RESUMEN
We report on femto- to nanosecond studies of the excited state intermolecular proton transfer (ESPT) reaction of trisodium 8-hydroxypyrene-1,3,6-trisulfonate (pyranine, HPTS) with the human serum albumin (HSA) protein. The formed robust 1:1 complexes (K(eq) = (2.6 ± 0.1) × 10(6) M(-1)) show both photoacid (â¼430 nm) and conjugated photobase (â¼500 nm) emissions of the caged HPTS in its protonated structure. The proton-transfer reactions in these complexes proceed in a large time window, spanning from 150 fs to â¼1.2 ns. The ultrafast component reflects a direct H-bond breaking and making in the robust complexes, involving the carboxylate groups of the amino acids, while the slowest one is arising from the slow dynamics of the so-called biological water. Additional time constants of the caged photoacid to give the conjugated photobase are observed, assigned to the ESPT reaction within "loose" complexes (3 to tens of picoseconds), and 130 ps and 1.2 ns due to the slow dynamics of the water molecules around the protein residues and involved in the proton transfer. The fs-ns anisotropy measurements confirm the robustness of the HPTS:HSA complexes. Our results indicate that, even though robust 1:1 complexes between HPTS and the HSA are formed, the system is heterogeneous, due to different possible interactions of the dye with the inside/outside parts of the protein. Furthermore, we find lower values of the initial anisotropy (r(0)) in the protein (0.33) and in γ-CD (0.28) in comparison with buffered aqueous solution (0.385). We propose that caging HPTS by the HSA protein and by the cyclodextrin affects the electronic redistribution in a different degree of mixing between the (1)L(a) and (1)L(b) states in the formed deprotonated form, for which the interactions of the sulfonate groups with the surroundings should play a key role.
Asunto(s)
Arilsulfonatos/química , Protones , Albúmina Sérica/química , Humanos , Modelos Moleculares , Espectrometría de FluorescenciaRESUMEN
Single molecule studies of the free DY-630-MI and interacting with MCM-41 and (Al)MCM-41, show the conformational diversity of the molecule. The free dye is characterized by a single broad (fwhm = 0.7 ns) lifetime distribution histogram centered on 1.47 ns, which is also reflected in the broadness of the polarization value distribution histogram, covering almost the full range of values from -1 to 1. The fluorescence intensity traces of the free DY-630-MI show strong blinking behavior and weak photostability. Upon interaction with the mesoporous silica nanomaterials, MCM-41 and (Al)MCM-41, the dye molecule becomes more stable, with less blinking present in the fluorescence traces. The lifetime distribution histogram in the case of DY-630-MI/MCM-41 complexes is fitted by 3 Gaussians, indicating 3 distinct interaction sites. The Gaussian with the largest amplitude is centered on 2.19 ns, consistent with the confinement effect of MCM-41 and in agreement with the ensemble average studies. The polarization value distribution histogram becomes narrower in comparison with the free molecule and is more biased towards the positive limit. Replacing few Si(4+) ions with Al(3+) ones in the regular MCM-41 changes the local electrostatic field within the nanotube. This atomic substitution in the nanohosts results in a more selective orientation of the dye molecules, giving two populations with time constants 1.56 and 2.10 ns.