α-Hemolysin
(HlyA) is an extracellular protein toxin secreted
by uropathogenic strains of Escherichia coli that
inserts into membranes of eukaryotic cells. The main goal of this
work was to investigate the involvement of tryptophan (W) residues
in the hemolytic activity of HlyA. We investigated the hemolytic activity
of six single-point mutant proteins, in which one of the four Ws was
replaced by cysteine (C) or leucine (L). We also analyzed the photoinactivation
of HlyA with pterin (Ptr), an endogenous photosensitizer, as a method
of unspecific oxidation of W and tyrosine (Y) residues. HlyA photoinactivation
was analyzed by ultraviolet–visible spectrophotometry, hemolytic
activity measurement, fluorescence spectroscopy, and electrophoretic
analysis. The results indicate that Ws are important in the hemolytic
process. Specifically, the chemical structure of the amino acid at
position 578 is important for the acylation of HlyA at residue K563.
Furthermore, the exposure of HlyA to ultraviolet radiation, with energy
similar to that experienced under sun exposure, in the presence of
Ptr induces the inactivation of the toxin, causing chemical changes
in, at least, W and Y, the rate of damage to W residues being faster
than that observed for Y residues. This work not only deepens our
understanding of the structure–function relationship of the
toxin but also introduces the possibility of using photoinactivation
of HlyA for potential applications such as obtaining innocuous molecules
for vaccine production and the elimination of the toxin from contaminated
surfaces and drinking water.