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J R Lakowicz

Publications and source records attributed to J R Lakowicz.

At least 199 records · Page 11Linked to original sources
PubMed1984

Time-dependent rotational rates of excited fluorophores. A linkage between fluorescence depolarization and solvent relaxation.

It is generally assumed that the rotational diffusion coefficients of fluorophores are independent of time subsequent to excitation, and that the rotational diffusion coefficients of the ground and the excited states are the same. We now describe a linkage between the extent of solvent relaxation and the rate of fluorescence depolarization. Specifically, if a fluorophore displays time-dependent solvent relaxation it may also show a time-dependent decrease in its rotational rate. A decreased rate of rotation could result from the increased interaction with polar solvent molecules which occurs as a result of solvent relaxation. The decays of anisotropy predicted from our model closely mimic those often observed for fluorophores which are bound to macromolecules. For example, the decays are more complex than a single exponential, and the time-resolved anisotropy can display a limiting value which does not decay to zero. The effect of solvent relaxation upon the rates of rotational diffusion is expected to be most dramatic for solvent-sensitive fluorophores in a viscous environment. These conditions are frequently encountered for fluorophore-macromolecule complexes. Consideration of the linkage between solvent relaxation and rotational diffusion leads to two unusual predictions. First even spherical fluorophores in an isotropic environment could display multi- or nonexponential decays of fluorescence anisotropy. Secondly, for the special case in which the fluorophore dipole moment decreases upon excitation, the theory predicts that the anisotropy decay rate may increase with time subsequent to pulsed excitation. The predictions of this theory are consistent with published data on the effects of red-edge excitation upon the apparent rotational rates of fluorophores in polar solvents.

Journal Article↗
PubMed1984

Torsional rearrangements of aryl-substituted anthracenes measured by phase fluorometry.

We examined the fluorescence phase angle spectra of aryl-substituted anthracenes in viscous solvents. These phase angles reflect the wavelength-dependent lifetimes displayed by the fluorophores. At intermediate viscosities the phase angles increased and decreased in accordance with the valleys and peaks of the structured emission spectra. At lower and higher temperatures the phase angles were independent of emission wavelength, which is equivalent to decay times which are independent of emission wavelength. We attribute the wavelength-dependent phase angles to rotation of the unsaturaled side chains towards a conformation more coplanar with the anthracene ring. By comparative studies with 9,10-di(alpha-naphthylanthracene), 9,10-di-phenylanthracene, and 9-vinylanthracene we showed that lower temperatures are required to inhibit rotation of the smaller phenyl and vinyl substitutents. Since these excited-state rearrangements are sensitive to the motional freedom allowed by the surrounding solvent, we suggest that this phenomenon may serve as a probe of volume fluctuations in macromolecules.

Journal Article↗
PubMed1984

Effect of ligand binding and conformational changes in proteins on oxygen quenching and fluorescence depolarization of tryptophan residues.

The rotational freedom of tryptophan residues in protein-ligand complexes was studied by measuring steady-state fluorescence anisotropies under conditions of oxygen quenching. There was a decrease in the oxygen bimolecular quenching constant upon complexation of trypsin and alpha-chymotrypsin with proteinaceous trypsin inhibitors, of lysozyme with N-acetylglucosamine (NAG) and di(N-acetyl-D-glucosamine) ((NAG)2) and of hexokinase with glucose. Binding of the bisubstrate analogue N-phosphonacetyl-L-aspartate (PALA) to aspartate transcarbamylase (ATCase) and binding of biotin to avidin resulted in increased oxygen quenching constants. The tryptophan of human serum albumin (HSA) in the F state was more accessible to oxygen quenching than that in the N state. With the exception of ATCase, the presence of subnanosecond motions of the tryptophan residues in all the proteins is suggested by the short apparent correlation times for fluorescence depolarization and by the low apparent anisotropies obtained by extrapolation to a lifetime of zero. Complex formation evidently resulted in more rigid structures in the case of trypsin, alpha-chymotrypsin and lysozyme. The effects of glucose binding on hexokinase were not significant. Binding of biotin to avidin resulted in a shorter correlation time for the tryptophan residues. The N --> F transition in HSA resulted in a more rigid environment for the tryptophan residue. Overall, these changes in the dynamics of the protein matrix and motional freedom of tryptophan residues due to complex formation and subsequent conformational changes are in the same direction as those observed by other techniques, especially hydrogen exchange. Significantly, the effects of complex formation on protein dynamics are variable. Among the limited number of cases we examined, the effects of complex formation were to increase, decrease or leave unchanged the apparent dynamics of the protein matrix.

