Johnson, Bob D.

Landberg, Karl H.

Sifford, Dewey H.

English

UARK (University of Arkansas )

Journal of the Arkansas Academy of Science Volume 34 Article 40 1980 Isolation of Phospholipase A2 from Agkistrodon bilineatus Venom Karl H. Landberg Arkansas State University Bob D. Johnson Arkansas State University Dewey H. Sifford Arkansas State University Follow this and additional works at: https://scholarworks.uark.edu/jaas  Part of the Zoology Commons Recommended Citation Landberg, Karl H.; Johnson, Bob D.; and Sifford, Dewey H. (1980) "Isolation of Phospholipase A2 from Agkistrodon bilineatus Venom," Journal of the Arkansas Academy of Science: Vol. 34 , Article 40. Available at: https://scholarworks.uark.edu/jaas/vol34/iss1/40 This article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able to read, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. This General Note is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the Arkansas Academy of Science by an authorized editor of ScholarWorks@UARK. For more information, please contact scholar@uark.edu. Arkansas Academy of Science Proceedings, Vol.XXXIV,1980 119General NotesBAUMGARTNER, A. M., F. M. BAUMGARTNER. 1944. Hawksand owls inOklahoma 1939-1942: Food habits and populationchanges. Wilson Bull.56:209-215.ERRINGTON, P. L. 1932. Studies on the behavior of the GreatHorned Owl. WilsonBull. 44:212-220.MILLER,L. 1930. The territorial concept in the Great Horned Owl.Condor 32:290-291.BENT, A.C. 1938. Life histories of North American birds of prey.Part II,U.S. Natl. Museum Bull.170:295-357.ERRINGTON, P. L. 1930. The pellet analysis method of raptor foodhabits study. Condor 32:292-296.RODNEY HARRIS, Science Dept., Southside HighSchool, Batesville Arkansas 72501 and EARLL.HANEBRINK. Div. ofBiological Science,Arkansas State University, State University, Arkansas 72467.ISOLATION OF PHOSPHOLIPASE A2 FROM AGKISTRODON BILINEATUS VENOMVenom produced by the Mexican mocassin, A.bilineatus, contains phosphomonoesterase, phosphodiesterase, 5-nucleotidase, esterases,thrombin-like, L-amino acid oxidase, protease, phospholipase A.., and NADnucleosidase activities (Tu et al.. 1967; Denson et al.. 1972; Siffordand Johnson, 1978;Brunson et al., 1978). Of these enzymes, phospholipase A2(PhL-A2) was chosen in this work forpossible isolation. This choiceof 1'hL- A > for isolation was due primarily toitsheat stability and toits distribution inthe eluates obtained byionexchange chromatography of thecrude venom as evidenced previously (Sifford and Johnson, 1978).Assay procedures with minor modifications (Sifford and Johnson, 1978) included phospholipase A2using the clearing ofan egg yolksuspen-sion (Marinetti, 1965), phosphomonoesterase and phosphodiesterase (Richards et al., 1965), esterase (Tu et al., 1965), 5'-nucleosidase (Lo et al.,1966; Ging, 1956), and L-amino acid oxidase (Paik and Kim, 1965). Hyaluronidase was assayed according to the turbidimetric procedures of Kassand Seastone (1944).A450 mg sample of crude venom (Sigma) was separated on Concanavalin Acovalently bound to Sepharose 4B gel (Con A)into glycopro-teins (anthrone reagent positive) and nonglycoproteins (anthrone reagent negative) by employing the methods ofIscove et al. (1974) and Aspbergand Porath (1970). In fractitmat ions by ion exchange chromatography employing DEAE Sephadex A-SO, the methods of Cheng and Ouyang(1967), Ouyang et al. (1971), and Johnson and Sifford(1978) were used. Proteins were desalted by using Sephadex G-10 columns at 4°C. Sephadex(5-75 and G-50 columns were used to separate I'hl.-A > from highermolecular weightmolecules.Animmunizing schedule was prepared according to Ownby et al. (1979). Preimmune serum was obtained from approximately 12 month oldNew Zealand white rabbits. An immunizing dose was prepared by dissolving 17 mg of lyophilized A. bilineatus crude venom in 20ml of sterilephysiological saline. A0.5 ml aliquot of this solution was then mixed with 0.5 ml of Freund's complete adjuvant. Injections of0.5 ml then wereE; subcutaneously into each thigh. Booster injections were prepared by mixing 0.5 ml of A. bilineatus venom (0.8 mg/ml) and 0.5 ml ofnd's complete adjuvant. One week later, subcutaneous injections of 0.5 ml of the solution were made in each shoulder. Four weeks after theter injections, antiserum via heart puncture was collected and stored at—20°C.kRabbitantiserum for the purified phospholipase A.