Thrombin-like activities were not observed inCrotalus basiliscus, C. molossus and C. scutulatus scutulatus crude venoms. 5\u27-Nucleotidase specific activities of 0.863, 0.273 and 5.520 units/mg of crude venom protein were observed inC. basiliscus, C. molossus and C. s. scutulatus venoms, respectively. Concanavalin ASepharose 4 B (Con A)affinitychromatography yielded two fractions from each of the crude venoms. Ineach instance, both fractions exhibited 5\u27-nucleotidase activities and the Con A-binding proteins had higher activities than the Con A-nonbinding proteins. 5\u27-Nucleotidase activities inthe DEAESephadex A-50 chromatographic fractions were localized in the first elution fraction and the last fraction(s) to elute. EDTAhad no effect on the 5\u27-nucleotidase activities ofthe crude venoms

Johnson, Bob D.

Shams, Ali M.S.

Sifford, Dewey H.

English

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Journal of the Arkansas Academy of ScienceVolume 49 Article 3619955'-Nucleotidase and Thrombin-Like Activities ofSelected Crotalid VenomsAli M.S. ShamsArkansas State UniversityDewey H. SiffordArkansas State UniversityBob D. JohnsonArkansas State UniversityFollow this and additional works at: http://scholarworks.uark.edu/jaasPart of the Animal Studies Commons, and the Terrestrial and Aquatic Ecology CommonsThis article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able toread, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking priorpermission from the publisher or the author.This Article is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the Arkansas Academyof Science by an authorized editor of ScholarWorks@UARK. For more information, please contact scholar@uark.edu, ccmiddle@uark.edu.Recommended CitationShams, Ali M.S.; Sifford, Dewey H.; and Johnson, Bob D. (1995) "5'-Nucleotidase and Thrombin-Like Activities of Selected CrotalidVenoms," Journal of the Arkansas Academy of Science: Vol. 49 , Article 36.Available at: http://scholarworks.uark.edu/jaas/vol49/iss1/361695' -Nucleotidase and Thrombin-LikeActivities of Selected Crotalid VenomsAliM.S. Shams, Dewey H.Sifford and BobD.JohnsonDepartment of Biological Sciences andDepartment of Chemistry, Biochemistry and PhysicsArkansas State UniversityState University, AR 72467AbstractThrombin-like activities were not observed inCrotalus basiliscus, C. molossus and C. scutulatus scutulatus crude venoms.5'-Nucleotidase specific activities of 0.863, 0.273 and 5.520 units/mg of crude venom protein were observed inC. basilis-cus, C. molossus and C. s. scutulatus venoms, respectively. Concanavalin ASepharose 4 B (Con A)affinitychromatographyyielded two fractions from each of the crude venoms. Ineach instance, both fractions exhibited 5'-nucleotidase activitiesand the Con A-binding proteins had higher activities than the Con A-nonbinding proteins. 5'-Nucleotidase activities intheDEAE Sephadex A-50 chromatographic fractions were localized in the first elution fraction and the last fraction(s) toelute. EDTAhad no effect on the 5'-nucleotidase activities of the crude venoms.IntroductionCrotalus basiliscus (Cope), the Mexican west-coast rat-tlesnake is one of the world's largest rattlesnakes.Specimens exceeding 150 cm are common, and the maxi-mum size reported is a little over 200 cm. The range ofthis species extends from the Rio del Fuerte drainage inextreme southern Sonora, southward along the plains,foothills, and valleys of Sinalo, Nayarit, Jalisco, Colima,and northwestern Michoacan, including the middle RioTapalcatepec Valley. The ranges of C. basiliscus and C.molossus overlap and they freely hybridize where theycome in contact (Campbell and Lamar, 1989).C. molossus, the blacktail rattlesnake, is one of themost beautiful rattlesnakes. Its maximum body length is126 cm. The darkest individuals are usually from habitatsfeaturing dark substrates. C. molossus is found from cen-tral and west-central Texas northwest through the south-ern half of New Mexico to northern and extreme westernArizona southward to the southern edge of MexicanPlateau and Mesa del sur, Ocaca. It also occurs on theTiburon and San Esteban islands in The Gulf ofCalifornia (Ernst, 1992).C. scutulatus, the Mojave rattlesnake, has a maximumlength of 129 