. 3 and 4). The results show that most of the proteins present in C. v. concolor venom are acidic, having pI's between pH 4 and 6. These data indicate that there is a great deal of overlap of proteins between adjacent column fractions. Isoelectric focusing of the venom and pooled eluates on a pH 4-6 gel greatly increased the resolution of the protein components. As can be seen in Fig. 4, the major lethal fraction, FD-7, is composed primarily of two acidic proteins . The major component in this fraction has a pI of 4~6-4~7. This protein is also present in FD-5 and FD-6 and is clearly a major component in the crude venom before fractionation. The minor component in fraction FD-7 has a pI of 4~4-4~5 and is present only in small amounts in the crude venom and fraction FD-6 . Polyacrylamide gel electrophoresis in the Tris-glycine system of fraction FD-1 b indicated the presence of two components. Electrophoresis of the same fraction in an acidic system also indicated the presence of two components, one migrating at the same position as the majority of the other components in the venom while the second migrated as a broad düfuse band immediately behind the tracking dye. Figures 5 and 6 are profiles of various enzymatic activities found in the eluates obtained by this fractionation. Table 1 summarizes the enzymatic activities in the crude venom and pooled eluate. Four of the five enzymes were present in significant amounts in the crude venom and fractions. Phosphomonoesterase was not present in the crude venom or any of the chromatographic fractions. The phospholipase activity occurs in the fractions that were most lethal when injected into mice. No other appreciable enzymatic activity was present in these fractions. DISCUSSION

As far as the authors know, there have not been any previous reports on the fractionation and characterization of the venom from the midget faded rattlesnake. This study has revealed the presence of three fractions which can elicit visible physiological responses when injected

Crotalus riri((ie conc'nlor Venom

FIG. 2(a) . TRIS-GLYCINE DISC ELECTROPHORESIS OF' CRUDE : VENOM AND VENOM FRACKIONS.

Conditions for electrophoresis : 7~5",; gels, pH 8~9, 0'2 watts/tube constant power. Samples : from IeR to right : 1, 150ug nude venom ; 2, SOpg FD-la ; 3, SOleg FD-lb ; 4, SOt(g FD-2 ; 5, SO~g FD-3 ; 6, 501(g FD-4 ; 7, 50Rg FD-5 ; 8, 50pg FD-6 ; 9, 50kg FD-7 ; 10, 50hg FD-8 ; 11, 50Ng FD-9 . Samples were applied at the cathodic end of the gels. FIG . 2(b) . ß-ALANINE DISC ELECTROPHORESIS OF CRUDE VENOM AND VENOM F~RACTIUNS .

Conditions for electrophoresis : 7~5 ", gels, pH 4~3, 0~2 watts/tube constant power, reversed polarity, tracking dye : methyl green. Samples : from left to right : 1, 150pg nude venom ; 2, SOpg FD-la ; 3, 50 kg FD-1 b; 4, 50 pg FD-2 ; 5, 50 t(g FD-3 ; 6, 50 ~g FD-4 ; 7, 50 pg FD-5 ; 8, 50 ug FD-6 ; 9, 50 ug FD7 ; 10, SOt(g FD-8 ; 11, SOteg FD-9. Samples were applied at the anodic end of the gels .

521

W. R. POOL and A . L. BIEBER

522

A

FIG .

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POLYACRYLAMIDE GEL 1SOELECCRIC FOCUSING PROFILES OF CRUDE VENOM AND VENOM FRACr10NS ON pH 2-11 AMPHOLYTPS .

Condirions for electrophoresis : 7~5 % gels, 0~2 watts/tube constant power. (A) Samples : from bottom to top : 1, ampholyte blank ; 2,100~g crude venom ; 3, SOpg FD-la ; 4, SOUg FD-Ib ; 5, SO~g FD-2 ; 6, 50 ~g FD-3 ; 7, 50 pg FD-4 ; 8, 50 ~g FD-5 ; 9, 50 hg FD-6 ; 10, 50 ~g FD-7 ; 11, 50 kg FD-8 ; 12, 50 ~g FD-9 . (B) Reference pH gradient for the gels in (A), obtained from 2-mm gel slices of a blank gel. Samples were applied at the cathodic end of the gels .

Crotulus rirírlis concolor Venom

523

B

6 a 5

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Slice Number

35

40

45

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POLYACRYLAMIDE GEL ISOELECTRIC h~OCUSING PROFILES OF CRUDE VENOM AND VENOM FRACTIONS ON pH 4-6 AMPHOLYTES .