> fraction was prepared by injecting an immunizing dose containing 0.3 ml of purifiedle(0.1 mg/ml) and 1.0 ml Freund's complete adjuvant. Subcutaneous injections of0.65 ml of this solution were made into each thigh. Onelater, booster injections of the same dose were administered into each shoulder. Four weeks later, antiserum was collected and stored at>Immunoelectrophoresis methods outlined by Campbell et al. (1963) and Garvey et al. (1977) were employed to determine PI1L-A2 purity,iecyl Sulfate-Polyacrylamide gel electrophoresis procedures of Weber and Osborn (1969) were used byDr.Collis Geren (University of Arkan-at Fayetteville) to assay crude venom and fraction samples.A. bilineatus crude venom contains nonglycoprotein and glycoprotein enzymes (Fig. 1). The larger nonglycoprotein fraction (Fraction I),comprising approximately 80% of the crude venom proteins, contained numerous enzyme activities. These included PhL-Ai, phosphomono-esterase, phosphodiesterase, 5 -nucleosidase, hyaluronidase, TAMEase, BAEEase, and L-amino acid oxidase activities.Fraction I,obtained by Con Achromatography, was pooled, lyophilized, and then desalted with Sephadex G-10. Fractionation of this de-fdnonglycoprotein fraction byion exchange chromatography (DEAE Sephadex A-50) yielded three large fractions and several minor fractions.•A2 activity was concentrated inthe second major fraction (Fig. 2). PI1L-A2 activity (14,000 units/mg) in this fraction was much higher thanof the crude venom (234 units/mg).§The PhL-Ai-containing fraction obtained by chromatography with DEAE Sephadex A-50 was divided into two samples. Even-numberedis (42 through 66) were pooled to from one sample while odd-numbered tubes (41 through 67) formed the other sample. These samples, afterhilization and desalting, were fractionated withSephadex G-75. Inboth instances, PI1L-A2activity (24,400 units/mg) was observed onlyinthemolecular weight fraction (Fig. 3).The low molecular weight PI1L-A2fraction (tubes 16-22) obtained by Sephadex G-75 chromatography was pooled, lyophilized, desalted, andapplied to a Sephadex G-50 column. This fractionation yielded a fraction free of the larger molecules (Fig. 4).Ilmmunoelectrophoresisand disc electrophoresis of crude venom samples indicated a complex mixture of proteins although the PI1L-A2:ion obtained by the sequence of Con A-Sepharose 4B, DEAE A-50, Sephadex G-75, and Sephadex G-50 chromatography procedures wastly purified(Figs. 5-8). Close examinations of both types of electrophoresis patterns, however, indicated trace contaminations. These con-nations could be due, inpart, to TAMEase and L-amino acid oxidase since the distributions of these enzymes inA.bilineatus venom overlap¦A2 after DEAE Sephadex A-50 chromatography (Sifford and Johnson, 1978). At present, work is directed toward purification of largeunts ofPhL-A2inorder that more enzyme characteristics may be obtained.We thank Dr. Collis Geren for his work withthe gel electrophoresis, Dr.L.W. Hinck for his assistance withthe im-munological procedures, John Ruff forhis assistance throughout this work, and Mrs. Alice Chandler for typing the manu-script.119Journal of the Arkansas Academy of Science, Vol. 34 [1980], Art. 40Published by Arkansas Academy of Science, 1980't•II•I'120 Arkansas Academy of Science Proceedings, Vol.XXXIV,1980Figure 1. Chromatography of Agkistrodon bilineatus crude venom(450 mg) on a Concanavalin ASepharose 4B column (2.5 X 15 cm)at4°C by two stage elution. The arrow indicates the start of the secondstate elution (using o-methyl-D-mannoside). Eluates were collectedat a flow rate of 17 ml/hr.50 100 150 200 250Figure 2. Chromatography of Agkistrodon bilineatus venom non-glycoproteins on a DEAE Sephadex A-50 column (2.5 X 56 cm) at4°C by two stage elution with ammonium acetate buffer. Eluates of5.0 ml each were collected at a flow rate of 17 ml/hr.Figure 3. Chromatography of the phospholipase Aicontaining frac-tion, collected from DEAE A-50 ion exchange chromatography, onaSephadex G-75 column (1 X 100 cm) at 4°C. Eluates of 4.0 ml/tubewere collected.TUBE NUMBERFigure 4. Chromatography ofphospholipase Aicontaining fraction,from Sephadex G-75 chromatography, on a Sephadex G-50 column(1 X 100 cm) at 4°C. Eluates of 4.0 ml/tube were collected.120Journal of the Arkansas Academy of Science, Vol. 34 [1980], Art. 40https://scholarworks.uark.edu/jaas/vol34/iss1/40'Arkansas Academy of Science Proceedings, Vol.XXXIV,1980 121Figure 5. Immunoelectrophoretic patterns obtained by reactinganti-crude Agkistrodon bilineatus venom serum with electrophoret-ically separated crude venom.Figure 6. Immunoelectrophoretic patterns obtained by reactinganti-crude Agkistrodon bilineatus venom serum with electrophoret-ically separated phospholipase A> containing fraction and crudevenom.Figure 7. Immunoelectrophoretic patterns obtained by reactinganti-phospholipase Ai fraction serum with electrophoreticallyseparated phospholipase Aj containing fraction and Agkistrodonhilineatus crude venom.Figure 8. Patterns obtained by Dodecyl Sulfate-Polyacrylamide GelElectrophoresis of Agkistrodon bilineatus crude venom and the phos-pholipase Aicontaining fraction.LITERATURE CITEDASPBERG, K.and J. PORATH. 1970. Group-specific adsorption ofglycoproteins. ActaChem. Scand. 24:1839-1841.VNSON, E. M., B. D. JOHNSON and D. H. SIFFORD. 