cm (Ernst, 1992). This crotalid species isprobably the most dangerous rattlesnake, as its venom isneurotoxic (Minton, 1956; Johnson et al., 1966). C. scutu-latus is found from southwestern Washington county,Utah, Lincoln and Clark counties, Nevada, and Kern, LosAngeles, and San Bernardino counties in California,southeast through Arizona and southward fromTranspeces, Texas, and southwestern Hildalgo and Oterocounties in New Mexico, to the southern edge of theMexican Plateau in Puebla and adjacent Vera Cruz(Campbell and Lamar, 1989).Crotalid venoms are very complex with the majorityof the venom consisting of numerous protein compo-nents having enzymatic activities. The concentrations ofproteins, and presumably of different toxins, varybetween individual snakes (Johnson et al., 1968). Atleast18 different enzymatic activities have been identified incrotalid venoms (Iwanaga and Suzuki, 1979). Amongthese, there are at least four enzymes involved in thehydrolysis of phosphate bonds. These include endonucle-ase, alkaline nonspecific phosphatase, 5'-nucleotidase,and phosphodiesterase (Tu, 1977). A specific phospho-monoesterase, 5'-nucleotidase, specifically hydrolyzesphosphate monoester which links at the 5'position ofDNA and RNA. Upon dialysis, crude venom 5'-nucleoti-dase activities increased two or three times that of thenondialyzed samples, suggesting that some substance,probably zinc ions which are abundant in crude venoms,inhibits the activity (Suzuki and Iwanaga, 1958).Many venoms exert profound effects on the bloodcoagulation system. Some accelerate the coagulationprocess and others retard it.Thus snake venoms are oftendivided into two types, coagulant and anticoagulant.Some venoms contain both coagulant and anticoagulantfactors simultaneously, and sometimes a venom becomescoagulant or anticoagulant depending on the concentra-tion used (Tu, 1977). Also, various mechanisms caninduce procoagulation or anticoagulation. A venom mayact as a coagulant for the following reasons: ithas throm-boplastin-like activity, it contains a factor X activator, itactivates factor V, it activates prothrombin, or it has athrombin-like activity. A venom may also act as an antico-Proceedings Arkansas Academy of Science, Vol.49, 1995169Journal of the Arkansas Academy of Science, Vol. 49 [1995], Art. 36Published by Arkansas Academy of Science, 19951705' -Nucleotidase and Thrombin-Like Activities of Selected Crotalid Venomsagulant for various reasons: it has fibrinolytic activity, itcan activate plasminogen to release plasmin, ithasantithrombic action, and it has inhibitory or destructiveaction toward any of the blood coagulation factors pre-ceding thrombin (Tu, 1977).The purpose of this work was to determine the distri-bution of 5'-nucleotidase and thrombin-like activities inthe Concanavalin A Sepharose 4B (Con A) affinity chro-matographic fractions and the DEAE Sephadex A-50 ionexchange chromatographic fractions of C. basiliscus, C.molossus and C. s. scutulatus crude venoms. This work is acontinuation of previous works centered on phosphodi-esterase (Beasley et al., 1993) and proteinase, N-benzoyl-L-arginine ethyl esterase and p-tosyl-L-arginine methylesterase (Stegall et al., 1994).Materials and MethodsLyophilized C. molossus and C. basiliscus venoms wereprovided by Dr.H.L. Stahnke of Arizona State University.C. s. scutulatus venoms, adenosine 5'-monophosphate, fib-rinogen, and thrombin were purchased from SigmaChemical Company. Ammonium molybdate, hydro-quinone, and sodium sulfite were purchased from FisherScientific Company. Magnesium sulfate, sulfuric acid, andtris (hydroxymethyl) aminomethane (TRIS) were pur-chased from Mallinckrodt Chemical Works. Glycine waspurchased from Matheson, Coleman and Bell. Sodiumsulfate was purchased from J.T. Baker ChemicalCompany. Sephadex G-25, Concanavalin A-Sepharose 4B(Con A), DEAE Sephadex A-50, and columns were pur-chased from Pharmacia, Uppsala 1, Sweden.Fractionations using Con Agel were performed using425 mg samples of whole venom on columns which hadbeen equilibrated with 0.05 Mammonium acetate buffer(pH 7.0) (Iscove et al., 1974; Aspberg and Porath, 1970;Hinson et al., 1985=5). The Con A-nonbinding proteins(FI) were eluted at 4 °C with 500 mL of 0.05 