ABSTRACT
The Micrurus genus is the American representative of Elapidae family. Micrurus spixii is endemic of South America and northern states of Brazil. Elapidic venoms contain neurotoxins that promote curare-mimetic neuromuscular blockage. In this study, biochemical and functional characterizations of M. spixii crude venom were performed and a new neurotoxic phospholipase A2 called MsPLA2-I was isolated. M. spixii crude venom caused severe swelling in the legs of tested mice and significant release of creatine kinase (CK) showing its myotoxic activity. Leishmanicidal activity against Leishmania amazonensis (IC50 1.24 µg/mL) was also observed, along with antiplasmodial activity against Plasmodium falciparum, which are unprecedented for Micrurus venoms. MsPLA2-I with a Mr 12,809.4 Da was isolated from the crude venom of M. spixii. The N-terminal sequencing of a fragment of 60 amino acids showed 80% similarity with another PLA2 from Micrurus altirostris. This toxin and the crude venom showed phospholipase activity. In a mouse phrenic nerve-diaphragm preparation, M. spixii venom and MsPLA2-I induced the blockage of both direct and indirect twitches. While the venom presented a pronounced myotoxic activity, MsPLA2-I expressed a summation of neurotoxic activity. The results of this study make M. spixii crude venom promising compounds in the exploration of molecules with microbicidal potential.
Subject(s)
Elapid Venoms/chemistry , Elapidae/metabolism , Neurotoxins/toxicity , Phospholipases A2/toxicity , Amino Acid Sequence , Animals , Antiparasitic Agents/pharmacology , Brazil , Creatine Kinase/metabolism , Inhibitory Concentration 50 , Leishmania/drug effects , Leishmania/growth & development , Mice , Molecular Sequence Data , Neurotoxins/isolation & purification , Phospholipases A2/isolation & purification , Plasmodium falciparum/drug effects , Plasmodium falciparum/growth & development , Protein Conformation , Toxins, BiologicalABSTRACT
Were analyzed poison samples as a pool extracted from B. alternatus and B. jararaca. The regional pool from different areas of Rio Grande do Sul. The national pool, poisons specially from São Paulo, Espírito Santo, Minas Gerais and Paraná states. Were analyzed: acute intraperitoneal median lethal doses (LD50), proteolytic activity on casein, coagulant effect on human plasma. The phospholipasic enzyme activities; the measurement of protein bands and the electrophoretic patterns were analyzed too. The analyses between the samples were made by comparison of biochemical actions and protein composition into the species, and inter-regionally between the pools. The comparison by electrophoresis, regional samples showed particularities in their venom protein composition sharing phospholipasic miotoxins, with 14 KDa, that were poorly observed in the national pool. However, we can observe other proteins with compatible size with phospholipases with miotoxic activities, with different molecular weigh (16 KDa), present in all of the samples. The major phospholipasic activity and protein quantity were verified in regional samples. The regional pool has differences in proteolytic and coagulant activities, when compared with the national pool.
Analisaram-se amostras de peçonha em forma de "pool", extraídas de B. alternatus, B. jararaca. O "pool"regional foi composto de peçonhas exclusivamente do Rio Grande do Sul, coletados de diversas regiões do estado. No "pool" nacional, peçonhas provenientes principalmente, de São Paulo, Espírito Santo, Minas Gerais e Paraná. O objetivo deste trabalho foi determinar a DL50 IP aguda, ação proteolítica da caseína, ação enzimática fosfolipásica, efeito coagulatório sobre o plasma humano, mensuração das bandas protéicas e perfil eletroforético, em camundongos. Quando comparadas pela eletroforese, as amostras regionais apresentaram particularidades, como a composição protéica da peçonha compartilhando miotoxinas fosfolipásicas com 14 KDa, que são pouco observadas nas amostras do "pool" nacional, sugerindo ser esta maior concentração a responsável pela maior ação do "pool" regional para estas peçonhas. Entretanto, observam-se outras proteínas com tamanhos compatíveis com as fosfolipases com atividade miotóxicas com peso molecular diferente (16KDa) presentes em todas as amostras. Também verificou- se maior atividade fosfolipásica e teor protéico nas amostras regionais. O "pool" regional teve diferenças nas atividades proteolítica e coagulante no plasma quando comparado com o "pool" nacional.