1978.NADnucleosidase of Agkistrodon bilineatus venom. Amer. J.Trop.Med. and Hyg. 27:365-368.CAMPBELL, D. H., J. S. GARVEY, N. E. CREMER and D. H.SUSSDORF. 1963. Immunoelectrophoresis. Pp. 149-155, InMethods inImmunology, ALaboratory Text for Instruction andResearch. W. A.BenjaminInc., New York.CHENG, H.and C. OUYANG. 1967. Isolation ofcoagulant and anti-coagulant principles from the venom of Agkistrodon acutus.Toxicon 4:235-243.KISON, K. W. E., F. E. RUSSELL, D. ALMAGRO and R. C.BISHOP. 1972. Characterization of the coagulant activity ofsome snake venoms. Toxicon 10:557-562.ItVEY, J. S., N.E. CREMER and D.H. SUSSDORF. 1977. Im-raunoelectrophoresis. Pp. 328-336, In Methods of Immunology,ALaboratory Text forInstruction and Research. W. A.Benja-min, Inc., Reading, Mass.IG, N. S. 1956. Extraction method of colorimetric determinationof phosphorus in microgram quantities. Anal. Chem. 28:1330-1333.IiVE, N. N.,F. SIEBER and K.H. WINTERHALTER. 1974.Erythroid colony formation in cultures of mouse and humanbone marrow: Analysis of the requirement for erythropoietinby gel filtration and affinity chromatography on Agarose-Concanavalin A.J. Cell. Physiol. 83:309-320.IS, E. H. and C. V.SEASTONE. 1944. The role of the mucoidpolysaccharide (Hyaluronic Acid) in the virulence of a group Ahemolytic streptococci. J. Exp. Med. 79:319-329.LO. T., Y.CHEN and C. LEE. 1966. Chemical studies of Formosancobra (Naja naja atra) venom. Part I.Chromatographic separa-tion of crude venom on CM-Sephadex and preliminary charac-terization of its components. J. Chinese ("hem. Soc. Ser. II13:25-37.MARINETTI,G. V. 1965. The action of phospholipase A on lipo-proteins. Biochim. Biophys. Acta. 98:554-565.OUYANG,C,J. HONGand C. TENG. 1971. Purification and prop-erties of the thrombin-like principle of Agkistrodon acutusvenom and its comparison with bovine thrombin. Thrombus.Diathes. haemorrh. (Stuttg.) 26:224-234.OWNBY, C. L., W. M. WOODS and G. V. ODELL. 1979.Antiserum to myotoxin from prairie rattlesnake (Crotalus viridisviridis) venom. Toxicon17:373-380.PAIK, K.and S. KIM.1965. pH substrate relationship of L-aminoacid oxidases from snake venom and rat kidney. Biochim. Bio-phys. Acta. 96:66-74.RICHARDS, G. M., G.DUVAIRand M.LASKOWSKI, SR. 1965.Comparison of the levels of phosphodiesterase, endonuclease,and monophosphatases in several snake venoms. Biochem.4:501-503.SIFFORD, D. H. and B. D. JOHNSON. 1978. Fractionation ofAgkistrodon bilineatus venom byion exchange chromatography,Pp. 231-242, In Toxins: Animal, Plant and Microbial (P. Ro-senberg, ed.) Pergamon Press, Oxfordand New York.TU,A. T., G. P. JAMES and A.CHUA. 1965. Some biochemicalevidence in support of the classification of venomous snakes.Toxicon 3:5-8.TU,A.T., P. M.TOOM and D.S. MURDOCK. 1967. Chemical dif-ferences in the venoms of genetically different snakes. Pp. 351-362, In Animal Toxins (Russell and Saunders, eds.) PergamonPress, Oxford and New York.121Journal of the Arkansas Academy of Science, Vol. 34 [1980], Art. 40Published by Arkansas Academy of Science, 1980122 Arkansas Academy of Science Proceedings, Vol.XXXIV,1980;¦Arkansas Academy of ScienceWEBER, K. and M. OSBORN. 1969. The reliability of molecularweight determinations by dodecyl sulfate-polyacrylamide gelelectrophoresis. J.Biol. Chem. 244:4406-4412.KARL H. LANDBERG, BOB D. JOHNSON and DEWEY H. SIFFORD. Department ofBiological Sciences and Department ofChemistry,Arkansas State University, State University, Arkansas 72467.\SAGE THRASHER (OREOSCOPTES MONTANUS), A NEW STATE RECORDOn 24 November 1979, Cheryl Lavers and Iwere looking forbirds inthe Farville area, about 6 miles NE of Jonesboro, Craighead County, iwhen we discovered a Sage Thrasher (Oreoscoptes montanus). The bird was rather Mockingbird-like (Mimus polyglottos) inshape, plain gray-brown crown and back, and bright yelloweye with ablack pupil. Other characters differed from a Mockingbird in that itwas smaller and shortertailed and it had a white throat with a black malar stripe. The underparts, witha ground color of warm buff, were densely streaked with blackchevrons arranged inlength-wise rows. There was a white wingbar, and a dark tail withwhite outer corners. The bill was slender, veryslightly de-curved, and dark bluish-black incolor, as were the rather short legs. The birdran along the ground inopen places, or under brush and stayed onornear the ground. Itwas observed catching, decapitating, then swallowing ground crickets (Acheta sp.). We have both observed Sage Thrashersin several western states. Anexamination of standard field guides (Peterson, R. T. 1961. A field guide to western birds. Houghton MifflinCo.,Boston; Robbins, C. S. et al. 1966. Birds of North America. Golden Press, N.Y.)further confirmed our identification. Adescription and slides ofthe bird have been sent to Charles Mills,Curator, the Arkansas Audubon Society, and Dr.Douglas James, Department of Zoology, University