Subject(s)
Liposomes , Myristates , Phosphatidylcholines , Spectrometry, Fluorescence , Time FactorsABSTRACT
Nanosecond time-resolved emission spectroscopy is used to characterize the complex fluorescence behavior of the probe 2-p-toluidinonaphthalene 6-sulfonate (2,6 p-TNS) when adsorbed to several bilayer membrane system. These include egg phosphatidylcholine vesicles with and without added cholesterol as well as erythrocyte ghost membranes. In each case a nanosecond time-dependent shift of the fluorescence emission to lower energy follows pulsed photoexcitation. The properties of the time-resolved surfaces obtained are consistent with a non-exponential decay law which describes a continuous interaction process of 2,6 p-TNS with its local environment in the membrane. This environment consists in part of polar residues (water plus polar head region) undergoing nanosecond motions. The pure phosphatidylcholine bilayer system was studied at four temperatures and electronic and spectral relaxation contributions to the total fluorescence decay were separated. Temperature coefficients for empirical rate parameters derived for the separated processes were obtained. It appears that a treatment of the fluorescence behavior of amphiphilic probes such as 2,6 p-TNS adsorbed to bilayer membranes at temperatures near ambient in which a single lifetime and radiative decay channel have been assumed is inappropriate.
Subject(s)
Cholesterol , Erythrocyte Membrane/ultrastructure , Erythrocytes/ultrastructure , Membranes, Artificial , Phosphatidylcholines , Humans , Kinetics , Membrane Lipids/blood , Naphthalenesulfonates , Spectrometry, FluorescenceABSTRACT
Excited state interactions of N-(p-tolyl)-2-aminonaphthalene-6-sulfonate (2, 6 p-TNS) bound to apomyoglobin were studied by nanosecond time-resolved emission spectroscopy. A dynamic interaction of the excited dye molecule with its binding site, associated with a significant change in the emission energy with time, was observed. The decay kinetics were found to be complex and consistent with the kinetic model for solvent relaxation as proposed by Bakhshiev et al. (Opt. Spectrosc. 21:307. 1966). The behavior of 2, 6 p-TNS bound to apomyoglobin was found to be qualitatively similar to that of the dye dissolved in a viscous solvent such as glycerol or adsorbed to egg lecithin vesicles. The detailed information obtained by following the changes in emission spectra of fluorescent probes on the nanosecond time scale leads to a better understanding of their interactions with biological systems.
Subject(s)
Apoproteins , Myoglobin , Anilino Naphthalenesulfonates , Animals , Binding Sites , Kinetics , Mathematics , Muscles , Naphthalenesulfonates , Protein Binding , Protein Conformation , Spectrometry, Fluorescence , Time Factors , WhalesABSTRACT
Nanosecond time-resolved emission spectra (TRES) are fluorescence emission spectra obtained at discrete times during the fluorescence decay. The complete data-set obtainable is a surface representing the intensity at all wavelengths and times during the emission decay time. When 2-p-toluidinonaphthalene-6-sulfonate (2,6 p-TNS) is adsorbed to egg lecithin vesicles, an excited-state reaction associated with energetic changes of the emitting species occurs on the nanosecond time scale. Convolution of the fluorescence decay with the excitation response introduces an artifact in the time-dependent spectra. A precedure is described by which this artifact can be eliminated. The data for the generation of time-resolved emission spectra are obtained with a computer-interfaced instrument based on the single-photon counting method.
