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Titulo corto: Metodologia para el aislamiento e identificacion  de LPS de periodonto-patogenos Titulo en ingles: Purification and characterization of lipopolysaccharide from Eikenella corrodens 23834 and Porphyromonas gingivalis W83 Resumen:  La purificacion de lipopolisacaridos (LPS) o endotoxinas y su caracterizacion es un aspecto esencial para estudios que buscan aclarar el papel de estas biomoleculas de bacterias Gram negativas presentes en la cavidad oral y su relacion con enfermedades locales periodontales y sistemicas. Este estudio implementa una metodologia para la extraccion, purificacion y caracterizacion de LPS a partir de bacteria completa de Eikenella corrodens 23834 y Porphyromonas gingivalis W83,  utilizando tecnicas previamente descritas. La extraccion cruda de LPS se realizo con fenol-agua caliente; la purificacion se realizo con tratamiento enzimatico con nucleasas y proteasa, seguido de cromatografia de exclusion por tamano (Sephacryl S-200 HR) con deoxicolato de sodio como fase movil. La caracterizacion de los extractos purificados se realizo por barrido espectrofotometrico, pruebas bioquimicas de electroforesis SDS-PAGE, ensayo Purpald y la prueba cromogenica de LAL. Como control para la identificacion y caracterizacion de los extractos purificados se utilizaron LPS comerciales de Escherichia coli, Salmonella typhimurium, Rodobacter sphaeroides y Porphyromonas gingivalis . La metodologia implementada permitio la obtencion de LPS de elevada pureza con la identificacion de KDO o heptosas, un quimiotipo de LPS-S (liso) para E. corrodens y LPS-SR (semi-rugoso) para P. gingivalis W83 . Ambos LPS purificados mostraron capacidad endotoxica a bajas concentraciones. La metodologia implementada en este estudio para la purificacion y caracterizacion de LPS a partir de bacteria completa  fue eficiente al compararla con los LPS comerciales. Palabras clave : endotoxinas, cromatografia, acido 2-ceto-3-desoxioctulosonico (KDO), test LAL, periodontitis. Abstract:  Purification of lipopolysaccharide (LPS) or endotoxins and its characterization is an important aspect for studies aimed at clarifying the role of these biomolecules from Gram negative bacteria present in the oral cavity and its relationship with periodontal and systemic diseases. This study describes an extraction, purification and characterization method of LPS from Eikenella corrodens 23834 and Porphyromonas gingivalis W83. LPS extraction was performed using hot phenol-water; the purification was done with nuclease and protease enzymatic treatment, followed by size-exclusion chromatography (Sephacryl S-200 HR) with sodium deoxycholate as mobile phase. The characterization of the purified extracts was performed by spectrophotometric scanning, SDS-PAGE biochemical tests, Purpald assay and chromogenic LAL test. As control, commercial LPS from Escherichia coli , Salmonella typhimurium , P. gingivalis , and Rodobacter sphaeroides were used. The methodology mentioned above had allowed obtaining high purity LPS by identifying KDO or heptoses, a chemotype S-LPS (smooth) to E. corrodens ; SR-LPS (semi-rough) for P. gingivalis W83. Both purified LPS showed endotoxic capacity at low concentrations. The methodology used in this study for purification and characterization of LPS from the whole bacteria was efficient when compared with commercial LPS. Key words : endotoxin, 2-keto-3-deoxyoctonate (KDO), Chromatography, Limulus test (LAL), periodontitis.

Purificación y caracterización de lipopolisacáridos de Eikenella corrodens 23834 y Porphyromonas gingivalis W83