English abstract
2-pyridone (2Py), 3-pyridone (3Py) and 4-pyridone (4Py) in solution, nanocavities of cyclodextrins, and their binding to human serum albumin (HSA) was studied using steady-state and time-resolved spectroscopic measurements and by ab initio calculations. In all solvents, 4Py was nonfluorescent, whereas 3Py was fluorescent in aqueous solvents only, and 2Py was fluorescent in all solvents. Solvation of 2Py and 3Py by water was examined in binary mixtures of 1,4-dioxane/water and 1,4 dioxane/methanol. Analysis of the absorption and fluorescence data reveals the solvation of the hydrogen bonding center in 2Py by one water molecule and in 3Py by three water molecules. Experimental results were complemented by calculations in which the ground state structures of the 2Py/H20 and 3Py/H20 complexes were calculated using ab intio methods at the MP2/6-311++G** level. In 3Py, a zwitterionic tautomer is formed in water only and shows distinct absorption peaks from the absorption of the neutral tautomer. This was further investigated by studying the caging effect of several cyclodextrins on the absorption behavior of 3Py. The results show a decrease in the zwitterionic absorption accompanied by an increase in the neutral absorption as the CD cavity. becomes smaller and/or more hydrophobic. This observation indicates that 3Py is a potential new photophysical probe to study supramolecular structures involving inclusion. The mechanism of binding of 2Py and 3Py as probe ligands to HSA was investigated by following the intensity change and lifetime of HSA fluorescence after excitation at 280 nm (the maximum absorption in HSA). The presence of 2Py and3Py causes a reduction in the fluorescence intensity and lifetime of HSA. This observation indicates that subdomain IIA binding site (Sudlow site I) is the host of the probes and the reduction in the fluorescence of HSA is due to energy transfer from the Trp-214 residue to the probe in each case. The distance between Trp-214 and each of the probes was calculated using Förster resonance energy transfer and found to be shorter for HSA/2Py. The calculated quenching rate constant (ka) and binding constant (K) were larger for HSA/2Py than the corresponding values for HSA/3Py. The results indicate more efficient energy transfer from HŞA to 2Py than to 3Py which is attributed to the larger extinction coefficient in 2Py. The calculated distances and the kg values indicate a static quenching mechanism operative in the two complexes. The number of binding sites of HSA was calculated to be one in both complexes. The latter results, along with the quenching results, indicate that both probes, 2Py and 3Py, bind only in Sudlow site I in subdomain IIA. Chemical unfolding of HSA in GuHCl shows fluorescence due to tryptophan and tyrosine. In the presence of 2Py and 3Py, quenching in the unfolded HSA indicates that the Tyr-263 residue, located in subdomain IIA, is responsible for the additional fluorescence peak. This was confirmed by lifetime measurements and by carrying out the study on free tryptophan and tyrosine in buffer. Finally, HSA refolding by dilution in buffer shows that refolding of subdomain IIA is not complete which is attributed to the presence of water in this subdomain during the unfolding process that prevents a complete refolding. The results obtained in HSA suggest that 2Py and 3Py can be used as potential probes to explore binding sites in proteins. 4Py probe did not show any quenching effect on HSA due to the poor overlap between its absorption spectrum with the fluorescence spectrum of HSA
