Johnson, Bob D.

Ruff, John D.

Sifford, Dewey H.

English

ScholarWorks@UARK

Journal of the Arkansas Academy of ScienceVolume 34 Article 481980Glycoprotein Proteinase in Agkistrodon bilineatusVenomJohn D. RuffArkansas 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 CitationRuff, John D.; Johnson, Bob D.; and Sifford, Dewey H. (1980) "Glycoprotein Proteinase in Agkistrodon bilineatus Venom," Journal ofthe Arkansas Academy of Science: Vol. 34 , Article 48.Available at: http://scholarworks.uark.edu/jaas/vol34/iss1/48130 Arkansas Academy of Science Proceedings, Vol.XXXIV,1980A GLYCOPROTEIN PROTEINASE INAGKISTRODON BILINEATUSVENOMSnake venoms are noted for their wide variety of proteolytic enzymes. The Mexican mocassin Agkistrodon bilineatus is apit viper whostcrude venom isno exception (Sifford and Johnson, 1978). The goal of this work was to determine ifA. bilineatus venom contains glycoproteinproteases.Assay procedures for protease, phosphomonoesterase, phosphodiesterase, 5-nucleotidase, phospholipase A2, N-benzoyl-L-arginineethylesterase (BAEEase) and p-toluenesuIfonyl-L-arginine methylesterase (TAMEase), thrombin-like, L-amino acid oxidase and NADnucleosidase were the same as those used in previous works (Sifford and Johnson, 1978; Brunson et al., 1978). Hyaluronidase activity wasmeasured by the turbidimetric method ofKass and Seastone (1944).Separations of crude venom intoproteins positive to the anthrone reagent (glycoproteins) and nonglycoproteins were performed using Con-canavalin A (Con A)covalently bound to Sepharose 4B gelas described by Iscove et al. (1974) and Aspberg and Porath (1970). The glycoproteinfraction was desalted witha column (1 X 90 cm) ofSephadex G-10 at 4°C. This desalted fraction was lyophilizedand then fractionated by ionex-change chromatography (DEAE Sephadex A-50). In this procedure the methods ofCheng and Ouyang (1967) and Ouyang et al. (1971) includingthe modifications by Sifford and Johnson (1978) were used. Sephadex G-100 at 4°C was then used to separate molecules according to their mole-cular weight. Dodecyl Sulfate-Polyacrylamide GelElectrophoresis separations were performed by Dr.Collis Geren, University of Arkansas atFayetteville, according to the procedures outlined byWeber and Osborn (1969).Antisera inNew Zealand white rabbits were developed according to Ownby et al. (1979). Immunoelectrophoresis procedures (Campbell etal., 1963; Buchler Instruments Manual, 1964; Garvey et al., 1977) were used to determine the purityof the glycoprotein protease enzyme.Fractionation of the crude venom withCon Ayielded two fractions; Fraction Icomposed of nonglycoproteins and Fraction IIcomposed ofglycoproteins (Fig. 1). The glycoprotein content of the crude venom was calculated as 17.4%. Enzyme activities of Fraction IIwere determined.Mean enzyme-specific activities included 11.79 for phosphomonoesterase; 3.54 for phosphodiesterase; 83units/mg forphospholipase Ai;2.0 timoles/min/mg for 5'-nucleotidase; 0.01 PUsf| forprotease; 66 TRU and 332 NF units forhyaluronidase;0.04 units/mg forNADase; 70 units/mg forBAEEase; 20 units/mg forTAMEase; and 1.5 for L-amino acid oxidase. Thrombin-likeactivity was notobserved inFraction II.Fraction IIwas pooled, lyophilized, desalted, and applied to a DEAE Sephadex A-50 column. This fractionation yielded several minor frac-tions and one major fraction, Fraction Pi (Fig. 2).Protease activity was present inFraction P|. This fraction was lyophilized and 5 ml of distilled water added. Fractionation of 1ml aliquotswithSephadex G-100 yielded a fraction (P2) having protease activity along withlow NADase, TAMEase, and BAEEase activities (Fig. 3). Assaysforphosphomonoesterase, phosphodiesterase, phospholipase Ai,5-nucleotidase, hyaluronidase, and L-amino acid oxidase in the fraction werenegative.Disc electrophoresis of the crude venom produced 12 fractions. Fraction P.>, containing the glycoprotein protease, contained 5 components(Fig.4). The trace activities ofNADase, BAEEase, and TAMEase inFraction P? may account for these discs.Seven precipitin arcs were produced withcrude venom as the electrophoretically separated antigens and withthe crude venom antiserum inthe trough (Fig. 5). By using Fraction P> in one welland crude venom in the other well as the electrophoretically separated antigens and crudevenom antiserum inthe trough, one precipitinarc (A)was produced byFraction P? and the antiserum. The crude venom antigens again producedseven precipitin arcs (Fig. 6). ByusingFraction P.