ofArkansas at Fayetteville. This is a firstrecorded instance of the Sage Thrasher inArkansas.Inits normal range, the Sage Thrasher breeds in the sagebrush (Artemisia) deserts of western North America, coming as far east as westernOklahoma. Itwinters in the southern part of its range and Mexico,occurring as far east as central and southern Texas, witha small isolated wintercolony in extreme southwestern Louisiana (A.O.U. check-list ofNorth American birds, 1957). The species is seldom recorded as a vagrant. Thefollowingextralimital occurrences to the east of its range have been recorded: Florida, 2;Illinois, 2;Maryland, 1;New York, 2;North Carolina.1;South Dakota, 1.NORMAN LAVERS,DivisionofEnglish, Philosophy, and Languages, Arkansas State University. State University, Arkansas 72467.:SOME EFFECTS OF METHYL GREEN ON EXPRESSION OF DAMAGEINDUCED INGl XENOPUS CELLS BY ULTRAVIOLETLIGHTMethylgreen shows a high degree of specificity forDNA and is a component of many nuclear stains. Kurnick (1952) observed that this basicdye was readily bound by polymerized DNA;two amino groups of the dye binding to twophosphoric acid groups of DNA.Errera (1951) demon-strated that the affinity ofDNA for methyl green is influenced byradiation and other agents which depolymerize DNA,or alter its internal con-figuration, Doudney and Haas (1958) showed that methyl green significantly influenced metabolic activities, such as DNA and RNA synthesis, inEscherichia coli. These results suggested that appropriate experiments, involving methyl green treatments coupled with germicidal UV expo-sures, might aid indescribing the expression and repair of UV-induced lethal and rautational damage inprokaryotic cells. Experiments of thisnature were carried out byinvestigators, such as Witkins (1961), which led to the notion that UV induces lesions inbacterial DNA that either areremoved by repair systems or are converted to permanent structural changes during the first DNA synthetic period following the exposure. Wereport here an extension ofsuch experiments to eukaryotic cells, in which some effects of methyl green on the repair of UV (254 nm(-induceddamage inGl phase Xenopus cells are examined.Routine maintenance of log phase A8W243 Xenopus cultures, incubations, cell synchronizations, irradiations, mitotic index determina-tions, survival determinations (colony counts), and chromosome analysis have been described in detail (Griggs and Bender, 1971; Payne andGriggs, 1977; Griggs and Orr, 1979).The basic dye used, methylgreen, was obtained fromDifco.Figure 1 shows results of a series of mitotic index experiments performed to examine the effects of methyl green on, egression of UVirradiated Gl cells through interphase and the first succeeding mitosis, (Ml).These data described the appropriate time intervals forcollection ofthe sets of mitotic cells analyzed foreffects of methylgreen on UV-induced aberration production (Table 1). Concentrations of the dye in therange 0.0-0.003 g/1 appeared to induce little delay inprogression of the irradiated cells above that induced by the UV alone. The similarity inaverage height and widthof these mitotic peaks indicated that the dye did not significantly reduce the number of irradiated cells that reached G1.The average cell cycle for non-irradiated Xenopus cells was approximately 26 hours (not shown); eight hours Gl, 13 hours S, three hours G2,and two hours Ml.Payne and Griggs (1977) carried out autoradiographic studies which indicated that early Glphase cells, exposed to low dosesof UV (0 - 150 ergs mm" 2 range), are not delayed in their progression through Gl, but experience rather lengthy S phase delays. These facts,coupled withthe data of Figure 1 and Table 1, indicate that chromatid aberration frequencies, resulting from UVexposure ofearly Gl cells, aresignificantly altered by methyl green only when the dye is incontact withthe exposed cells as they pass through early S phase. Some relationshipbetween the aberrant intracellular mechanism, by which methylgreen augments chromatid type aberration production, and DNAsynthesis is sug-gested by the fact that both mechanisms appear to function withpeak efficiency in early S phase.The data of Figure 2 indicate that a methyl green concentration of 0.003 g/ 1has virtually no effect on the expression of UV-induced lethaldamage, nomatter where in the cell cycle the dye is applied. These data suggest that the mechanism which expresses UV-induced aberrationaldamage in Xenopus cells differs significantly from the mechanism which expresses lethal damage, supporting results of previous studies of theoverlap ofUV-induced lethal and aberrational lesions inXenopus cells (Griggs and Orr, 1979; Payne and Griggs, 1977).122Journal of the Arkansas Academy of Science, Vol. 34 [1980], Art. 40https://scholarworks.uark.edu/jaas/vol34/iss1/40

Isolation of Phospholipase A2 from Agkistrodon bilineatus Venom

