Purificação e caracterização biológica de uma nova serinoprotease com atividade trombina

José Antonio Valeriano Zapana

In this word, a new serine protease with thrombin "like" activity the venom of Bothrops andianus (TLBan), snake of the Andes of Peru, was isolated by two steps: molecular exclusion chromatography G-75 and liquid chromatography in reversed-phase HPLC; with a high degree of purity and molecular homegenidade. Through of electrophoresis on SDS-PAGE shows the TLBan have a relative mass of 29 kDa under reducing conditions and 26 kDa in reducing conditions that was not confirmed for precision by mass spectrometry MALDI-TOF, with a molecular mass of 25 835.65 Da after when was sometida a desglycosilation with PNGase F and neuraminidase, The TLBan relative mass decreased 22 kDa and 25 kDa, respectively. The kinetic studies of the activity showed that the serine protease thrombin "like" activity features a opposite behavior michaeliano substrate DL-BApNA, recording the kinetic constants of Vmax = 5.4 x 10-1 nmol p-NA/min and Km = 7.9 x 10 -1 mM had to be stable between 25 ° C and 60 ° C and at pH between 4 and 10. In the presence of different ions (Mg2 +, Ca2 +, Mn2 +, Cd2 + and Zn2 +) and inhibitors (PMSF, EDTA and SBTI), its proteolytic activity and fibrinogenolítica was maintained, except for the ions Cd2 +, Zn2 + and inhibitor PMSF. TLBan was able to enhance its activity against the fibrinogen fibrinogenolítica veal, hydrolyzing the alpha cha thrombin "like" type venobim AB, presented a CI of 144.93 s-1, a minimum coagulant dose (DMC) of 1.33 ± 0.25 mg / mL and induces platelet aggregation. The structural characterization of TLBan was determined by its molecular mass via mass spectrometry. Structural analysis of the sequence was deduced using the database: http://www.expasy.ch/sprot/, with the help of the sequence of tryptic peptides, showing a high homology with other amino acid sequence of the serine protease from snake venom. Its polypeptide chain TLBan showed the presence of a catalytic triad in positions His (44), Asp (90) and Ser (185). The reproducibility of the biological activity through the pharmacological effects is only possible with the use of chemically homogeneous fractions to maintain the integrity of the molecule. These fractions are obtained with high efficiency methods: HPLC and mass spectrometry. The results may be associated with its biological activity, eliminating the subjectivity caused by poison or impure fractions. This approach will be applied to govern the biochemical studies, structure-function, physiological and pharmacological, may also reveal unknown mechanisms in the structure-function relationship of serine protease with thrombin "like" activity from snake venom. In the case of the snake Bothrops andianus, studies are valued due to the fact that there is no work done, probably because this is a species not yet studied, but that is of interest to the scientific field of venom Resumo: No presente trabalho, uma nova serinoprotease com atividade trombina "like" do veneno de Bothrops andianus (TLBan), serpente dos Andes do Perú, foi isolada mediante duas etapas: cromatografia de exclusão molecular G-75 e cromatografia líquida em HPLC de fase reversa; com um alto grau de pureza e homogeneidade molecular. Através da eletroforese em SDS-PAGE a TLBan mostrou ter uma massa relativa de 29 kDa sob condições redutoras e 26 kDa em condições não redutoras que foi confirmada com exatidão pela espectrometria de massa MALDI-TOF com uma massa molecular de 25 835,65 Da, após de ser submetida à glicosilação com a PNGase F e a neuraminidase, a massa relativa de TLBan diminuiu a 22 kDa e 25 kDa, respectivamente. Os estudos da atividade cinética mostraram que a serinoprotease com atividade trombina "like" possui um comportamento michaeliano frente ao substrato DL-BApNA, registrando as constantes cinéticas de Vmax = 5.4 x 10-1 nmoles p-NA/min e Km = 7.9 x 10-1 mM apresentou ser estável entre 25 ºC e 60 ºC e na faixa de pH entre 4 e 10. Na presença de diferentes íons (Mg2+, Ca2+, Mn2+, Cd2+ e Zn2+) e inibidores (PMSF, EDTA e SBTI), sua atividade proteolítica e fibrinogenolítica foi mantida, com exceção para os íons Cd2+, Zn2+ e inibidor PMSF. TLBan foi capaz de evidenciar sua atividade fibrinogenolítica frente ao fibrinogênio bovino, hidrolisado a cadeia alfa ( alfa ) e beta ( beta ), comportando-se como uma trombina "like" de tipo venobim AB, apresentou um IC de 144,93 s-1, uma dose mínima coagulante (DMC) de 1,33 ± 0,25 ?g/mL e induz á agregação plaquetária. A caracterização estrutural de