Nanosecond Time-resolved Fluorescence Research Articles

Nanosecond time-resolved fluorescence measurements during protein denaturation

A procedure is described by which the information available from nanosecond time-resolved fluorescence measurements can be used to study rates of reactions taking place on time scales of seconds to hours. A pulse fluorometer was modified so as to obtain a series of short sequential data collections which were rapidly stored on computer disk files. As an application of this new methodology, the unfolding of horse liver alcohol dehydrogenase under acid conditions was monitored by changes in the decay parameters of the intrinsic fluorescence. Although the individual decay curves each had relatively few counts (330 to 160 counts at the peak), a series of decay curves obtained as a function of time could be analyzed in terms of a biexponential function. It was found that the decrease in steady-state fluorescence could be explained most simply by a decrease in the amplitude associated with the longer of the two decay constants.

On the kinetics of formation of an excited charge-transfer state in 4-(9-anthryl)N,N-dimethylaniline

The kinetics of formation of an excited charge-transfer (CT) state in 4-(9-anthryl)-N,N-dimethylaniline have been studied by means of steady-state and nanosecond time-resolved fluorescence. It is concluded that the excited CT state is formed irreversibly at room temperature in ethyl acetate on a timescale shorter than 54 picoseconds.

Excimer formation and energy transfer in vinyl(aromatic polymers)

This paper reviews the results of many time-resolved fluorescence measurements upon vinyl(aromatic polymers) in dilute solution excited by picosecond lasers. The complex kinetics of excimer formation and decay in such polymers revealed by these measurements are discussed, and some preliminary results on the models for these polymers, the dinaphthyl propanes, presented. The kinetics of population and decay of a phenyl oxazole trap in poly(styrene) have been studied and results interpreted in terms of energy migration within the poly(styrene) system. Support for this contentious proposal is gained from measurements of nanosecond time-resolved fluorescence anisotropy.

Reevaluation of the wobbling dynamics of diphenylhexatriene in phosphatidylcholine and cholesterol/phosphatidylcholine membranes

The dynamics of lipid hydrocarbon chains in phosphatidylcholine (dimyristoyl- or dipalmitoyl-) and cholesterol/dimyristoylphosphatidylcholine membranes were investigated by nanosecond time-resolved fluorescence depolarization measurements on a lipophilic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene embedded in the membranes. In the pure lipid membranes, both the range (amplitude) and the rate of the wobbling motion of the probe increased sigmoidally with temperature reflecting the thermotropic phase transition of the lipid. The rise in the rate slightly preceded the increase in the range, suggesting that the fluctuation of lipid chains is activated to a high level before the ordered array of chains melt into the liquid-crystalline phase. Above the transition temperature, incorporation of cholesterol resulted in a dramatic decrease in the range of wobbling motion while the rate remained high. Below the transition, on the other hand, cholesterol had little effect on the range, whereas the rate was greatly increased. These effects of cholesterol are remarkably similar to the effects of cytochrome oxidase on lipid chain dynamics (Kinosita, K., Jr., Kawato, S., Ikegami, A., Yoshida, S. and Orii, Y. (1981) Biochim. Biophys. Acta 647, 7–17).

Nanosecond time-resolved fluorescence investigations of temperature-induced conformational changes in cytochrome oxidase in phosphatidylcholine vesicles and solubilized systems

Intrinsic and lipid phase transition-induced conformational changes in cytochrome oxidase in phosphatidylcholine vesicle and solubilized systems were examined by the fluorescence lifetime of N-(1-anilinonaphthyl-4)-maleimide conjugated with the enzyme. The time-dependent fluorescence intensity of N-(1-anilinonaphthyl-4)-maleimide attached to cytochrome oxidase was described as a triple exponential decay. Both the intrinsic and lipid phase transition-induced conformational changes were detectable in plots of the average lifetime against temperature. In most cases a peak occurred at the temperature of the conformational change. The time-dependent emission anisotropy showed that N-(1-anilinonaphthyl-4)-maleimide embedded in cytochrome oxidase in phosphatidylcholine vesicles underwent a rapid restricted wobbling within a cone. The half-angle of the cone was around 30° for cytochrome oxidase in dimyristoyl phosphatidylcholine vesicles.

Fluorescence Decay Studies of 9-Aminoacridine Bound to Polyriboadenylic Acid

Abstract The interaction of the mutagenic dye 9-aminoacridine (9AA) with polyriboadenylic acid (poly rA) was studied by both steady-state and transient fluorescence measurements. It was found that the fluorescence decay of 9AA bound to poly d(A-T) follows a single-exponential decay law but that the decay data of 9AA bound to poly rA can be well described as a sum of two-exponential functions. The fact that nanosecond time-resolved fluorescence spectra are independent of time suggests that the double-exponential decay may be attributed to different microenvironments at the binding sites. The decay parameters, fluorescence emission maxima, and fluorescence quantum yields obtained at acid pH differ from those obtained at neutral pH. The fluorescence behavior of 9AA is discussed in connection with the conformational change of poly rA.