> inone well and crude venom in the other well as the electrophoretically separated antigens andFraction P? antiserum in the trough, one precipitin arc was produced against Fraction P> and the crude venom (Fig. 7). Although this arc isprobably due to the glycoprotein protease more data are required forsubstantiation.Figure 1. Chromatography of Agkistrodon bilineatus crude venom(450 mg) on a Concanavalin ASepharose 4-B column (2.5 X 15 cm)at 4°C by two stage elution. The arrow indicates the start of thesecond stage elution (using a-methyl-D-mannoside). Eluates (5 ml/tube) were collected at aflow rate of 17 ml/hr.Figure 2. Chromatography of Agkistrodon bilineatus venom glyco-proteins on a DEAE Sephadex A-50 column (2.5 X 56 cm) at 4°Cusing an ammonium acetate buffer gradient. Eluates of 5.0 ml/tubewere collected at a flow rate of 17 ml/hr.130Journal of the Arkansas Academy of Science, Vol. 34 [1980], Art. 48Published by Arkansas Academy of Science, 1980''Arkansas Academy of Science Proceedings, Vol.XXXIV,1980 131E5. Composite slide of immuno>phoretic patterns obtained by re-anti-crude Agkistrodon bilineatusiserum with electrophoreticallyted crude venom. Figure 6. Composite slide of immuno-electrophoretic patterns obtained by re-acting anti-crude Agkistrodon bilineatusvenom serum with electrophoreticallyseparated proteinase containing fraction(P2) and/4, bilineatus crude venom.Figure 4. Comparison of patterns ob-tained by Dodecyl Sulfate-Polyacryla-raide GelElectrophoresis of Agkistrodonbilineatus crude venom and the proteinasecontaining fraction (P?).Figure 3. Chromatography of Fraction Pi (Fig. 2)on a Sephadex G-100 column (1 X 90 cm) at 4°C. Eluates of 4.0 ml/tube were col-lected at a flowrate of 16 ml/hr.Figure 7. Immunoelectrophoretic pat-terns obtained by reacting anti-FractionP> serum with electrophoreticallyseparated proteinase containing fraction(P2) and Agkistrodon bilineatus crudevenom.We thank Dr.Collis Geren forhis work withthe gel electrophoresis, Dr.L.W. Hinck forhis assistance withthe immu-nological procedures, KarlLandberg for his assistance throughout this work, and Mrs. Alice Chandler for typing the manu-script.LITERATURE CITEDrBERG, K.and J. PORATH. 1970. Group-specific adsorption ofglycoproteins. Acta Chem. Scand. 24:1839-1841.r'NSON, E. M., B. D. JOHNSON and D. H. SIFFORD. 1978.NADnucleosidase of Agkistrodon bilineatus venom. Amer. J.Trop.Med. and Hyg.27:365-368.r'HLER. 1964. Routine Clinical Analysis using the Buchler Elec-trophoresis Apparatus. Buchler Instruments Division Nuclear-Chicago Corporation.flPBELL, D. H., J. S. GARVEY, N. E. CREMER and D. H.SUSSDORF. 1963. Immunoelectrophoresis. Pp. 149-155, InMethods in Immunology, A Laboratory Text for Instructionand Research. W. A.Benjamin, Inc., New York.rNG, H.andC. OUYANG.1967. Isolation ofcoagulant and anti-coagulant principles from the venom of Agkistrodon acutus.Toxicon 4:235-243.rtVEY, J.S., N. E. CREMER and D. H.SUSSDORF. 1977. Im-munoelectrophoresis. Pp. 328-336, InMethods of Immunology,ALaboratory Text for Instruction and Research. W. A. Benja-min, Inc.,Reading, Mass.ISCOVE, 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 chroraatography on Agarose-Con-canavalin A.J. Cell. Physiol. 83:309-320.KASS, 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.OUYANG,C,J. HONG andC. TENG. 1971. Purification and prop-erties of the thrombin-like principle of Agkistrodon acutusvenom and its comparison with bovine thrombin. Thrombos.Diathes. haemorrh. (Stuttg.) 26:224-234.OWNBY, C. L., W. M.WOODS and G. V.ODELL. 1979. Antiserumto myotoxin from prairie rattlesnake (Crotalus viridis viridis)venom. Toxicon 17:373-380.SIFFORD, D. H. and B. D. JOHNSON. 1978. Fractionation ofAgkistrodon bilineatus venom by ion exchange chromatography.Pp. 231-242, In Toxins: Animal, Plant and Microbial (P.Rosenberg, ed.) Pergamon Press, Oxfordand New York.WEBER, K. and M. OSBORN. 1969. The reliability of molecularweight determinations by dodecyl sulfate-polyacrylaraide gelelectrophoresis. J. Biol. Chem. 244:4406-4412.JOHN D. RUFF, BOB D. JOHNSON and DEWEY H. SIFFORD. Department ofBiologicalSciences and Department ofChemistry. ArkansasState University, State University, Arkansas 72467.131Journal of the Arkansas Academy of Science, Vol. 34 [1980], Art. 48http://scholarworks.uark.edu/jaas/vol34/iss1/48

