INIBIOLP   05426
INSTITUTO DE INVESTIGACIONES BIOQUIMICAS DE LA PLATA "PROF. DR. RODOLFO R. BRENNER"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Inactivation of E. coli a-hemolysin photoinduced by pterin
Autor/es:
DANTOLA, MARIA LAURA; HERLAX, VANESA; REID, LARA O.; THOMAS, ANDRÉS H.
Lugar:
La Plata
Reunión:
Congreso; XLVII Reunión Anual de la Sociedad Argentina de Biofísica; 2018
Institución organizadora:
Sociedad Argentina de Biofísica
Resumen:
Pterinsare a family of heterocyclic compounds widespread in living systems. Thesemolecules are photochemically active under UV-A excitation (320? 400 nm), canfluoresce, undergo photooxidation to produce various products and generate reactiveoxygen species. Due to the photochemical features of these compounds, they arepotential photosensitizers of biomolecules, such as DNA1 and proteins. 2 α-Hemolysin (HlyA) is an exotoxin, memberof the pore-forming Repeat in Toxin (RTX) family, secreted by some pathogenicstrains of Escherichia coli. The mechanism of action of this toxin seems toinvolve three stages that ultimately lead to cell lysis: binding, insertion andoligomerization of the toxin within the membrane.3Theaim of this work is to investigate the capability of pterin (Ptr), the parentand unsubstituted 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 byUV/visible spectrophotometry, fluorescence spectroscopy and gel electrophoresis(SDS-PAGE). The hemolytic activity of the irradiated toxin was analyzed by lightscattering at 595 nm in time. Results indicate that HlyA can be inactivated byPtr through a photosensitized process. The photodamage to HlyA results in theoxidation of the toxin in at least two different and specific sites: tryptophan(Trp) and tyrosine (Tyr) residues. The Trp degradation results in a fastdecrease of the fluorescence intensity, but the hemolytic activity remainsconstant until 10 minutes of irradiation. In addition, Tyr residues contributeto dimerization of the protein, since Tyr dimers were detected by fluorescence.The electrophoresis analysis indicates unequivocally that Ptr photoinducescross-linking of HlyA.References1. 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)