Inactivation of E. coli α-hemolysin photoinduced by pterin

REID LO; DÁNTOLA ML; HERLAX VANESA; THOMAS AH

Congreso; Reunion Anual de la sociedad Argentina de biofisica; 2018

Pterins are a family of heterocyclic compounds widespread in living systems.These molecules are photochemically active under UV-A excitation (320? 400 nm),can fluoresce, undergo photooxidation to produce various products and generatereactive oxygen species. Due to the photochemical features of these compounds,they are potential photosensitizers of biomolecules, such as DNA1 and proteins.2α-Hemolysin (HlyA) is an exotoxin, member of the pore-forming Repeat in Toxin(RTX) family, secreted by some pathogenic strains of Escherichia coli. Themechanism of action of this toxin seems to involve three stages that ultimatelylead to cell lysis: binding, insertion and oligomerization of the toxin within themembrane.3The aim of this work is to investigate the capability of pterin (Ptr), the parent andunsubstituted compound of oxidized pterins, to photoinduce chemical changesand inactivation of HlyA.Air equilibrated aqueous solution of HlyA and Ptr wereexposed to UV-A radiation for different periods of time and were analyzed by UV/visible spectrophotometry, fluorescence spectroscopy and gel electrophoresis(SDS-PAGE). The hemolytic activity of the irradiated toxin was analyzed by ligthscattering at 595 nm in time.Results indicate that HlyA can be inactivated by Ptr through a photosensitizedprocess. The photodamage to HlyA results in the oxidation of the toxin in at leasttwo different and specific sites: tryptophan (Trp) and tyrosine (Tyr) residues. TheTrp degradation results in a fast decrease of the fluorescence intensity, but thehemolytic activity remains constant until 10 minutes of irradiation. In addition, Tyrresidues contribute to dimerization of the protein, since Tyr dimers were detectedby fluorescence. The electrophoresis analysis indicates unequivocally that Ptrphotoinduces cross-linking of HlyA.Contact: laraoreid@inifta.unlp.edu.arReferences1. K. Ito, et. al. Biochemistry 36, 1774 (1997)2. L. O. Reid, et. al. Biochemistry, 55, 4777 (2016)3. S. M. Maté, et. al. Biomembranes, 1832, 1838 (2014)