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-pyreny l)propane (1Py-(3)-1Py) incorporated into macromolecules of human seru m albumin (HSA), into micelles of dodecyltrimethylammonium chloride (D TAC),and dissolved in 1,4-dioxane were compared. The steady-state fluo rescence spectra indicated that in all the mentioned environments, upo n 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, respec tively. However, when the 1Py-(3)-1Py probe was located inside of the macromolecules of HSA, only the decay of emission was observed for exc imer with our set-up (t > 2 ns after excitation). The instantaneous fo rmation of excimer, unrelated to the decay of monomer excitation, indi cates that the considerable fraction of 1Py-(3)-1Py in the hydrophobic pockets of HSA is present as the ground state dimer. The red shift (D elta 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 co mparison of excitation spectra of 1Py-(3)-1Py in HSA and in 1,4-dioxan e also indicate that label molecules bound to some sites of HSA are in the ground state in the dimer conformation. Moreover, the close value s 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 interconvers ion 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 monome r and excimer emissions of 1Py-(3)-1Py in HSA do not allow to obtain a ny information on the local microfluidity inside of the protein macrom olecules but could be used for discrimination between different confor mations of the probe, possibly located in different protein pockets.