Download PDF
Thromb Haemost 2000; 83(03): 438-444
DOI: 10.1055/s-0037-1613834
Review Article
Schattauer GmbH

A Novel Fibrinogen-clotting Enzyme, TL-BJ, from the Venom of the Snake Bothrops jararaca: Purification and Characterization

Solange M. T. Serrano
1   From the Laboratório de Bioquímica e Biofísica, Instituto Butantan, São Paulo
,
Claudio A. M. Sampaio
2   From the Departamento de Bioquímica, EPM-UNIFESP, São Paulo, Brazil
,
Reinhard Mentele
3   From the Abteilung für Klinische Chemie und Klinische Biochemie, Chirurgische Klinik, Klinikum Innenstadt, Ludwig-Maximilians-Universität, München, Germany
,
Antonio C. M. Camargo
1   From the Laboratório de Bioquímica e Biofísica, Instituto Butantan, São Paulo
,
Edwin Fink
3   From the Abteilung für Klinische Chemie und Klinische Biochemie, Chirurgische Klinik, Klinikum Innenstadt, Ludwig-Maximilians-Universität, München, Germany
› Author Affiliations
Further Information

Publication History

Received 05 April 1999

Accepted after resubmission 19 November 1999

Publication Date:
14 December 2017 (online)

Also available at
    Permissions and Reprints

Summary

Three chromatographically distinct forms of a novel fibrinogenclotting serine endopeptidase, TL-BJ 1, 2 and 3, were purified from the venom of Bothrops jararaca by a combination of ammonium sulfate precipitation and chromatographic steps. The three forms of TL-BJ have similar amidolytic and plasma coagulating activities. TL-BJ 1, TL-BJ 2 and TL-BJ 3 cause the specific clotting of fibrinogen with release of fibrinopeptide A, the specific activities are 16.8 NIH U/mg (TL-BJ 1), 16.7 NIH U/mg (TL-BJ 2) and 20.8 NIH U/mg (TL-BJ 3). The most sensitive chromogenic substrates for measuring the amidolytic activity of TL-BJ 3 were D-Pro-Phe-Arg-pNA, D-Phe-pipecolyl-ArgpNA and Z-D-Arg-Gly-Arg-pNA. The amidolytic and coagulant activities of TL-BJ were inhibited by phenylmethylsulfonyl fluoride but not by hirudin. Benzamidine derivatives, which are competitive inhibitors of trypsin-like serine endopeptidases, also inhibited the amidolytic activity of TL-BJ. In SDS/PAGE the main bands of TL-BJ 1, 2 and 3 showed molecular masses of 30 kDa, 31 kDa and 32 kDa. Upon incubation with N-glycosidase F only TL-BJ 3 remained unchanged, whereas TL-BJ 1 and TL-BJ 2 showed products with molecular masses around 23 kDa. Thus, TL-BJ 3 does not seem to be N-glycosylated. The N-terminal amino acid sequences of TL-BJ 2 and TL-BJ 3 are identical while TL-BJ 1 has five substitutions.

