Isolation and functional characterization of a new acidic PLA2 Ba SpII RP4 of the Bothrops alternatus snake venom from Argentina |
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Authors: | Marí a E. Garcia Denegri,Salomó n Huancahuire-Vega,Sergio Marangoni,Gladys P. Teibler,Luis A. Ponce-Soto |
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Affiliation: | a Laboratorio de Química Biológica, Departamento de Bioquímica, Facultad de Ciencias Exactas y Naturales y Agrimensura, Universidad Nacional del Nordeste (UNNE), Av. Libertad 5470, Campus Universitario, CP 3400, Corrientes, Argentina b Laboratorio de Farmacología, Facultad de Ciencias Veterinarias, Universidad Nacional del Nordeste (UNNE), Sargento Cabral 2139, CP 3400, Corrientes, Argentina c Laboratório de Química de Proteínas, Departamento de Bioquímica, Instituto de Biología, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil d Max Planck Institute of Psychiatry, Munich, Germany e Departamento de Farmacología, Faculdade de Ciencias Médicas, Universidade Estadual de Campinas (UNICAMP), CP 6109, CEP 13083-970, Campinas, SP, Brazil |
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Abstract: | An acidic protein with phospholipase A2 activity was purified to homogeneity from the venom of the Northeast Argentinian viperid Bothrops alternatus by two chromatographic steps: a conventional gel filtration on Sephadex G-75 and reversed phase on C18 HPLC column.A molecular mass of 14185.48 Da was determined by mass spectrometry, displaying a homodimer conformation. The kinetic assay demonstrated a catalytically active phospholipase A2 in correspondence with Asp49 PLA2 group. The enzyme designated Ba SpII RP4 contains an amino acid composition of 121 residues and a calculated theoretical pI value of 4.88. Amino acid sequence alignments with other Bothrops PLA2 revealed a high degree of homology sequence (90-56%). Ba SpII RP4 did not show myotoxic activity upon muscular fibers at doses up to 100 μg i.m. route injection or lethal response when it was i.p. injected at the hightest dose of 200 μg. This toxin generates slight biological activities like paw edema inflammation and a delay in the clotting time, although Ba SpII RP4 exhibited catalytic activity. The primary amino acid sequence, determined a quadruple-time of flight (Q-TOF) hybrid mass spectrometer Q-TOF Ultima from Micromass (Manchester, UK) equipped with a nano Zspray source operating in a positive ion mode and tandem mass spectrum, an ESI/MS mass spectrum (TOF MS mode) “de novo amino acid sequencing”, also provides more database about the small group of the non-myotoxic PLA2s isolated up to the present. |
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Keywords: | Phospholipase A2 Bothrops alternatus Acidic Asp49 Amino-acid sequence Non-myotoxic Non-lethal |
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