Conditions for electrophoresis : 7~5 ~ gels, 0~2 watts/tube constant power. (A) Samples from bottom to top. 1, ampholyte blank ; 2,100 ug crude venom ; 3, 50 hg FD-la ; 4, 50 (~g FD-lb ; 5, 50 pg FD-2 ; 6, 50 hg FD-3 ; 7, 50 Kg FD-4 ; 8, 50 pg FD-5 ; 9, 50 hg FD-6 ; 10, 50 kg FD-7 ; 11, 50 hg FD-8 ; 12, 50 Ng FD-9. (B) Reference pH gradient for the gels in (A) obtained from 2-mm gel slices of a blank gel . Samples were applied at the cathodic end of the gels.

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FIG. S. LOCATION OF PHOSPHODIESTERASE ANDARGININE FS'rEA HYDROLASE (TAME) ACI'MTIES IN THE DEAE-SEPHADFJ( A-SO ELU770N PROFILE .

The protein elution represented by the solid line is the same as shown in Fig. 1 . Every fourth fraction was assayed for enzymatic activity exocpt in areas of peak activity, where every fraction was assayed . Fractionation conditions are the same as in Fig. 1 . See Table 1 for definition of international units.

into white mice. Two ofthese fractions result in death of the test animals when injected i.p. at concentrations of 0'75 pg/g . The major lethal component is present in fraction FD-7. The chromatographic procedure used to purify this toxin from the venom is a modification ofthe procedure used by BYEt3ER et al. (1975) for the purification of Mojave (Crotalus scutulatus scutulatus) toxin. A comparison ofthe position ofelution of the major lethal component present in C. v. concolor venom with that of Mojave toxin reveals that both toxins elute in similar positions. The pI of the major component present in C. v. concolor lethal fraction FD-7 (4'6-4'7) is lower than that of Mojave toxin (5'S) (CATE and BIESER, 1978). Further, C. v. concolor toxin, like Mojave toxin, elutes coincidentally with phospholipase activity (CATS and BIEBER, 1976). The reported LD SO value for midget faded rattlesnake venom is 0'25 Rg/g (GLENN and STxAtctrr,1977). Mojave toxin has been characterized as an acidic lethal protein with an i.v. ~so of 0'056 hglg in white mice (CATS and BIEHER, 1978). The evidence presented here indicates that C. v. concolor also has an acidic toxin possessing phospholipase activity. The reported LDSO of the lethal component present in C. v. concolor venom is only an

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FIG. Ó. LOCATION OF L-AMINO ACID OXIDASEAND PHOSPHOLIPASE ACTIVTTTES IN THE DEAFrSEPHADEX A-SO ELUTION PROFILE. The protein elution profile represented by the solid line is the same as shown in Fig. 1. Every fourth fraction was assayed for enzymatic activity except in areas of peak activity where every fraction was assayed. Fractionation conditions are the same as in Fig. l. SeeTable 1 for definition of international units.

approximation of the actual LD SO since the material used was contaminated with at least one other protein, as indicated by disc gel electrophoresis and isoelectricfocusing. It is possible that the actual LD SO of purified C. u. concolor toxin will be lower than that of the partially purified toxin and may equal or even exceed the reported LD SO for Mojave toxin. GONCALVES (1956) purified and described the properties of crotamine, a myotoxin fromthe South American rattlesnake Crotalus durissus terrificus. The pharmacological properties of this substance in mice are characterized by spastic paralysis of the hind limbs ofthe mice with the legs remaining stretched backwards (C~YiKOr. et a1.,1971). Similar symptomatology has been reported by GR> 9) as a broad diffuse band at the basicend of the geL

Crotales oiridis concolor Venom

527

In a wide variety of snake venom, multiple forms of a particular enzyme are common. Some of these are phospholipase A (KAwAUCIIl et al., 1971 ; SHILOAH et al., 1973), arginine ester hydrolaxes (DELPIERRE et a1.,1973) and L-amino acid oxidase (SHAHAIII et a1.,1973) . The enzyme elution profiles from the fractionation of C. v. concolor venom (Figs. 5 and 6) show multiple peaks of phospholipase, arginine ester hydrolase and ~-amino acid oxidase activity, indicating the presence of different species of these enzymes in this venom or incomplete resolution of a single form We are presently purifying and characterizing the major lethal component in fraction FD7 and the paralytic toxin present in fraction FD-lb. Our goal is to elucidate the mechanism by which these toxins exert their physiological actions. Acknowkdgenrnts-This work was supported by NIH Grant 1RO1GM-24566 . The authors are grateful to Dr. Rtctt~an Srtwattr of the Venom Research Laboratory for his gift of Crotales viridis corrcolor venom.