Glycoprotein Proteinase in Agkistrodon bilineatus Venom

UARK (University of Arkansas )

Journal of the Arkansas Academy of Science Volume 34 Article 48 1980 Glycoprotein Proteinase in Agkistrodon bilineatus Venom John D. Ruff 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 Ruff, John D.; Johnson, Bob D.; and Sifford, Dewey H. (1980) "Glycoprotein Proteinase in Agkistrodon bilineatus Venom," Journal of the Arkansas Academy of Science: Vol. 34 , Article 48. Available at: https://scholarworks.uark.edu/jaas/vol34/iss1/48 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. 130 Arkansas Academy of Science Proceedings, Vol.XXXIV,1980A GLYCOPROTEIN PROTEINASE INAGKISTRODON BILINEATUSVENOMSnake venoms are noted for their wide variety of proteolytic enzymes. The Mexican mocassin Agkistrodon bilineatus is apit viper whostcrude venom isno exception (Sifford and Johnson, 1978). The goal of this work was to determine ifA. bilineatus venom contains glycoproteinproteases.Assay procedures for protease, phosphomonoesterase, phosphodiesterase, 5-nucleotidase, phospholipase A2, N-benzoyl-L-arginineethylesterase (BAEEase) and p-toluenesuIfonyl-L-arginine methylesterase (TAMEase), thrombin-like, L-amino acid oxidase and NADnucleosidase were the same as those used in previous works (Sifford and Johnson, 1978; Brunson et al., 1978). Hyaluronidase activity wasmeasured by the turbidimetric method ofKass and Seastone (1944).Separations of crude venom intoproteins positive to the anthrone reagent (glycoproteins) and nonglycoproteins were performed using Con-canavalin A (Con A)covalently bound to Sepharose 4B gelas described by Iscove et al. (1974) and Aspberg and Porath (1970). The glycoproteinfraction was desalted witha column (1 X 90 cm) ofSephadex G-10 at 4°C. This desalted fraction was lyophilizedand then fractionated by ionex-change chromatography (DEAE Sephadex A-50). In this procedure the methods ofCheng and Ouyang (1967) and Ouyang et al. (1971) includingthe modifications by Sifford and Johnson (1978) were used. Sephadex G-100 at 4°C was then used to separate molecules according to their mole-cular weight. Dodecyl Sulfate-Polyacrylamide GelElectrophoresis separations were performed by Dr.Collis Geren, University of Arkansas atFayetteville, according to the procedures outlined byWeber and Osborn (1969).Antisera inNew Zealand white rabbits were developed according to Ownby et al. (1979). Immunoelectrophoresis procedures (Campbell etal., 1963; Buchler Instruments Manual, 1964; Garvey et al., 1977) were used to determine the purityof the glycoprotein protease enzyme.Fractionation of the crude venom withCon Ayielded two fractions; Fraction Icomposed of nonglycoproteins and Fraction IIcomposed ofglycoproteins (Fig. 1). The glycoprotein content of the crude venom was calculated as 17.4%. Enzyme activities of Fraction IIwere determined.Mean enzyme-specific activities included 11.79 for phosphomonoesterase; 3.54 for phosphodiesterase; 83units/mg forphospholipase Ai;2.0 timoles/min/mg