Keywords

Snake venom - purification - serine peptidase - fibrinogen-clotting
 

  • References

  • 1 Macfarlane RG. Russell’s viper venom. Br J Haematol 1967; 13: 437-51.
    CrossrefPubMedSearch in Google Scholar
  • 2 Stocker KF. In: Medical use of snake venom proteins, Stocker KF. ed. Boca Raton; CRC Press: 1990: 97-160.
    Search in Google Scholar
  • 3 Hutton RA, Warrel DA. Action of venom components on the haemostatic system. Blood Rev 1993; 07: 176-89.
    CrossrefPubMedSearch in Google Scholar
  • 4 Pirkle H. Thrombin-like enzymes from snake venoms: an updated inventory. Thromb Haemost 1998; 79: 675-83.
    Thieme ConnectPubMedSearch in Google Scholar
  • 5 von Klobusitzky D, König P. Estudos biochimicos sobre os venenos das serpentes do gênero Bothrops. . Mem. Inst. Butantan (São Paulo) 1935/36; 10: 223-36.
    PubMed
  • 6 Denson KWE, Rousseau WE. Separation of the coagulant components of Bothrops jararaca venom. Toxicon 1970; 08: 15-9.
    CrossrefPubMedSearch in Google Scholar
  • 7 Furukawa Y, Hayashi K. Factor X converting and thrombin-like activities of Bothrops jararaca snake venom. Toxicon 1977; 15: 107-14.
    CrossrefPubMedSearch in Google Scholar
  • 8 Nahas L, Kamiguti AS, Barros MAR. Thrombin-like and factor X-activator components of Bothrops snake venoms. Thromb Haemost 1979; 41: 314-28.
    Thieme ConnectPubMedSearch in Google Scholar
  • 9 Nishida S, Fujimura Y, Miura S, Ozaki Y, Usami Y, Suzuki M, Titani K, Yoshida E, Sugimoto M, Yoshioka A, Fukui H. Purification and characterization of bothrombin, a fibrinogen-clotting serine protease from the venom of Bothrops jararaca. . Biochemistry 1994; 33: 1843-9.
    CrossrefPubMedSearch in Google Scholar
  • 10 Serrano SMT, Mentele R, Sampaio CAM, Fink E. Purification, characterization, and amino acid sequence of a serine endopeptidase, PA-BJ, with platelet-aggregating activity from the venom of Bothrops jararaca. . Biochemistry 1995; 34: 7186-93.
    CrossrefPubMedSearch in Google Scholar
  • 11 Serrano SMT, Hagiwara Y, Murayama N, Higuchi S, Mentele R, Sampaio CAM, Camargo ACM, Fink E. Purification and characterization of a kinin-releasing and fibrinogen-clotting serine endopeptidase (KN-BJ) from the venom of Bothrops jararaca, and molecular cloning and sequence analysis of its cDNA. Eur J Biochem 1998; 251: 845-53.
    CrossrefPubMedSearch in Google Scholar
  • 12 Henriques OB, Fichman M, Henriques SB. Partial purification and some properties of the blood-clotting factor from the venom of Bothrops jararaca. . Biochem J 1960; 75: 551-6.
    CrossrefPubMedSearch in Google Scholar
  • 13 Fink E, Schill W-B, Fiedler F, Krasnigg G, Geiger R, Shimamoto K. Tissue kallikrein of human seminal plasma is secreted by the prostate gland. Biol Chem Hoppe-Seyler 1985; 366: 917-24.
    PubMedSearch in Google Scholar
  • 14 Mustard JF, Perry DW, Ardlie NG, Packham MA. Preparation of suspensions of washed platelets from humans. Br J Haematol 1972; 22: 193-204.
    CrossrefPubMedSearch in Google Scholar
  • 15 Fiedler F, Geiger R, Hirschauer C, Leysath G. Peptide esters and nitroanilides as substrates for the assay of human urinary kallikrein. Hoppe- Seyler’s Z Physiol Chem 1978; 259: 1667-73.
    PubMedSearch in Google Scholar
  • 16 Jameson GW, Roberts DV, Adams RW, Kyle WSA, Elmore DT. Determination of the operational molarity of solutions of bovine α-chymotrypsin, trypsin, thrombin and factor Xa by spectrofluorimetric titration. Biochem J 1973; 131: 107-17.
    CrossrefPubMedSearch in Google Scholar
  • 17 Stürzebecher J, Stürzebecher U, Markwardt F. Inhibition of batroxobin, a serine endopeptidase from Bothrops snake venom, by derivatives of benzamidine. Toxicon 1986; 24: 585-95.
    CrossrefPubMedSearch in Google Scholar
  • 18 Dixon M. The determination of enzyme inhibitor constants. Biochem J 1953; 55: 170-1.
    CrossrefPubMedSearch in Google Scholar
  • 19 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteoriophage T4. Nature 1970; 227: 680-5.
    CrossrefPubMedSearch in Google Scholar
  • 20 Heukeshoven J, Dernick R. Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis 1985; 06: 103-12.
    CrossrefPubMedSearch in Google Scholar
  • 21 Friedman M, Krull LH, Cavins JF. The chromatographic determination of cystine and cysteine residues in proteins as S-beta-(4-pyridylethyl)cysteine. J Biol Chem 1970; 245: 3868-71.
    PubMedSearch in Google Scholar
  • 22 Lis H, Sharon N. Protein glycosylation. Structural and functional aspects. Eur J Biochem 1993; 218: 1-27.
    CrossrefPubMedSearch in Google Scholar
  • 23 Pirkle H. Thrombin-like enzymes from snake venoms: an updated inventory. Thromb Haemost 1998; 79: 675-83.
