CHARACTERIZATION OF SECRETED PRODUCTS FROM Corynebacterium diphtheriae, Bordetella pertussis AND Clostridium tetani (DPT) BACTERIA IN GROWTH MEDIA - PRELIMINARY DATA

PERPÉTUO, E.A.(1), JULIANO, L. (2), FRATELLI, F. (3), PRADO, S.M.A. (3), LEBRUN, I.(1)

(1)Laboratory of Biochemistry and Biophysics, Butantan Institute, (2) Department of Biophysics, UNIFESP, (3)Section of Anaerobic Vaccines, Butantan Institute, SP, Brasil.

Objectives: To characterize secreted proteins from Corynebacterium diphtheriae, Bordetella pertussis and Clostridium tetani bacteria, to observe the production process steps, to identify possible production markers and new products. For these purposes, different fluorescent substrates were used to determine new enzymatic activities present in production batch. Methods and Results:1-Purification of DPT toxins by gel filtration chromatography 2-Characterization of DPT toxins by electrophoresis and enzymatic activities, using fluorescent substrates. Four fractions were obtained by chromatography of tetanus toxin and the main active fraction was assayed on different fluorescent substrates, all based on synaptobrevin sequence (a natural substrate cleaved by tetanus toxin). The results showed that the tetanus toxin can hydrolyze substrates with 4 to 10 amino acid residues at different rates. The toxins from.C. diphtheriae and B. pertussis were also fractionated and assayed on different fluorescent substrates. Both toxins (D and P) presented enzymatic activities. The best substrate for active acellular pertussis toxin was Abz LYLVEGQ EDDnp and for diphtheria toxin was AbzLYTPKAQ EDDnp. Electrophoresis of concentrated and purified toxins also were carried out. Conclusions: The fluorescent substrates represent a good tool to identify and characterize new enzymatic activities which could help to follow the production process (Perpetuo et al , Biotec. and Appl. Biochem., in press). In addition, these activities will be better characterized to search new enzymes which could be used for other purposes than antigen for vaccine production.

Financial support: FAPESP

CORRESPONDENCE TO:

Elen Aquino Perpetuo, Avenida Vital Brasil 1500, Laboratório de Bioquimica, São Paulo, SP, CEP: 05503-900, Brasil, Email: perpetuoe@hotmail.com