Nanosecond time-resolved fluorescence of phototautomeric lumichrome

Nanosecond time-resolved fluorescence of phototautomeric lumichrome

Rotational relaxation of the “microviscosity” probe diphenylhexatriene in paraffin oil and egg lecithin vesicles.

The rotational relaxation of the widely used "microviscosity" probe, 1,6-diphenyl-1,3,5-hexatriene, was examined by the technique of nanosecond time-resolved fluorescence depolarization. The decays of the emission anisotropy were determined at five temperatures in the range 3-31 degrees both in a reference paraffin oil and in sonicated egg lecithin vesicles. These decays were complex in both media. Marked qualitative as well as quantitative differences were observed in the rotational behavior of the probe in the complex bilayer medium as opposed to the homogeneous reference solvent. The results are discussed in relation to the structure of the hydrophobic bilayer membrane interior and the concept of its "microviscosity".

Time-Resolved Fluorescence Study of Exciplex and Triple Exciplex in 1,4-Dicyanobenzene and Alkylnaphthalenes

Abstract Exciplex (DA)* and triple exciplex (DDA)* formation between 1,4-dicyanobenzene (DCB) and alkylnaphthalenes was investigated by steady-state and nanosecond time-resolved fluorescence spectroscopies at room temperature. Typical two-component decay of the exciplex and the fluorescence rise of the triple exciplex were observed in dioxane solutions of DCB and several alkylnaphthalenes. The results imply a significant dissociation process of (DDA)* to (DA)*. Triple exciplex formation via excimer (DD)* was proposed to be significant in a concentrated solution of alkylnaphthalene (>10−1 M), while the triple exciplex was formed via the exciplex in a dilute solution of the electron donor.

Time-resolved fluorescence study of triple exciplex in 1,3-dinaphthylpropane and 1,4-dicyanobenzene

The triple exciplex formation between two naphthyl moieties in 1,3-dinaphthylpropane (ββ- and βα-DNP) and 1,4-dicyanobenzene (DCB) was studied by a nanosecond time-resolved fluorescence spectroscopy. The fluorescence of a dioxane or chloroform solution of ββ-DNP and DCB consists of three fluorescence spectra of the intramolecular excimer, the exciplex (DA)* and the triple exciplex (DDA)*. The triple exciplex was not formed via the excimer, but formed via the exciplex.

The Effect of High Density Excitation on the Fluorescence Spectrum of the Anthracene-Tetracene Mixed Crystal

Abstract The nanosecond time-resolved fluorescence spectra of anthracene-tetracene mixed crystals were studied under low and high density excitations. By measuring the time-resolved spectra with low density excitation, the energy-transfer process in the mixed crystal was directly observed. The spectral change due to high density excitation was observed and was explained in terms of the saturation effect on energy transfer from the host to the guest. The effect was found to be related with another nonlinear phenomenon, the bimolecular deactivation of the excited state.

Nanosecond time-resolved emission spectroscopy of a fluorescence probe bound to L-α-EGG lecithin vesicles

The monophoton counting technique was used to measure nanosecond time-resolved fluorescence emission spectra of 2-p-toluidino-naphthalene-6-sulfonate (2,6 p-TNS) adsorbed to lipid bilayer vesicles. A time-dependent red shift in the emission maximum was observed and the rate of this red shift was shown to be temperature dependent. Analysis of fluorescence decay curves obtained at different emission wavelengths indicates that the time-dependent spectral shifts are due to an excited-state reaction such as solvent relaxation or an excited-state interaction between the chromophore and a polar residue of the phospholipid.

Nanosecond Time-Resolved Emission Spectroscopy of 1-Anilino-8-Naphthalene Sulfonate

Nanosecond time-resolved emission spectroscopy and fluorescence lifetime measurements are reported for 1-anilino-8-naphthalene sulfonate. Time-dependent spectral shifts are observed that are interpreted in terms of dipole reorientation of the solvent around the electronically excited molecule. Both the radiative and nonradiative transitions probabilities are observed to be strongly solvent dependent.

Nanosecond Time-resolved Fluorescence Spectra of a Protein-Dye Complex

Abstract Fluorescence lifetime measurements have been used to obtain nanosecond time-resolved emission spectra of 2-p-toluidinylnaphthalene-6-sulfonate in ethanol, glycerol, and when adsorbed to bovine serum albumin. Red shifts with time were observed in the glycerol and bovine serum albumin experiments but not in ethanol. The effect of temperature between 4 and 60° supports the interpretation that the spectral shifts reflect nanosecond relaxations of polar groups around the dye during the lifetime of the excited state. It is suggested that this experimental approach has potential value for the investigation of relaxation kinetics on the nanosecond time scale.