ScholarWorks@UARK

Journal of the Arkansas Academy of ScienceVolume 34 Article 401980Isolation of Phospholipase A2 from Agkistrodonbilineatus VenomKarl H. LandbergArkansas State UniversityBob D. JohnsonArkansas State UniversityDewey H. SiffordArkansas State UniversityFollow this and additional works at: http://scholarworks.uark.edu/jaasPart of the Zoology 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 General Note is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the ArkansasAcademy of Science by an authorized editor of ScholarWorks@UARK. For more information, please contact scholar@uark.edu, ccmiddle@uark.edu.Recommended CitationLandberg, Karl H.; Johnson, Bob D.; and Sifford, Dewey H. (1980) "Isolation of Phospholipase A2 from Agkistrodon bilineatusVenom," Journal of the Arkansas Academy of Science: Vol. 34 , Article 40.Available at: http://scholarworks.uark.edu/jaas/vol34/iss1/40Arkansas Academy of Science Proceedings, Vol.XXXIV,1980 119General NotesBAUMGARTNER, A. M., F. M. BAUMGARTNER. 1944. Hawksand owls inOklahoma 1939-1942: Food habits and populationchanges. Wilson Bull.56:209-215.ERRINGTON, P. L. 1932. Studies on the behavior of the GreatHorned Owl. WilsonBull. 44:212-220.MILLER,L. 1930. The territorial concept in the Great Horned Owl.Condor 32:290-291.BENT, A.C. 1938. Life histories of North American birds of prey.Part II,U.S. Natl. Museum Bull.170:295-357.ERRINGTON, P. L. 1930. The pellet analysis method of raptor foodhabits study. Condor 32:292-296.RODNEY HARRIS, Science Dept., Southside HighSchool, Batesville Arkansas 72501 and EARLL.HANEBRINK. Div. ofBiological Science,Arkansas State University, State University, Arkansas 72467.ISOLATION OF PHOSPHOLIPASE A2 FROM AGKISTRODON BILINEATUS VENOMVenom produced by the Mexican mocassin, A.bilineatus, contains phosphomonoesterase, phosphodiesterase, 5-nucleotidase, esterases,thrombin-like, L-amino acid oxidase, protease, phospholipase A.., and NADnucleosidase activities (Tu et al.. 1967; Denson et al.. 1972; Siffordand Johnson, 1978;Brunson et al., 1978). Of these enzymes, phospholipase A2(PhL-A2) was chosen in this work forpossible isolation. This choiceof 1'hL- A > for isolation was due primarily toitsheat stability and toits distribution inthe eluates obtained byionexchange chromatography of thecrude venom as evidenced previously (Sifford and Johnson, 1978).Assay procedures with minor modifications (Sifford and Johnson, 1978) included phospholipase A2using the clearing ofan egg yolksuspen-sion (Marinetti, 1965), phosphomonoesterase and phosphodiesterase (Richards et al., 1965), esterase (Tu et al., 1965), 5'-nucleosidase (Lo et al.,1966; Ging, 1956), and L-amino acid oxidase (Paik and Kim, 1965). Hyaluronidase was assayed according to the turbidimetric procedures of Kassand Seastone (1944).A450 mg sample of crude venom (Sigma) was separated on Concanavalin Acovalently bound to Sepharose 4B gel (Con A)into glycopro-teins (anthrone reagent positive) and nonglycoproteins (anthrone reagent negative) by employing the methods ofIscove et al. (1974) and Aspbergand Porath (1970). In fractitmat ions by ion exchange chromatography employing DEAE Sephadex A-SO, the methods of Cheng and Ouyang(1967), Ouyang et al. (1971), and Johnson and Sifford(1978) were used. Proteins were desalted by using Sephadex G-10 columns at 4°C. Sephadex(5-75 and G-50 columns were used to separate I'hl.-A > from highermolecular weightmolecules.Animmunizing schedule was prepared according to Ownby et al. (1979). Preimmune serum was obtained from approximately 12 month oldNew Zealand white rabbits. An immunizing dose was prepared by dissolving 17 mg of lyophilized A. bilineatus crude venom in 20ml of sterilephysiological saline. A0.5 ml aliquot of this solution was then mixed with 0.5 ml of Freund's complete adjuvant. Injections of0.5 ml then wereE; subcutaneously into each thigh. Booster injections were prepared by mixing 0.5 ml of A. bilineatus venom (0.8 mg/ml) and 0.5 ml ofnd's complete adjuvant. One week later, subcutaneous injections of 0.5 ml of the solution were made in each shoulder. Four weeks after theter injections, antiserum via heart puncture was collected and stored at —20°C.kRabbit antiserum for the purified phospholipase A.