    Thieme ConnectPubMedSearch in Google Scholar
  • 24 Tanaka N, Nakada H, Itoh N, Mizuno Y, Takanishi M, Kawasaki T, Tate S, Inagaki F, Yamashina I. Novel structure of the N-acetylgalactosamine containing N-glycosidic carbohydrate chain of batroxobin, a thrombin-like snake venom enzyme. J. Biochem 1992; 112: 68-74.
    CrossrefPubMedSearch in Google Scholar
  • 25 Blombäck B, Blombäck M, Hessel B, Iwanaga S. Structure of N-terminal fragments of fibrinogen and specificity of thrombin. Nature 1967; 215: 1445-8.
    CrossrefPubMedSearch in Google Scholar
  • 26 Nolan C, Hall LS, Barlow GH. Ancrod, the coagulant enzyme from Malayan pit viper (Agkistrodon rhodostoma) venom. Meth Enzymol 1976; 45B: 205-13.
    PubMedSearch in Google Scholar
  • 27 Stocker K, Barlow GH. The coagulant enzyme from Bothrops atrox venom (batroxobin). Meth Enzymol 1976; 45B: 214-23.
    PubMedSearch in Google Scholar
  • 28 Hung CC, Huang KF, Chiou SH. Characterization of one novel venom protease with beta fibrinogenase activity from the Taiwan habu (Trimeresurus mucrosquamatus): purification and cDNA sequence analysis. Biochem Biophys Res Commun 1994; 205: 1707-15.
    CrossrefPubMedSearch in Google Scholar
  • 29 Iwasaki A, Shieh TC, Shimohigashi Y, Waki M, Kihara H, Ohno M. Purification and characterization of a coagulant enzyme, okinaxobin I, from the venom of Trimeresurus okinavensis (Himehabu snake) which releases fibrinopeptide. B J Biochem 1990; 108: 822-8.
    PubMedSearch in Google Scholar
  • 30 Faure G, Bon C. Crotoxin, a phospholipase A2 neurotoxin from the South American rattlesnake Crotalus durissus terrificus: purification of several isoforms and comparison of their molecular structure and of their biological activities. Biochemistry 1988; 27: 730-8.
    CrossrefPubMedSearch in Google Scholar
  • 31 Faure G, Guillaume J-L, Camoin L, Saliou B, Bon C. Multiplicity of acidic subunit isoforms of crotoxin, the phospholipase A2 neurotoxin from Crotalus durissus terrificus venom, results from posttranslational modifications. Biochemistry 1991; 30: 8074-83.
    CrossrefPubMedSearch in Google Scholar
  • 32 Tokunaga F, Nagasawa K, Tamura S, Miyata T, Iwanaga S, Kisiel W. The factor V-activating enzyme (RVV-V) from Russell’s viper venom. Identification of isoproteins RVV-V alpha, -V beta, and V-gamma and their complete amino acid sequences. J Biol Chem 1988; 263: 17471-81.
    PubMedSearch in Google Scholar
  • 33 Randolph A, Chamberlain SH, Chu H-LC, Retzios AD, Markland FS, Masiarz FR. Amino acid sequence of fibrolase, a direct-acting fibrinolytic enzyme from Agkistrodon contortrix contortrix venom. Protein Sci 1992; 01: 590-600.
    CrossrefPubMedSearch in Google Scholar
  • 34 Deshimaru M, Ogawa T, Nakashima K-I, Nobuhisa I, Chijiwa T, Shimohigashi Y, Furumaki Y, Niwa M, Yamashina I, Hattori S, Ohno M. Accelerated evolution of crotalinae snake venom gland serine proteases. FEBS Letters 1996; 397: 83-8.
    CrossrefPubMedSearch in Google Scholar
  • 35 Hahn B-S, Yang K-Y, Park E-M, Chang I-M, Kim Y-S. Purification and molecular cloning of calobin, a thrombin-like enzyme from Agkistrodon caliginosus (Korean viper). J Biochem 1996; 119: 835-43.
    CrossrefPubMedSearch in Google Scholar
  • 36 Shieh T-C, Kawabata S-I, Kihara H, Ohno M, Iwanaga S. Amino acid sequence of a coagulant enzyme, flavoxobin, from Trimeresurus flavoviridis venom. J Biochem 1988; 103: 596-605.
    CrossrefPubMedSearch in Google Scholar
  • 37 Pirkle H, Theodor I, Lopez R. Catroxobin, a weakly thrombin-like enzyme from the venom of Crotalus atrox. NH2-terminal and active site amino acid sequences. Thromb Res 1989; 56: 159-68.
    CrossrefPubMedSearch in Google Scholar
  • 38 Pirkle H, Theodor I, Miyada D, Simmons G. Thrombin-like enzyme from the venom of Bitis gabonica. Purification, properties and coagulant actions. J Biol Chem 1986; 261: 8830-5.
    PubMedSearch in Google Scholar
  • 39 Au L-C, Lin S-B, Chou J-S, Teh G-W, Chang K-J, Shih C-M. Molecular cloning and sequence analysis of the cDNA for ancrod, a thrombin-like enzyme from the venom of Calloselasma rhodostoma. . Biochem J 1993; 294: 387-90.
    CrossrefPubMedSearch in Google Scholar
  • 40 Bode W, Mayr I, Baumann U, Huber R, Stone SR, Hofsteenge J. The refined 1.9 Å X-ray crystal structure of human α.-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-ProTrp insertion segment. Embo J 1989; 08: 3467-75.
    PubMedSearch in Google Scholar
  • 41 Mandelbaum FR, Henriques OB. Purification and properties of Bothrops protease A. Arch Biochem Biophys 1964; 104: 369-73.
    CrossrefPubMedSearch in Google Scholar