Subdomain IIA binding site of human serum albumin (HSA) was characterized by examining the change in HSA fluorescence in the native, unfolded, and refolded states. The study was carried out in the absence and presence of small molecular probes using steady-state and time-resolved fluorescence measurements. 2-Pyridone, 3-pyridone, and 4-pyridone bear similar molecular structures to those found in many drugs and are used here as probes. They are found to specifically bind in subdomain IIA and cause a reduction in the fluorescence intensity and lifetime of the Trp-214 residue in native HSA which is located in the same subdomain. The efficiency of energy transfer from Trp-214 fluorescence to the probes was found to depend on the degree of the spectral overlap between the donor's fluorescence and the acceptor's absorption. After probe binding in subdomain IIA, the distance between the donor and acceptor was calculated using Forster theory. The calculated quenching rate constants and binding constants were also shown to depend on the degree of spectral overlap. The results point to a static quenching mechanism operating in the complexes. Denaturation of HSA in the presence of guanidine hydrochloride (GdnHCl) starts at [GdnHCl] > 1.0 M and is complete at [GdnHCl] > or = 6.0 M. Upon unfolding, two fluorescence peaks were observed. One peak was assigned to the fluorescence of Trp-214 in a polar environment, and the other peak was assigned to tyrosine fluorescence. A reduction of the fluorescence intensity of the two peaks upon binding of the probes to the denatured HSA indicates that Tyr-263 in subdomain IIA is one of the tyrosine residues responsible for the second fluorescence peak. The results were confirmed by measuring the fluorescence spectra and lifetimes of denatured HSA at different excitation wavelengths, and of L-tryptophan and L-tyrosine free in buffer. The measured lifetimes of denatured HSA are typical of tryptophan in a polar environment and are slightly reduced upon probe binding. Dilution of the denatured HSA by buffer results in a partial refolding of subdomain IIA. This partial refolding is attributed to some swelling of the binding site caused by water. The swelling prevents a full recovery from the denatured state.

The solvent-dependent ground-state conformational equilibrium and excited-state dynamics of 2-(2′-hydroxyphenyl)benzoxazole have been characterized in several solvents on the femtosecond to nanosecond time scales. The only observable ground-state tautomer is the enol, which exists in equilibrium between the syn-and anti-rotational isomers. In the anti-enol isomer, the phenyl hydroxyl group appears to not interact strongly with solvent but rather forms a strong intramolecular hydrogen bond with the benzoxazole oxygen atom. In the syn-enol isomer, the phenyl hydroxyl proton may interact with solvent or form an internal hydrogen bond with the benzoxazole nitrogen atom. Upon excitation, the proton is transferred from the oxygen atom to the nitrogen atom of the internally hydrogen bonded syn-enol isomer in 170 fs, regardless of the solvent. The lifetime of the resulting excited keto tautomer is solvent dependent and on the order of picoseconds. In addition to these dynamics, several additional dynamic processes are detected which may correspond to relaxation of a distorted excited keto tautomer.

The ground- and excited-state tautomerization of the 2,2'-bipyridine-3,3'-diol molecule (BP(OH)(2)) was studied in different solvents and in confined nanocavities of cyclodextrins (CDs) using steady-state and lifetime spectroscopic measurements. In all solvents, a dizwitterion (DZ) tautomer is produced in the excited state after intramolecular double-proton transfer. This tautomer is stabilized in the ground state in water only and produces two unique absorption peaks in the region of 400-450 nm. The DZ tautomer fluoresces in the green and as the solvent polarity increases, the fluorescence peak is blue-shifted (498 nm in cyclohexane versus 462 nm in water), and the fluorescence lifetime gets shorter (3.10 ns in cyclohexane versus 0.65 ns in water). The results indicate the sensitivity of this tautomer to solvent polarity, particularly the solvent's hydrogen-bonding capability. In water, another photoinduced tautomerization mechanism takes place via a water network solvating each of the two hydrogen-bonding centers of the molecule. The second tautomer is detected as a small shoulder in the blue side of the fluorescence peak and has a lifetime of 5.40 ns. Using BP(OH)(2) to probe the nanocavities of aqueous CDs reveals the degree of hydrophobicity of the cavities and the different mechanisms of probe encapsulation. As the cavity size decreases in the order gamma-CD to beta-CD to alpha-CD, the cavity is more hydrophobic, which is reflected in an intensity decrease of the absorbance of the DZ tautomer and a red shift in its fluorescence peak. The measured lifetimes show the same trend and reveal how the probe interacts with the CD moiety. In gamma-CD, the probe is located near the secondary rim of the CD annulus, whereas in alpha-CD, the probe is completely sequestered between two CDs, and the hydrophobicity is close to that observed in cyclohexane. In beta-CD and its derivatives, the spectral changes and the measured lifetimes indicate that the CD cavity gets more hydrophobic as a result of methyl substitution of the primary and secondary hydroxyls of the beta-CD rims. In the fully methylated 2,3,6-tri-O-methyl-beta-CD, the probe is exposed to water near the secondary rim due to the steric effect at the entrance rim that prevents the probe from full encapsulation.

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