Electrophoretic characterization of the 900 kDa type A botulinum neurotoxin.

PATRICIA CABALLERO; MIGUEL A. SOSA; L.DE JONG; MI BIANCO; AS CICCARELLI; RA FERNANDEZ

Mendoza (Argentina)

Congreso; XXIV Reunión Científica Anual Sociedad de Biología de Cuyo. IV Reunión Anual de la Sociedad Argentina de Microscopía.; 2006

Secretaría de Ciencia y Técnica de la Universidad Nacional de Cuyo

Electrophoretic characterization of the 900 kDa type A botulinum neurotoxin. Caballero PA, Sosa MA*, de Jong LIT, Bianco MI, Ciccarelli AS, Fernández R A. Área Microbiología, Dto. Patología, Instituto de Histología y Embriología* (IHEM), FCM, UN Cuyo, Mendoza, CC33, 5500, Argentina. pacaballero@yahoo.com The type A botulinum neurotoxin (NTBo), produced by Clostridium botulinum, is one of seven antigenic types recognized so far. This toxin is synthesized as a native simple chain, where the toxic fraction (S), of approximately 150 kDa, associates with non toxic haemagglutinin subcomponent and with other non toxic non haemagglutinin one by covalent bonds. All these components may be associated, forming three different protein complexes of approximately 900, 500 and 300 kDa (named LL, L and M respectively). The aim of this work was to characterize the LL NTBo by electrophoresis and what is the sensitivity of the complex to chemical and physical agents. The toxin was produced and purified from an A Hall strain-stock prototype of Clostridium botulinum, cultured in liquid yeast trypticase medium. Purification was carried out by acid precipitation followed by dialysis.  Samples were submitted to different temperatures and the products were analyzed by SDS-PAGE on 6% acrlylamide gels, under reducing or non reducing conditions. We observed that the integrity of the complex LL is affected by high temperature,  since the gels were depleted of 900 kDa band. Moreover, reducing conditions induced changes in electrophoretic mobility. We conclude that 900 kDa NTBo is highly sensitive to physical and chemical agents, and we should take in account this behavior for ulterior manipulations of the toxin. The study will be extended to other possible conditions that may affect the protein complex.