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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.

The two-state excited-state proton-transfer process for d-equilenin [d-3-hydroxyestra-1,3,-5(10),6,8-pentaen-17-one] and dihydroequilenin is found to depend both on pH and on proton acceptor concentration. Both the protonated and deprotonated forms of the excited molecule are fluorescent. As is the case for 2-naphthol, the excited-state pKa (pKa*) is substantially lower than the ground-state pKa. Fluorescence decay studies have been performed as a function of emission wavelength in aqueous solutions at pH 6.9 in the presence of acetate anion (0.1 M). At this pH, both back-reaction from the excited-state and ground-state heterogeneity are minimal. A monoexponential decay is found in the blue region of the spectrum and a biexponential decay on the red edge. The lifetimes measured across both regions are constant, with a negative preexponential term, characteristic of an excited-state reaction, evident at longer wavelengths. Decay-associated spectra (DAS), the preexponential terms associated with the measured lifetimes, have been acquired for these aqueous solutions. Equilenin and dihydroequilenin are found to adsorb to dimyristoyllecithin (DML) vesicles. Rates for excited-state proton transfer are greatly reduced when dihydroequilenin adsorbs to vesicles. The accessibility of the bound probe to acetate as a proton acceptor depends on the cholesterol content of the vesicles.

When 5(6)-carboxyfluorescein (6CF) is encapsulated in liposomes at 0.2 M, 97-98% of the fluorescence is quenched. We have studied the mechanism of this effect. The dye-liposome system is a special case of concentration quenching of dyes, a phenomenon recognized for 100 years. Absorption spectra of encapsulated dye show that 6CF dimerizes, and the dimer is nonfluorescent. The dimerization constant was estimated, and it was concluded that dimerization can account for only part of the quenching. In 6CF solutions, the fluorescence lifetime decreased drastically as concentration was changed over the narrow range 0.02-0.05 M, a finding which was attributed to energy transfer to dimers. Inhibition of dimerization by propylene glycol also inhibited the shortening of lifetime. Förster critical transfer distances were calculated to be 51 and 57 A for monomer-monomer and monomer-dimer transfer, respectively. Monomer-monomer transfer was demonstrated directly by steady-state or time-resolved anisotropy experiments, while transfer to dimer was modeled by using sulforhodamine B, which has a critical transfer distance like that for the dimer and also quenches 6CF emission. No direct evidence for collisional self-quenching of 6CF could be found, although a model compound, salicylate, did quench weakly. For xanthene dyes, the rate of energy transfer is much faster than that for quenching collisions, implying that collisional quenching in the usual 6CF-liposome system is insignificant. The reason why 6CF is not 100% quenched in liposomes is attributed to dye interaction with lipid as evidenced by (i) multiexponential decay of 6CF in liposomes with a long component of 3-4 ns, (ii) inhibition of dimerization in liposomes, (iii) partial protection of dye from quenching by KI, (iv) differing amounts of dimerization in liposomes made from different kinds of phospholipid, and (v) enhancement of fluorescence lifetime in the presence of Triton X-100.

The application of a new spectroscopic tool [Knutson, J. R., Davenport, L., & Brand, L. (1986) Biochemistry (preceding paper in this issue)] for studying rotational microheterogeneity of probe location in lipid bilayer systems is described. Anisotropy decay associated spectra are derived from experimentally obtained polarized emission components. "Early" difference spectra (IV - IH) contain contributions from both fast and slow rotors, while "late" difference spectra predominantly reflect the emission from slowly rotating fluorophores. Anisotropy decay associated spectra have been used to resolve the emission spectra of 1,6-diphenyl-1,3,5-hexatriene (DPH) imbedded within a known rotationally heterogeneous mixture of two vesicle types (L-alpha-dimyristoyllecithin and L-alpha-dipalmitoyllecithin). At 29 degrees C, diphenylhexatriene within pure dimyristoyllecithin vesicles rotates rapidly, with a small r infinity, while diphenylhexatriene in dipalmitoyllecithin vesicles exhibits a large r infinity. Spectra for diphenylhexatriene imbedded in the two vesicle types show small but significant spectral differences. A spectrum of a mixture of the two vesicle types with DPH lies between these characteristic component spectra. The spectrum extracted for "immobilized" probes in the mixture correctly overlays the dipalmitoyllecithin spectrum. Further studies have shown that diphenylhexatriene exhibits more than one emission anisotropy decay associated spectrum in vesicles of a single lipid type, when that lipid is near its phase transition temperature. Diphenylhexatriene apparently inhabits more than one rotational environment even in these "homogeneous" vesicle preparations.

Oxygen quenching of perylene fluorescence was used to vary its fluorescence lifetime. Steady-state fluorescence anisotropy measurements under these quenching conditions were used to investigate the diffusive motions of perylene in the isotropic solvent propylene glycol and in lipid bilayers. These lifetime-resolved anisotropy measurements indicate that the anisotropy of perylene in propylene glycol decays to zero at times long compared to its fluorescence lifetime. In contrast, the asymptotic or "limiting" anistropy values at these long times (r infinity) are nonzero in vesicles of dimyristoyl-phosphatidylcholine (DMPC). r infinity values are largest at temperatures below the DMPC phase transition temperature of 23 degrees C. Representative values of r infinity for perylene in DMPC vesicles are 0.16 and 0.02 at 5 and 47 degrees C, respectively. Thus, in contrast to the free rotations observed for perylene in propylene glycol, perylene rotations are hindered in lipid bilayers. Less marked, yet significant, rotational hindrance was observed in dioleoylphosphatidylcholine (DOPC) vesicles. Representative values for r infinity in this unsaturated lipid are 0.05 and 0.01 at 2 and 45 degrees C, respectively. Steady-state anisotropy measurements with short-wavelength excitations were used to investigate whether the in-plane or out-of-plane rotations of perylene were responsible for the observed r infinity values. In DMPC vesicles we conclude that both rotations are partially hindered. In DOPC vesicles we can only conclude that one or both of these rotations are partially hindered, but both are not free. Most importantly, the existence of fundamentally different diffusive behavior for perylene in solvents and in lipids calls into question the meaning of membrane microviscosities which are derived via such comparisons.

Enzyme I of the bacterial phosphotransferase system is a protein component which undergoes a temperature-dependent monomer/dimer equilibrium. Reaction of sulfhydryl residues with SH-specific reagents inhibits both activity and dimerization. There are four cysteine residues available in each subunit, one of which (Cys 502) is proximate to one of the two tryptophan residues (Trp 498). Previous studies revealed two major lifetimes and spectra, suggesting distinct environments for tryptophan. In this paper, we examine the dynamic quenching of tryptophanyl fluorescence that occurs when an energy transfer acceptor, thio-2-nitrobenzoic acid (TNB), is covalently attached to the sulfhydryl groups. More precisely, we have traced the recovery of nativelike fluorescence lifetime components (and the concomitant loss of "reduced lifetime" amplitudes) that accompanies TNB release. The course of lifetime changes seen when a reducing reagent removes the quencher may be sensitive to a variety of effects, including different SH affinities, different proximities to Trp, changing availability for dimerization, or conformational changes. The prospective value of separating each lifetime component from the mixture is illustrated.