The fluorescence time decay parameters of the
β-lactoglobulin-1-anilinonaphthalene-8-sulfonate
complex have been investigated under physical and chemical
perturbations (2 < pH < 8 and added electrolyte 0
< NaCl < 0.5 M) to obtain new insight on the nature
of the protein binding interactions. A double exponential
decay of the bound probe lifetime has been confirmed by
the presence of a longer component, 11 to 14.5 ns, and
a shorter component, 2.5 to 3.5 ns. The two lifetimes are
ascribed to different binding modes associated also with
different exposure to the solvent; in particular, the longer
component is attributed to binding inside the hydrophobic
beta barrel, while a “surface” site is suggested
for the shorter component. A detailed analysis of the lifetime
fractional intensities correlates the binding constants
with ionic strength and supports the presence of electrostatic
effects at both sites. A Debye–Hückel approach,
applied to extrapolate the electrostatic free energy contribution
vs. pH at vanishing ionic strength, gives interesting clues
on the effective charge felt by the ANS ligands in the
proximity of each site. In particular, binding is found
to parallel the aspartate and glutamate titrations between
pH 3 and pH 4.5; the “surface” site mainly
responds to the presence of these local titrating charges
while the “internal” site more closely follows
the overall protein net charge.