> fraction was prepared by injecting an immunizing dose containing 0.3 ml of purifiedle(0.1 mg/ml) and 1.0 ml Freund's complete adjuvant. Subcutaneous injections of0.65 ml of this solution were made into each thigh. Onelater, booster injections of the same dose were administered into each shoulder. Four weeks later, antiserum was collected and stored at>Immunoelectrophoresis methods outlined by Campbell et al. (1963) and Garvey et al. (1977) were employed to determine PI1L-A2 purity,iecyl Sulfate-Polyacrylamide gel electrophoresis procedures of Weber and Osborn (1969) were used byDr.Collis Geren (University of Arkan-at Fayetteville) to assay crude venom and fraction samples.A. bilineatus crude venom contains nonglycoprotein and glycoprotein enzymes (Fig. 1). The larger nonglycoprotein fraction (Fraction I),comprising approximately 80% of the crude venom proteins, contained numerous enzyme activities. These included PhL-Ai, phosphomono-esterase, phosphodiesterase, 5 -nucleosidase, hyaluronidase, TAMEase, BAEEase, and L-amino acid oxidase activities.Fraction I,obtained by Con Achromatography, was pooled, lyophilized, and then desalted with Sephadex G-10. Fractionation of this de-fdnonglycoprotein fraction by ion exchange chromatography (DEAE Sephadex A-50) yielded three large fractions and several minor fractions.•A2 activity was concentrated inthe second major fraction (Fig. 2). PI1L-A2 activity (14,000 units/mg) in this fraction was much higher thanof the crude venom (234 units/mg).§The PhL-Ai-containing fraction obtained by chromatography with DEAE Sephadex A-50 was divided into two samples. Even-numberedis (42 through 66) were pooled to from one sample while odd-numbered tubes (41 through 67) formed the other sample. These samples, afterhilization and desalting, were fractionated withSephadex G-75. Inboth instances, PI1L-A2activity (24,400 units/mg) was observed onlyinthemolecular weight fraction (Fig. 3).The low molecular weight PI1L-A2fraction (tubes 16-22) obtained by Sephadex G-75 chromatography was pooled, lyophilized, desalted, andapplied to a Sephadex G-50 column. This fractionation yielded a fraction free of the larger molecules (Fig. 4).Ilmmunoelectrophoresis and disc electrophoresis of crude venom samples indicated a complex mixture of proteins although the PI1L-A2:ion obtained by the sequence of Con A-Sepharose 4B, DEAE A-50, Sephadex G-75, and Sephadex G-50 chromatography procedures wastly purified(Figs. 5-8). Close examinations of both types of electrophoresis patterns, however, indicated trace contaminations. These con-nations could be due, inpart, to TAMEase and L-amino acid oxidase since the distributions of these enzymes inA.bilineatus venom overlap¦A2 after DEAE Sephadex A-50 chromatography (Sifford and Johnson, 1978). At present, work is directed toward purification of largeunts ofPhL-A2inorder that more enzyme characteristics may be obtained.We thank Dr. Collis Geren for his work withthe gel electrophoresis, Dr.L.W. Hinck for his assistance withthe im-munological procedures, John Ruff forhis assistance throughout this work, and Mrs. Alice Chandler for typing the manu-script.119Journal of the Arkansas Academy of Science, Vol. 34 [1980], Art. 40Published by Arkansas Academy of Science, 1980't•II•I'120 Arkansas Academy of Science Proceedings, Vol.XXXIV,1980Figure 1. Chromatography of Agkistrodon bilineatus crude venom(450 mg) on a Concanavalin ASepharose 4B column (2.5 X 15 cm)at4°C by two stage elution. The arrow indicates the start of the secondstate elution (using o-methyl-D-mannoside). Eluates were collectedat a flow rate of 17 ml/hr.50 100 150 200 250Figure 2. Chromatography of Agkistrodon bilineatus venom non-glycoproteins on a DEAE Sephadex A-50 column (2.5 X 56 cm) at4°C by two stage elution with ammonium acetate buffer. Eluates of5.0 ml each were collected at a flow rate of 17 ml/hr.Figure 3. Chromatography of the phospholipase Aicontaining frac-tion, collected from DEAE A-50 ion exchange chromatography, onaSephadex G-75 column (1 X 100 cm) at 4°C. Eluates of 4.0 ml/tubewere collected.TUBE NUMBERFigure 4. Chromatography ofphospholipase Aicontaining fraction,from Sephadex G-75 chromatography, on a Sephadex G-50 column(1 X 100 cm) at 4°C. Eluates of 4.0 ml/tube were collected.120Journal of the Arkansas Academy of Science, Vol. 34 [1980], Art. 40http://scholarworks.uark.edu/jaas/vol34/iss1/40'Arkansas Academy of Science Proceedings, Vol.XXXIV,1980 121Figure 5. Immunoelectrophoretic patterns obtained by reactinganti-crude Agkistrodon bilineatus venom serum with electrophoret-ically separated crude venom.Figure 6. Immunoelectrophoretic patterns obtained by reactinganti-crude Agkistrodon bilineatus venom serum with electrophoret-ically separated phospholipase A> containing fraction and crudevenom.Figure 7. Immunoelectrophoretic patterns obtained by reactinganti-phospholipase Ai fraction serum with electrophoreticallyseparated phospholipase Aj containing fraction and Agkistrodonhilineatus crude venom.Figure 8. Patterns obtained by Dodecyl Sulfate-Polyacrylamide GelElectrophoresis of Agkistrodon bilineatus crude venom and the phos-pholipase Aicontaining fraction.LITERATURE CITEDASPBERG, K.and J. PORATH. 1970. Group-specific adsorption ofglycoproteins. ActaChem. Scand. 24:1839-1841.VNSON, E. M., B. D. JOHNSON and D. H. SIFFORD. 1978.NADnucleosidase of Agkistrodon bilineatus venom. Amer. J.Trop.Med. and Hyg. 27:365-368.CAMPBELL, D. H., J. S. GARVEY, N. E. CREMER and D. H.SUSSDORF. 