Mammoni-um acetate (pH 7.0) containing 0.5 MNaCl. The Con A-binding proteins (FII) were eluted at 4 °C with 500 mL of0.05 Mammonium acetate buffer containing 0.5 MNaCland 0.25 Ma- methyl-D-mannoside. Eluates of 110 drops(-4 mL) per tube were collected and stored at -12 *C with-in 2 hrs after collection (Beasley et al., 1993 and Stegall etal., 1994).DEAE Sephadex A-50 ion exchange chromatographywas performed on 400 mg samples of whole venom dis-solved in 0.05 M ammonium acetate buffer (pH 8.0)(Cheng and Ouyang, 1967; Ouyang et al., 1971; Siffordand Johnson, 1978; Hintson et al., 1985). Two stage gra-dient elutions were performed on each venom usingammonium acetate buffers. The first stage gradient elu-tion was performed with 250 mL of 0.05 Mammoniumacetate (pH 8.0) in the mixing vessel and 310 mL of 0.9 Mammonium acetate (pH 6.0) in the reservoir. The secondstage gradient elution was performed with 240 mL of 0.3Mammonium acetate (pH 6.0) in the mixing vessel and310 mL of 0.9 Mammonium acetate (pH 5.4) in thereservoir. The column (2.5 cm X 56 cm) was maintainedat 4 *C and eluates were collected in5 mL fractions. Allfractions were stored at -12 *C within 2 hrs of collection(Beasley et al., 1993).For protein concentration estimations and 5'-nucleoti-dase assays, a Beckman DB spectrophotometer and aSpectronic 1201 were used. Protein concentrations of thefractions were estimated from their absorbances at 280nm. An absorbance at 280 nm of 1.0 for 1.0 mg of venomper mL was used (Sifford and Johnson, 1978).5'-Nucleotidase activities were determined by themethod of Ging (1956), Lo et al. (1966), Sifford andJohnson (1978) and Hinson et al. (1985). The factor(0.250) used by Ging (1956) and Lo et al. (1966) for con-verting 5'-nucleotidase activities which were measured at37 *C to values at 25 *C was used. Thrombin-like activi-ties were determined by the method ofSato et al. (1965)with the minor modification used in the works by Siffordand Johnson (1978) and Hinson et al. (1985).Metal requirements for each enzyme were investigat-ed by incubating 0.3 mL of venom or venom fractionwith an equal volume of ethylenediaminetetraacetic acid(EDTA) solution for 10 minutes at 37 *C (Friederich andTu, 1971; Goucher and Flowers, 1964; Stegall et al.,1994). This mixture was diluted with water to yield a finalconcentration of 0.6 m EDTA in a total volume of 1.0mL. Aliquots of this mixture were assayed for 5'-nucleoti-dase withcontrol samples having water inplace of EDTA.Results and DiscussionThrombin-like activities were not observed in C.basiliscus, C. molossus, and C. s. scutulatus crude venoms.Thus, subsequent assays for thrombin-like activities werenot performed on the venom fractions obtained by affini-ty and ion exchange chromatography. Thrombin-likeactivities have been reported in other crotalid venoms,e.g., Agkistrodon acutus, Trimeresurus gramineus, T. oki-navensis, T. flavoriridis, C. horridus horridus,snid C.adamanteus (Tu, 1977). Markland and Damus (1971) haveextensively studied the chemical properties of purified C.adamanteus venom thrombin-like enzyme.Con Afractionation yielded two fractions for each ofthe whole venoms: Fraction I(FI) was composed of ConA-nonbinding proteins and Fraction II(FII) was com-posed of Con A-binding proteins. In each instance, thecrude venom 5'-nucleotidase activities were higher thanthe activities in the corresponding FIand FIIproteinsProceedings Arkansas Academy of Science, Vol.49, 1995170Journal of the Arkansas Academy of Science, Vol. 49 [1995], Art. 36http://scholarworks.uark.edu/jaas/vol49/iss1/36171(Table 1).Table 1. 5'-Nucleotidase specific activities of selected cro-talid venoms.Venom Activity*Crotalus basiliscus:Crude Venom 0.863Concanavalin A-nonbinding proteins (Fl) 0.265Concanavalin A-binding proteins (Fl1) 0.740Crotalus molossusCrude venom 0.273Concanavalin A-nonbinding proteins (Fl) 0.024Concanavalin A-bindingproteins (Fl1) 0.036Crotalus scutulatus scutulatusCrude venom 5.520Concanavalin A-nonbinding proteins (Fl) 0.131Concanavalin A-bindingproteins (Fl1) 1.010*5'-Nucleotidase activities are reported as mean jimoles sub-strate hydrolyzed/min/mg venomDEAE Sephadex A-50 chromatography of C. basilis-cus, C. molossus, and C. s. scutulatus venoms yielded severalfractions from each of the whole venoms. 