Ligand-dependent structural changes in serum albumin are suggested to underlie its role in physiological solute transport and receptor-mediated cellular selection. Evidence of ligand-induced (oleic acid) structural changes in serum albumin are shown in both time-resolved and steady-state fluorescence quenching and anisotropy measurements of tryptophan 214 (Trp214). These studies were augmented with column chromatography separations. It was found that both the steady-state and time-resolved Stern-Volmer collisional quenching studies of Trp214 with acrylamide pointed to the existence of an oleate-dependent structural transformation. The bimolecular quenching rate constant of defatted human serum albumin, 1.96 x 10(9) M-1 s-1, decreased to 0.94 x 10(9) M-1 s-1 after incubation with oleic acid (9:1). Furthermore, Stern-Volmer quenching studies following fractionation of the structural forms by hydrophobic interaction chromatography were in accordance with this interpretation. Time-resolved fluorescence anisotropy measurements of the Trp214 residue yielded information of motion within the protein together with the whole protein molecule. Characteristic changes in these motions were observed after the binding of oleate to albumin. The addition of oleate was accompanied by an increase in the rotational diffusion time of the albumin molecule from approximately 22 to 33.6 ns. Within the body of the protein, however, the rotational diffusion time for Trp214 exhibited a slight decrease from 191 to 182 ps and was accompanied by a decrease in the extent of the angular motion of Trp214, indicating a transition after oleate binding to a more spatially restricted but less viscous environment.

Articles you may be interested inTwo-photon-excited fluorescence resonance energy transfer in an aqueous system of CdTe quantum dots and Rhodamine BNew polarized time resolved fluorescence techniques are implemented to determine the full angular motion of a probe molecule in an anisotropic environment. Studies of rhodamine 6G and resorufin molecules aligned in a free ethylene glycol jet show that the presence of net molecular order is accompanied by a distinct anisotropy in alignment relaxation following photoselection. Diffusion coefficients for and motion ͑D ʈ and D Ќ ͒ in a jet fixed axis system are determined from the cylindrically symmetric and asymmetric alignment relaxation rates for the isotropic and anisotropic regions of the jet. The presence of net negative molecular alignment as the free jet is formed is seen to correspond to restricted motion (D ʈ ϽD Ќ ), with a net positive steady state alignment the anisotropy in the diffusion dynamics is reversed. The differences in D ʈ and D Ќ are attributed to anisotropy in the solvent viscosity as a consequence of flow. The combination of linear and circular polarization techniques is seen to provide useful information on cylindrical asymmetry and relaxation dynamics hitherto unobserved by conventional fluorescence polarization techniques.

New polarized time resolved fluorescence techniques are implemented to determine the full angular motion of a probe molecule in an anisotropic environment. Studies of rhodamine 6G and resorufin molecules aligned in a free ethylene glycol jet show that the presence of net molecular order is accompanied by a distinct anisotropy in alignment relaxation following photoselection. Diffusion coefficients for and motion ͑D ʈ and D Ќ ͒ in a jet fixed axis system are determined from the cylindrically symmetric and asymmetric alignment relaxation rates for the isotropic and anisotropic regions of the jet. The presence of net negative molecular alignment as the free jet is formed is seen to correspond to restricted motion (D ʈ ϽD Ќ), with a net positive steady state alignment the anisotropy in the diffusion dynamics is reversed. The differences in D ʈ and D Ќ are attributed to anisotropy in the solvent viscosity as a consequence of flow. The combination of linear and circular polarization techniques is seen to provide useful information on cylindrical asymmetry and relaxation dynamics hitherto unobserved by conventional fluorescence polarization techniques.

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We present results of single and three pulse photoselection studies of the dynamics of a fluorescent probe (Oxazine 4) in the nematic phase of the liquid crystal 4-n-pentyl-4'-cyanobiphenyl (5CB). The combination of these techniques with time resolved fluorescence anisotropy measurements allows the unambiguous determination of the full angular motion of the probe together with the ground and excited state degrees of equilibrium (steady state) alignment. The restricted JHRPHWU\ LPSRVHG E\ WKH QHPDWLF KRVW LV VHHQ WR LPSDUW D PDUNHG DQLVRWURS\ LQ DQG φ GLIIXVLRQ ! ~4 ).