TLBan foi determinada por sua massa molecular via espectrometria de massa. A análise estrutural da sequência foi deduzida utilizando a base de dados: http://www.expasy.ch/sprot/, com a ajuda da sequência dos peptídeos trípticos, mostrando uma alta homologia seqüêncial dos aminoácidos com outras serinoproteases de veneno de serpente. Sua cadeia polipeptídica da TLBan mostrou a presença da tríade catalítica nas posições de His (44), Asp (90) e Ser (185). A reprodutibilidade da atividade biológica por meio dos efeitos farmacológicos só é possível com a utilização de frações quimicamente homogêneas para manter a integridade da molécula. Essas frações são obtidas com metodologias de alta eficiência: HPLC e espectrometria de massa. Os resultados podem ser associados com sua atividade biológica, eliminando a subjetividade causada pelo veneno ou frações impuras. Este tipo de abordagem será aplicado para pautar os estudos bioquímicos, estrutura-função, fisiológico e farmacológico; pode ainda revelar mecanismos desconhecidos na relação estrutura-função da serinoprotease com atividade trombina "like" do veneno de serpente. No caso da serpente Bothrops andianus, são valorizados os estudos devido ao fato de não haver trabalhos realizados, provavelmente pelo fato de tratar-se de uma espécie ainda não estudada, mas que tem interesse para o campo científico no campo da venômica. Abstract: In this word, a new serine protease with thrombin "like" activity the venom of Bothrops andianus (TLBan), snake of the Andes of Peru, was isolated by two steps: molecular exclusion chromatography G-75 and liquid chromatography in reversed-phase HPLC; with a high degree of purity and molecular homegenidade. Through of electrophoresis on SDS-PAGE shows the TLBan have a relative mass of 29 kDa under reducing conditions and 26 kDa in reducing conditions that was not confirmed for precision by mass spectrometry MALDI-TOF, with a molecular mass of 25 835.65 Da after when was sometida a desglycosilation with PNGase F and neuraminidase, The TLBan relative mass decreased 22 kDa and 25 kDa, respectively. The kinetic studies of the activity showed that the serine protease thrombin "like" activity features a opposite behavior michaeliano substrate DL-BApNA, recording the kinetic constants of Vmax = 5.4 x 10-1 nmol p-NA/min and Km = 7.9 x 10 -1 mM had to be stable between 25 ° C and 60 ° C and at pH between 4 and 10. In the presence of different ions (Mg2 +, Ca2 +, Mn2 +, Cd2 + and Zn2 +) and inhibitors (PMSF, EDTA and SBTI), its proteolytic activity and fibrinogenolítica was maintained, except for the ions Cd2 +, Zn2 + and inhibitor PMSF. TLBan was able to enhance its activity against the fibrinogen fibrinogenolítica veal, hydrolyzing the alpha cha thrombin "like" type venobim AB, presented a CI of 144.93 s-1, a minimum coagulant dose (DMC) of 1.33 ± 0.25 mg / mL and induces platelet aggregation. The structural characterization of TLBan was determined by its molecular mass via mass spectrometry. Structural analysis of the sequence was deduced using the database: http://www.expasy.ch/sprot/, with the help of the sequence of tryptic peptides, showing a high homology with other amino acid sequence of the serine protease from snake venom. Its polypeptide chain TLBan showed the presence of a catalytic triad in positions His (44), Asp (90) and Ser (185). The reproducibility of the biological activity through the pharmacological effects is only possible with the use of chemically homogeneous fractions to maintain the integrity of the molecule. These fractions are obtained with high efficiency methods: HPLC and mass spectrometry. The results may be associated with its biological activity, eliminating the subjectivity caused by poison or impure fractions. This approach will be applied to govern the biochemical studies, structure-function, physiological and pharmacological, may also reveal unknown mechanisms in the structure-function relationship of serine protease with thrombin "like" activity from snake venom. In the case of the snake Bothrops andianus, studies are valued due to the fact that there is no work done, probably because this is a species not yet studied, but that is of interest to the scientific field of venom.

Purificação e caracterização biológica de uma nova serinoprotease com atividade trombina"like" do veneno total de Brothrops andianus (TLBan) / Purification and biological characterization of a new serine protease with thrombin "like" activity the whole venom of Brothrops andianus (TLBan)

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