REFERENCES BtEBEa, A. L., Tu, T. and Tu, A. T. (1975) Studies of an acidic cardiotoxin isolated from the venom of Mojave rattlesnake (Crotahu scetelates). Blodhün. btophys. Acts 11, 481. HoxtLtw, C. A. and FIERO, M. K. (1971) Comparative biochemistry and pharmacology of salivary gland secretions II . Chromatographic separation of the basic proteins from some North American rattlesnake venoma J. Chromatog. 56, 253. CITE, R. L. and HIEHER, A. L. (1976) Effectsof Mojave toxin on rat skeletal muscle sarcoplasmic reticulum. Biochtm. blophys. Res. Commun. 72, 295. CATS, R. L. and BtesEa, A. L. (1978) Purification and characterv8tion of Mojave (Crotales scetulates scetulates) toxin and its subunib. Arche. Btochem. Blophys. 189, 397. CttEt~ot, J, GortcwLV~es, J. M, BouwLLer F. andRoctt-AAVEaLea, M. (1971) Action neuromusculaite comparée de la crotamine et du venin de Crotales derlssec takes, var. crotaminices - I. Sur préparations aeuromueculaire to süe. Toxicon 9, 279. Dpi vmQa~ G. R, RoeExTSOtv, S. S. D. and STEnv, K. (1973) Proteolytic and related enzymes in venom of African makes. In : Toxins of Anünals and Plant Origin, pp. 483-489 (DE VatES, A. and KocttvA, E., Eda) . New York : Gardon & Breach . FooTt; R. and MACMAxox, J. A. (1977) F.lectrophoretic studies of rattlesnake (Crotales and Sütreres) venom : taxonomic implications . Comp . Blocran. Physiol. S1B, 235. GLExet, J. L.and SraAtottT, R. (1977) Themidget faded rattlesnake (Crotalesvirldis concolor) venom: lethal toxicity and individual variability. Toxicon 15, 129. GoxcALVes, J. M.(1936) Purification and properties ofaotamine. In : Vsnoms, p.261. Washington, D.C.:American Association for the Adveaeement of Science. GaILLO-Ronatauez, O., SCANNONE, H. R. andPAaaA, N. D. (1974) Enzymatic activities and other characteristics of Crotales dtvisaes cenwnertria venom. Toxkon 12, 297. HoFSrEE, B. H. J. (1954) Direct and continuous spectrophototnetric assay of phosphomonaesterasea Arclu. Biochern. Blophys. Sl, 139. HUMMEL, H. C. W. (1959) A modified epectrophotomctric determination of chymotrypsin, trypsin, and thrombin. Can. J. Biochem. Physiol. 37, 1393. KAwAUCta, S., IwAxAaA, S, SAMFJIMA, Y. and Suzutct, T. (1971) Isolation and characterization of two phospholipese A's from the venom of Agklstrodon halys blornl%~i. Biochim. biophys. Acts 136, 142. KocxoUTV, W. F., L®rottq E. H, DALY, J. G. and Hn.Lnvos, T.A. (1971) Toxicity andsome enzymaticproperties and activities in the venom of Crotaltdac, Elapidae and Vipaddae . Toxicon 9, 131. Ntcxotsatv, J. A. and Ktut, T. S. (1973) A ose-step L-amino acid oxidase assay for intestinal peptide hydrofase activity. Analyt. Blodhem. 63, 110 . R~~ W. E. and KxoaArtA, H. G. (1959) Studies ao polynucbotides III. Enzymatic degradation. Substrate specificity and properties of snake venom phosphodieaterase . J. 6ío1. Chan. 234, 2105. REED, L J. and Muenctt, H. (1938) Simpb method of estimating 50/ endpoints. AnL J. Hyg. 27, 493. $FtAitAt~á, N., Bnoiwtt, A. and KocttVA,13 (1973) Isolation of L-amino acid oxidase fran Vipers pakstbwt venom and preparation of a monospociflc antiserum in rabbits. Ice : Toxins of Münal and Plant Origin pp . 919-925 (De votes, A. and KocxvA, E, Eds.). New York : Gardon & Breach. StttLOAx,J, ICtlsArts¢v, C. sad DE V'RIE3, A.(1973) Pho:pholipase isoenzymes from Naja naja venom. I. Purification and partial characterization. Toxlcon il, 481. StILL1VAN, J. A, FAax, E. andGEaEtv,C. R. (1979) Fractionation andpartialcharacterization of toxic components of timber rattlesnake venom. Toxida~ 17, 269. Tu, A. T. (1977) V~nans : ChemLury and Molecular Biology, pp. 211-233. New York : John Wiley. WOODHURY, A. M. (1929) A new rattlesnake from Utah . Bull. Unü. Utar 211, 1. WooDeuav, A. M. (1958) The name Crotales vtridis concolor Woodbury. Copcia 2, 151.