5 -Nucleotidaseactivities were localized, for the most part, in the firstfraction obtained during the first stage elution and inthelast few fractions obtained during the second stage elu-tion. The highest specific activities occurred in the firstfraction (around tube 20) to elute (Figs. 1-3). Theseresults were comparable to distributions of phosphodi-esterase and 5'-nucleotidase activities in the DEAESephadex A-50 fractions of A. bilineatus venom in whichthese enzymes eluted in the first fraction (Sifford andJohnson, 1978). C. basiliscus and C. s. scutulatus venomsyielded only two peaks of activity for 5'-nucleotidase. C.molossus venom's 5'-nucleotidase activity was much lowerthan the activities observed inC. basiliscus and C. s. scutu-latus venoms.The peak activities for 5'-nucleotidase observed inthe DEAE Sephadex A-50 fractions were less than theactivities in the crude venoms, suggesting that thisenzyme is heat labile or that a necessary activator mighthave been removed. The mean S'-nucleotidase activity forC. s. scutulatus crude venom in this work was somewhathigher than the activities reported byHinson et al. (1985)and Childs et al. (1986). The 5'-nucleotidase activities inour Con A fractions, however, were lower than thosereported by either Hinson et al. (1985) or Childs et al.(1986).EDTA had no effect on the 5'-nucleotidase activitiesof the crude venoms. Thus, no EDTA assays were per-formed on the venom fractions.Fig. 1. 5'-Nucleotidase activities in the eluates fromDEAE Sephadex A-50 column chromatography of wholeCrotalus basiliscus venom. The arrow indicates the start ofthe second stage elution.Fig. 2. 5'-Nucleotidase activities in the eluates fromDEAE Sephadex A-50 column chromatography of wholeCrotalus molossus venom. The arrow indicates the start ofthe second stage elution.Fig. 3. 5'-Nucleotidase activities in the eluates for DEAESephadex A-50 column chromatography of wholeCrotalus scutulatus scutulatus venom. The arrow indicatesthe start of the second stage elution.Additional purification willbe necessary to deter-mine whether the 5'-nucleotidase activities observed inthe DEAE Sephadex A-50 fractions were the result of oneProceedings Arkansas Academy of Science, Vol.49, 1995171Journal of the Arkansas Academy of Science, Vol. 49 [1995], Art. 36Published by Arkansas Academy of Science, 1995172enzyme or several. This question was also raised byStegall et al. (1994) in their work with proteinases andesterases in C. basiliscus, C. molossus, and C. s. scutulatusvenoms.Literature CitedAspberg, K.and J. Porath. 1970. Group-specific absorp-tionof glycoproteins. Acta. Chem. Scand. 24:1829-1841.Beasley, R.J., E.L.Ross, D.H. Sifford, P.M. Nave andB.D.Johnson. 1993. Phosphodiesterase activities ofselected crotalid venoms. Southern Association ofAgricultural Scientists. Bull.:Biochem. and Biotech.6:48-53.Campbell, J.A.and W.W. Lamar. 1989. Pp. 334-335, 361,InThe Venomous Reptiles of Latin America.Comstock Publishing Association, New York.Cheng, H.and C. Ouyang. 1967. Isolation of coagulantand anticoagulant principle from the venom ofAgkistrodon acutus. Toxicon 4:235-243.Childs, C.K., M.W. Hinson, D.H. Sifford and B.D.Johnson. 1986. Concanavalin A-nonbinding enzymesof Crotalus scutulatus scutulatus venom. Proc. ArkansasAcad. Sci. 40:20-21.Ernst, C.H. 1992. Venomous reptiles of North America.Smithsonian Institution Press, Washington andLondon.rederich, C. and A.T.Tu. 1971. Role of metals insnakevenoms for hemorrhagic, esterase and proteolyticactivities. Biochem. Pharmacol. 20:1549-1556.rg,N.S. 1956. Extraction method of colorimeter deter-mination of phosphorus inmicrogram quantities.Anal. Chem. 28:1330-1333.Goucher, C.R. and H.H.Flowers. 1964. The chemicalmodification of necrogenic and proteolytic activitiesof Agkistrodon piscivorus venom and the use of EDTAto produce a venom toxoid. Toxicon 2:139-147.rson, M.W., C.K. 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5\u27-Nucleotidase and Thrombin-Like Activities of Selected Crotalid Venoms

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5\u27-Nucleotidase and Thrombin-Like Activities of Selected Crotalid Venoms

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