1963. Immunoelectrophoresis. Pp. 149-155, InMethods inImmunology, ALaboratory Text for Instruction andResearch. W. A.BenjaminInc., New York.CHENG, H.and C. OUYANG. 1967. Isolation ofcoagulant and anti-coagulant principles from the venom of Agkistrodon acutus.Toxicon 4:235-243.KISON, K. W. E., F. E. RUSSELL, D. ALMAGRO and R. C.BISHOP. 1972. Characterization of the coagulant activity ofsome snake venoms. Toxicon 10:557-562.ItVEY, J. S., N.E. CREMER and D.H. SUSSDORF. 1977. Im-raunoelectrophoresis. Pp. 328-336, In Methods of Immunology,ALaboratory Text forInstruction and Research. W. A.Benja-min, Inc., Reading, Mass.IG, N. S. 1956. Extraction method of colorimetric determinationof phosphorus in microgram quantities. Anal. Chem. 28:1330-1333.IiVE, N. N.,F. SIEBER and K.H. WINTERHALTER. 1974.Erythroid colony formation in cultures of mouse and humanbone marrow: Analysis of the requirement for erythropoietinby gel filtration and affinity chromatography on Agarose-Concanavalin A.J. Cell. Physiol. 83:309-320.IS, E. H. and C. V.SEASTONE. 1944. The role of the mucoidpolysaccharide (Hyaluronic Acid) in the virulence of a group Ahemolytic streptococci. J. Exp. Med. 79:319-329.LO. T., Y.CHEN and C. LEE. 1966. Chemical studies of Formosancobra (Naja naja atra) venom. Part I.Chromatographic separa-tion of crude venom on CM-Sephadex and preliminary charac-terization of its components. J. Chinese ("hem. Soc. Ser. II13:25-37.MARINETTI,G. V. 1965. The action of phospholipase A on lipo-proteins. Biochim. Biophys. Acta. 98:554-565.OUYANG,C,J. HONGand C. TENG. 1971. Purification and prop-erties of the thrombin-like principle of Agkistrodon acutusvenom and its comparison with bovine thrombin. Thrombus.Diathes. haemorrh. (Stuttg.) 26:224-234.OWNBY, C. L., W. M. WOODS and G. V. ODELL. 1979.Antiserum to myotoxin from prairie rattlesnake (Crotalus viridisviridis) venom. Toxicon17:373-380.PAIK, K.and S. KIM.1965. pH substrate relationship of L-aminoacid oxidases from snake venom and rat kidney. Biochim. Bio-phys. Acta. 96:66-74.RICHARDS, G. M., G.DUVAIRand M.LASKOWSKI, SR. 1965.Comparison of the levels of phosphodiesterase, endonuclease,and monophosphatases in several snake venoms. Biochem.4:501-503.SIFFORD, D. H. and B. D. JOHNSON. 1978. Fractionation ofAgkistrodon bilineatus venom byion exchange chromatography,Pp. 231-242, In Toxins: Animal, Plant and Microbial (P. Ro-senberg, ed.) Pergamon Press, Oxfordand New York.TU,A. T., G. P. JAMES and A.CHUA. 1965. Some biochemicalevidence in support of the classification of venomous snakes.Toxicon 3:5-8.TU,A.T., P. M.TOOM and D.S. MURDOCK. 1967. Chemical dif-ferences in the venoms of genetically different snakes. Pp. 351-362, In Animal Toxins (Russell and Saunders, eds.) PergamonPress, Oxford and New York.121Journal of the Arkansas Academy of Science, Vol. 34 [1980], Art. 40Published by Arkansas Academy of Science, 1980122 Arkansas Academy of Science Proceedings, Vol.XXXIV,1980;¦Arkansas Academy of ScienceWEBER, K. and M. OSBORN. 1969. The reliability of molecularweight determinations by dodecyl sulfate-polyacrylamide gelelectrophoresis. J.Biol. Chem. 244:4406-4412.KARL H. LANDBERG, BOB D. JOHNSON and DEWEY H. SIFFORD. Department ofBiological Sciences and Department ofChemistry,Arkansas State University, State University, Arkansas 72467. \SAGE THRASHER (OREOSCOPTES MONTANUS), A NEW STATE RECORDOn 24 November 1979, Cheryl Lavers and Iwere looking forbirds inthe Farville area, about 6 miles NE of Jonesboro, Craighead County, iwhen we discovered a Sage Thrasher (Oreoscoptes montanus). The bird was rather Mockingbird-like (Mimus polyglottos) inshape, plain gray-brown crown and back, and bright yelloweye with ablack pupil. Other characters differed from a Mockingbird in that itwas smaller and shortertailed and it had a white throat with a black malar stripe. The underparts, witha ground color of warm buff, were densely streaked with blackchevrons arranged inlength-wise rows. There was a white wingbar, and a dark tail withwhite outer corners. The bill was slender, veryslightly de-curved, and dark bluish-black incolor, as were the rather short legs. The birdran along the ground inopen places, or under brush and stayed onornear the ground. Itwas observed catching, decapitating, then swallowing ground crickets (Acheta sp.). We have both observed Sage Thrashersin several western states. Anexamination of standard field guides (Peterson, R. T. 1961. A field guide to western birds. Houghton MifflinCo.,Boston; Robbins, C. S. et al. 1966. Birds of North America. Golden Press, N.Y.)further confirmed our identification. Adescription and slides ofthe bird have been sent to Charles Mills,Curator, the Arkansas Audubon Society, and Dr.Douglas James, Department of Zoology, University ofArkansas at Fayetteville. This is a firstrecorded instance of the Sage Thrasher inArkansas.Inits normal range, the Sage Thrasher