Fluorescence of 1,3-di(1-pyrenyl)propane probe incorporated into human serum albumin protein enforced conformations of the probe

Steady-state and time-resolved fluorescence spectra of 1,3-di(1-pyrenyl)propane (1Py-(3)-1Py) incorporated into macromolecules of human serum albumin (HSA), into micelles of dodecyltrimethylammonium chloride (DTAC),and dissolved in 1,4-dioxane were compared. The steady-state fluorescence spectra indicated that in all the mentioned environments, upon excitation of 1Py-(3)-1Py, light was emitted from the single pyrene chromophores (1Py*) and from the 1Py, 1Py* excimers. The time-resolved fluorescence emission registered at 480 nm (excimer emission) for 1Py-(3)-1Py in the DTAC micelles and dissolved in 1,4-dioxane allowed to monitor formation of excimer with time constant tau(1) = 40.0 ns and 9.6 ns, for 1Py-(3)-1Py in the DTAC micelles and in 1,4-dioxane, respectively. However, when the 1Py-(3)-1Py probe was located inside of the macromolecules of HSA, only the decay of emission was observed for excimer with our set-up (t > 2 ns after excitation). The instantaneous formation of excimer, unrelated to the decay of monomer excitation, indicates that the considerable fraction of 1Py-(3)-1Py in the hydrophobic pockets of HSA is present as the ground state dimer. The red shift (Delta lambda = 8 nm) and broadening of UV absorption for 1Py-(3)-1Py in HSA (when compared with absorption 1Py-(3)-1Py in 1,4-dioxane) and comparison of excitation spectra of 1Py-(3)-1Py in HSA and in 1,4-dioxane also indicate that label molecules bound to some sites of HSA are in the ground state in the dimer conformation. Moreover, the close values of the ratios of intensities of monomer emission to excimer emission, registered 2 ns (5 ns gate) after excitation pulse with duration 300 ps and at the steady-state conditions, indicate that the interconversion between conformers of 1Py-(3)-1Py inside of the macromolecules of HSA is slow in comparison with the decay time of Py chromophore in the excited state in HSA (two-exponential decay with decay times tau(1) = 2.41 ns, tau(2) = 69.0 ns). Thus, ratios of the intensities of monomer and excimer emissions of 1Py-(3)-1Py in HSA do not allow to obtain any information on the local microfluidity inside of the protein macromolecules but could be used for discrimination between different conformations of the probe, possibly located in different protein pockets.