for 5'-nucleotidase; 0.01 PUsf| forprotease; 66 TRU and 332 NF units forhyaluronidase;0.04 units/mg forNADase; 70 units/mg forBAEEase; 20 units/mg forTAMEase; and 1.5 for L-amino acid oxidase. Thrombin-likeactivity was notobserved inFraction II.Fraction IIwas pooled, lyophilized, desalted, and applied to a DEAE Sephadex A-50 column. This fractionation yielded several minor frac-tions and one major fraction, Fraction Pi (Fig. 2).Protease activity was present inFraction P|. This fraction was lyophilized and 5 ml of distilled water added. Fractionation of 1ml aliquotswithSephadex G-100 yielded a fraction (P2) having protease activity along withlow NADase, TAMEase, and BAEEase activities (Fig. 3). Assaysforphosphomonoesterase, phosphodiesterase, phospholipase Ai,5-nucleotidase, hyaluronidase, and L-amino acid oxidase in the fraction werenegative.Disc electrophoresis of the crude venom produced 12 fractions. Fraction P.>, containing the glycoprotein protease, contained 5 components(Fig.4). The trace activities ofNADase, BAEEase, and TAMEase inFraction P? may account for these discs.Seven precipitin arcs were produced withcrude venom as the electrophoretically separated antigens and withthe crude venom antiserum inthe trough (Fig. 5). By using Fraction P> in one welland crude venom in the other well as the electrophoretically separated antigens and crudevenom antiserum inthe trough, one precipitinarc (A)was produced byFraction P? and the antiserum. The crude venom antigens again producedseven precipitin arcs (Fig. 6). ByusingFraction P.> inone well and crude venom in the other well as the electrophoretically separated antigens andFraction P? antiserum in the trough, one precipitin arc was produced against Fraction P> and the crude venom (Fig. 7). Although this arc isprobably due to the glycoprotein protease more data are required forsubstantiation.Figure 1. Chromatography of Agkistrodon bilineatus crude venom(450 mg) on a Concanavalin ASepharose 4-B column (2.5 X 15 cm)at 4°C by two stage elution. The arrow indicates the start of thesecond stage elution (using a-methyl-D-mannoside). Eluates (5 ml/tube) were collected at aflow rate of 17 ml/hr.Figure 2. Chromatography of Agkistrodon bilineatus venom glyco-proteins on a DEAE Sephadex A-50 column (2.5 X 56 cm) at 4°Cusing an ammonium acetate buffer gradient. Eluates of 5.0 ml/tubewere collected at a flow rate of 17 ml/hr.130Journal of the Arkansas Academy of Science, Vol. 34 [1980], Art. 48Published by Arkansas Academy of Science, 1980''Arkansas Academy of Science Proceedings, Vol.XXXIV,1980 131E5.Composite slide of immuno>phoretic patterns obtained by re-anti-crude Agkistrodon bilineatusiserum with electrophoreticallyted crude venom.Figure 6. Composite slide of immuno-electrophoretic patterns obtained by re-acting anti-crude Agkistrodon bilineatusvenom serum with electrophoreticallyseparated proteinase containing fraction(P2) and/4, bilineatus crude venom.Figure 4. Comparison of patterns ob-tained by Dodecyl Sulfate-Polyacryla-raide