In vitro characterization of jellyfish venom fibrin(ogen)olytic enzymes from Nemopilema nomurai

Background: Because jellyfish are capable of provoking envenomation in humans, they are considered hazardous organisms. Although the effects of their toxins are a matter of concern, information on the venom components, biological activity and pathological mechanisms are still scarce. Therefore, the aim of the present study was to investigate a serine protease component of Nemopilema nomurai jellyfish venom (NnV) and unveil its characteristics. Methods: To determine the relationship between fibrinolytic activity of NnV and the serine protease, fibrin zymography was performed using metalloprotease and serine protease inhibitors. The biochemical characterization of serine proteases of NnV were determined by the amidolytic assay. Fractions with fibrinolytic activity were obtained by DEAE cation exchange column. Results: NnV displayed fibrinolytic activities with molecular masses of approximately 70, 35, 30, and 28 kDa. The fibrinolytic activity of NnV was completely obliterated by phenylmethylsulfonyl fluoride, a prototype serine protease inhibitor. Based on amidolytic assays using chromogenic substrates specific for various kinds of serine proteases, NnV predominantly manifested a chymotrypsin-like feature. Its activity was completely eliminated at low pH (< 6) and high temperatures (> 37 °C). Some metal ions (Co2+, Cu2+, Zn2+ and Ni2+) strongly suppressed its fibrinolytic activity, while others (Ca2+ and Mg2+) failed to do so. Isolation of a serine protease with fibrionolytic activity from NnV revealed that only p3 showed the fibrinolytic activity, which was completely inhibited by PMSF. Conclusion: The present study showed that N. nomurai jellyfish venom has a chymotrypsin-like serine protease with fibrinolytic activity. Such information might be useful for developing clinical management of jellyfish envenomation and pharmacological agents with therapeutic potential for thrombotic diseases in the future.(AU)

In vitro characterization of jellyfish venom fibrin(ogen)olytic enzymes from Nemopilema nomurai

Background: Because jellyfish are capable of provoking envenomation in humans, they are considered hazardous organisms. Although the effects of their toxins are a matter of concern, information on the venom components, biological activity and pathological mechanisms are still scarce. Therefore, the aim of the present study was to investigate a serine protease component of Nemopilema nomurai jellyfish venom (NnV) and unveil its characteristics. Methods: To determine the relationship between fibrinolytic activity of NnV and the serine protease, fibrin zymography was performed using metalloprotease and serine protease inhibitors. The biochemical characterization of serine proteases of NnV were determined by the amidolytic assay. Fractions with fibrinolytic activity were obtained by DEAE cation exchange column. Results: NnV displayed fibrinolytic activities with molecular masses of approximately 70, 35, 30, and 28 kDa. The fibrinolytic activity of NnV was completely obliterated by phenylmethylsulfonyl fluoride, a prototype serine protease inhibitor. Based on amidolytic assays using chromogenic substrates specific for various kinds of serine proteases, NnV predominantly manifested a chymotrypsin-like feature. Its activity was completely eliminated at low pH (< 6) and high temperatures (> 37 °C). Some metal ions (Co2+, Cu2+, Zn2+ and Ni2+) strongly suppressed its fibrinolytic activity, while others (Ca2+ and Mg2+) failed to do so. Isolation of a serine protease with fibrionolytic activity from NnV revealed that only p3 showed the fibrinolytic activity, which was completely inhibited by PMSF. Conclusion: The present study showed that N. nomurai jellyfish venom has a chymotrypsin-like serine protease with fibrinolytic activity. Such information might be useful for developing clinical management of jellyfish envenomation and pharmacological agents with therapeutic potential for thrombotic diseases in the future.(AU)

Prospeção de inibidores de serinoproteinases em folhas de leguminosas arbóreas da floresta Amazônica / Prospecting serine proteinase inhibitors in leaves from leguminous trees of the Amazon forest

Os inibidores de proteinases são proteínas extensivamente investigadas nos tecidos de estocagem, mas pouco prospectadas em outros tecidos vegetais. O objetivo deste estudo foi detectar a presença de inibidores de serinoproteinases em extratos foliares de quinze espécies de leguminosas arbóreas da Amazônia. As espécies estudadas foram: Caesalpinia echinata, C. ferrea, Cedrelinga cateniformis, Copaifera multijuga, Dinizia excelsa, Enterolobium contortisiliquum, E. maximum, E. schomburgkii, Leucaena leucocephala, Ormosia paraensis, Parkia multijuga, P. pendula, P. platycephala, Swartzia corrugata e S. polyphylla. Folhas foram coletadas, secas a 30ºC durante 48 h, trituradas e submetidas à extração com NaCl (0,15 M, 10 por cento p/v) resultando no extrato total. Ensaios foram executados para determinar a concentração de proteínas e detectar a atividade inibitória contra a tripsina e quimotripsina bovina. Os teores de proteínas bruta e solúvel nos extratos foliares variaram de 7,9 a 31,2 por cento e 1,3 a 14,8 por cento, respectivamente. A atividade inibitória sobre a tripsina e quimotripsina foi observada em todos os extratos foliares. Contudo, nos extratos de E. maximum, L. leucocephala, P. pendula, S. corrugata e S. polyphylla a inibição foi maior sobre a tripsina, enquanto o extrato de P. multijuga foi mais efetivo contra a quimotripsina. Nós concluímos que nos extratos foliares de leguminosas arbóreas têm inibidores de serinoproteinases e exibem potencial aplicações biotecnológicas.

The proteinase inhibitors are proteins extensively investigated in tissue storage, but few prospected in other plant tissues. The aim of this study was to detect the presence of serine proteinase inhibitors in leaf extracts from fifteen species of leguminous trees of the Amazon forest. The species studied were Caesalpinia echinata, C. ferrea, Cedrelinga cateniformis, Copaifera multijuga, Dinizia excelsa, Enterolobium contortisiliquum, E. maximum, E. schomburgkii, Leucaena leucocephala, Ormosia paraensis, Parkia multijuga, P. pendula, P. platycephala, Swartzia corrugata and S. polyphylla. Leaves were collected, dried at 30ºC for 48 h, crushed and subjected to extraction with NaCl (0.15 M, 10 percent w/v), resulting in the total extract. Tests were performed to determine the concentration of proteins and to detect of inhibitory activity against bovine trypsin and chymotrypsin. The content of crude and soluble protein in leaf extracts varied from 7.9 to 31.2 percent and 1.3 to 14.8 percent, respectively. The inhibitory activity on trypsin and chymotrypsin was observed in all leaf extracts. However, in extracts of E. maximum, L. leucocephala, P. pendula, S. corrugata and S. polyphylla, the inhibition was greater on trypsin, while extract of P. multijuga was more effective against chymotrypsin. We conclude that leaf extracts of leguminous trees have serine proteinase inhibitors and show potential biotecnological applications.