breeds in the sagebrush (Artemisia) deserts of western North America, coming as far east as westernOklahoma. Itwinters in the southern part of its range and Mexico,occurring as far east as central and southern Texas, witha small isolated wintercolony in extreme southwestern Louisiana (A.O.U. check-list ofNorth American birds, 1957). The species is seldom recorded as a vagrant. Thefollowingextralimital occurrences to the east of its range have been recorded: Florida, 2;Illinois, 2;Maryland, 1;New York, 2;North Carolina.1;South Dakota, 1.NORMAN LAVERS,DivisionofEnglish, Philosophy, and Languages, Arkansas State University. State University, Arkansas 72467.:SOME EFFECTS OF METHYL GREEN ON EXPRESSION OF DAMAGEINDUCED INGl XENOPUS CELLS BY ULTRAVIOLETLIGHTMethylgreen shows a high degree of specificity forDNA and is a component of many nuclear stains. Kurnick (1952) observed that this basicdye was readily bound by polymerized DNA;two amino groups of the dye binding to twophosphoric acid groups of DNA.Errera (1951) demon-strated that the affinity ofDNA for methyl green is influenced byradiation and other agents which depolymerize DNA,or alter its internal con-figuration, Doudney and Haas (1958) showed that methyl green significantly influenced metabolic activities, such as DNA and RNA synthesis, inEscherichia coli. These results suggested that appropriate experiments, involving methyl green treatments coupled with germicidal UV expo-sures, might aid indescribing the expression and repair of UV-induced lethal and rautational damage inprokaryotic cells. Experiments of thisnature were carried out byinvestigators, such as Witkins (1961), which led to the notion that UV induces lesions inbacterial DNA that either areremoved by repair systems or are converted to permanent structural changes during the first DNA synthetic period following the exposure. Wereport here an extension ofsuch experiments to eukaryotic cells, in which some effects of methyl green on the repair of UV (254 nm(-induceddamage inGl phase Xenopus cells are examined.Routine maintenance of log phase A8W243 Xenopus cultures, incubations, cell synchronizations, irradiations, mitotic index determina-tions, survival determinations (colony counts), and chromosome analysis have been described in detail (Griggs and Bender, 1971; Payne andGriggs, 1977; Griggs and Orr, 1979).The basic dye used, methylgreen, was obtained fromDifco.Figure 1 shows results of a series of mitotic index experiments performed to examine the effects of methyl green on, egression of UVirradiated Gl cells through interphase and the first succeeding mitosis, (Ml).These data described the appropriate time intervals forcollection ofthe sets of mitotic cells analyzed foreffects of methylgreen on UV-induced aberration production (Table 1). Concentrations of the dye in therange 0.0 - 0.003 g/1 appeared to induce little delay inprogression of the irradiated cells above that induced by the UV alone. The similarity inaverage height and widthof these mitotic peaks indicated that the dye did not significantly reduce the number of irradiated cells that reached G1.The average cell cycle for non-irradiated Xenopus cells was approximately 26 hours (not shown); eight hours Gl, 13 hours S, three hours G2,and two hours Ml.Payne and Griggs (1977) carried out autoradiographic studies which indicated that early Glphase cells, exposed to low dosesof UV (0 - 150 ergs mm" 2 range), are not delayed in their progression through Gl, but experience rather lengthy S phase delays. These facts,coupled withthe data of Figure 1 and Table 1, indicate that chromatid aberration frequencies, resulting from UVexposure ofearly Gl cells, aresignificantly altered by methyl green only when the dye is incontact withthe exposed cells as they pass through early S phase. Some relationshipbetween the aberrant intracellular mechanism, by which methylgreen augments chromatid type aberration production, and DNAsynthesis is sug-gested by the fact that both mechanisms appear to function withpeak efficiency in early S phase.The data of Figure 2 indicate that a methyl green concentration of 0.003 g/ 1has virtually no effect on the expression of UV-induced lethaldamage, nomatter where in the cell cycle the dye is applied. These data suggest that the mechanism which expresses UV-induced aberrationaldamage in Xenopus cells differs significantly from the mechanism which expresses lethal damage, supporting results of previous studies of theoverlap ofUV-induced lethal and aberrational lesions inXenopus cells (Griggs and Orr, 1979; Payne and Griggs, 1977).122Journal of the Arkansas Academy of Science, Vol. 34 [1980], Art. 40http://scholarworks.uark.edu/jaas/vol34/iss1/40

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Isolation of Phospholipase A2 from Agkistrodon bilineatus Venom

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