GelElectrophoresis of Agkistrodonbilineatus crude venom and the proteinasecontaining fraction (P?).Figure 3. Chromatography of Fraction Pi (Fig. 2)on a Sephadex G-100 column (1 X 90 cm) at 4°C. Eluates of 4.0 ml/tube were col-lected at a flowrate of 16 ml/hr.Figure 7. Immunoelectrophoretic pat-terns obtained by reacting anti-FractionP> serum with electrophoreticallyseparated proteinase containing fraction(P2) and Agkistrodon bilineatus crudevenom.We thank Dr.Collis Geren forhis work withthe gel electrophoresis, Dr.L.W. Hinck forhis assistance withthe immu-nological procedures, KarlLandberg for his assistance throughout this work, and Mrs. Alice Chandler for typing the manu-script.LITERATURE CITEDrBERG, K.and J. PORATH. 1970. Group-specific adsorption ofglycoproteins. Acta Chem. Scand. 24:1839-1841.r'NSON, E. M., B. D. JOHNSON and D. H. SIFFORD. 1978.NADnucleosidase of Agkistrodon bilineatus venom. Amer. J.Trop.Med. and Hyg.27:365-368.r'HLER. 1964. Routine Clinical Analysis using the Buchler Elec-trophoresis Apparatus. Buchler Instruments Division Nuclear-Chicago Corporation.flPBELL, D. H., J. S. GARVEY, N. E. CREMER and D. H.SUSSDORF. 1963. Immunoelectrophoresis. Pp. 149-155, InMethods in Immunology, A Laboratory Text for Instructionand Research. W. A.Benjamin, Inc., New York.rNG, H.andC. OUYANG.1967. Isolation ofcoagulant and anti-coagulant principles from the venom of Agkistrodon acutus.Toxicon 4:235-243.rtVEY, J.S., N. E. CREMER and D. H.SUSSDORF. 1977. Im-munoelectrophoresis. Pp. 328-336, InMethods of Immunology,ALaboratory Text for Instruction and Research. W. A. Benja-min, Inc.,Reading, Mass.ISCOVE, 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 chroraatography on Agarose-Con-canavalin A.J. Cell. Physiol. 83:309-320.KASS, 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.OUYANG,C,J. HONG andC. TENG. 1971. Purification and prop-erties of the thrombin-like principle of Agkistrodon acutusvenom and its comparison with bovine thrombin. Thrombos.Diathes. haemorrh. (Stuttg.) 26:224-234.OWNBY, C. L., W. M.WOODS and G. V.ODELL. 1979. Antiserumto myotoxin from prairie rattlesnake (Crotalus viridis viridis)venom. Toxicon 17:373-380.SIFFORD, D. H. and B. D. JOHNSON. 1978. Fractionation ofAgkistrodon bilineatus venom by ion exchange chromatography.Pp. 231-242, In Toxins: Animal, Plant and Microbial (P.Rosenberg, ed.) Pergamon Press, Oxfordand New York.WEBER, K. and M. OSBORN. 1969. The reliability of molecularweight determinations by dodecyl sulfate-polyacrylaraide gelelectrophoresis. J. Biol. Chem. 244:4406-4412.JOHN D. RUFF, BOB D. JOHNSON and DEWEY H. SIFFORD. Department ofBiologicalSciences and Department ofChemistry. ArkansasState University, State University, Arkansas 72467.131Journal of the Arkansas Academy of Science, Vol. 34 [1980], Art. 48https://scholarworks.uark.edu/jaas/vol34/iss1/48

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Glycoprotein Proteinase in Agkistrodon bilineatus Venom

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