Journal Article↗
PubMed1984

Structural fluctuations in aspartate transcarbamylase. Succinimide quenching and fluorescence depolarization of tryptophan and tyrosine residues.

The effects of binding of various effector ligands on the dynamics of aspartate transcarbamylase (ATCase, c6r6) and on its regulatory (r2) and catalytic (c3) subunits were characterized by examining succinimide quenching of the intrinsic fluorescence, and by measurement of the lifetime-resolved anisotropies. The lifetimes of the tryptophan residues in c3 and c6r6 are about 1.7 ns while those of tyrosine residues in r2 are 2.7 ns. These lifetimes are not significantly altered by the binding of various substrates, substrate analogs and nucleotides. The effects of ligand binding on the accessibility of both tyrosine and tryptophan residues to the quencher are modest in all cases, though the changes are in the same direction as seen using other physicochemical techniques such as hydrogen exchange (M. Lennick and N.M. Allewell, Proc. Natl. Acad. Sci. U.S.A. 78 (1981) 6759). The tryptophan residues in both c3 and c6r6 are immobilized whereas the tyrosine residues of r2 have some motional freedom. Ligands have no effect on the immobilized tryptophan residues in c3 and c6r6, while binding of nucleotides to r2 results in a small decrease in the motional freedom of the tyrosine residues. These results suggest that the protein matrix around the aromatic amino acids in r2, c3 and c6r6 is rather rigid and that local effects of ligands on the dynamics of these residues, and that of the surrounding protein matrix, are minor. They are in general agreement with the results of the crystal structure determination (R.B. Honzatko et al., J. Mol. Biol. 160 (1982) 219).

Aspartate Carbamoyltransferase↗
PubMed1984

Analysis of fluorescence decay kinetics from variable-frequency phase shift and modulation data.

Recently it has become possible to measure fluorescence phase-shift and modulation data over a wide range of modulation frequencies. In this paper we describe the analysis of these data by the method of nonlinear least squares to determine the values of the lifetimes and fractional intensities for a mixture of exponentially decaying fluorophores. Analyzing simulated data allowed us to determine those experimental factors that are most critical for successfully resolving the emissions from mixtures of fluorophores. The most critical factors are the accuracy of the experimental data, the relative difference of the individual decay times, and the inclusion of data measured at multiple emission wavelengths. After measuring at eight widely spaced modulation frequencies, additional measurements yielded only a modest increase in resolution. In particular, the uncertainty in the parameters decreased approximately as the reciprocal of the square root of the number of modulation frequencies. Our simulations showed that with presently available precision and data for one emission bandpass, two decay times could be accurately determined if their ratio were greater than or equal to 1.4. Three exponential decays could also be resolved, but only if the range of the lifetimes were fivefold or greater. To reliably determine closely-spaced decay times, the data were measured at multiple emission wavelengths so that the fractional intensities of the components could be varied. Also, independent knowledge of any of the parameters substantially increased the accuracy with which the remaining parameters could be determined. In the subsequent paper we present experimental results that broadly confirm the predicted resolving potential of variable-frequency phase-modulation fluorometry.

Kinetics↗
PubMed1984

Resolution of mixtures of fluorophores using variable-frequency phase and modulation data.

We measured fluorescence phase shift and modulation data for one-, two- and, three-component mixtures of fluorophores at modulation frequencies ranging from 1 to 140 MHz. These data were analyzed using the least-squares procedure described in the preceding paper (Lakowicz, J. R., G. Laczko, M. Cherek, E. Gratton, and M. Limkeman, 1984, Biophys. J., 46:463-477). Using data obtained at a single emission bandpass, the lifetimes and preexponential factors of two-component mixtures could be easily resolved if the lifetimes differed by a factor of 2. With currently available instrumental stability, three-component mixtures could be resolved when the overall range of decay times was 10-fold, (e.g., 1.3, 4.4, and 12 ns). Measurement of phase and modulation data at several emission wavelengths, where the ratio of the preexponential factors varied, enhanced our ability to resolve closely spaced two and three-component decays. Two-component mixtures could then be resolved if the lifetimes differed by 30% (4.4 and 6.2 ns). Also, the multiple-wavelength data allowed the lifetimes and emission spectra of the three-components of a mixture to be resolved. These results demonstrated that resolution of multiexponential decay laws was possible using frequency-domain phase-modulation fluorometry.

Anthracenes↗
PubMed1983

Differential polarized phase fluorometric studies of phospholipid bilayers under high hydrostatic pressure.

Differential polarized phase fluorometry was used to quantify the rotational rate (R) and limiting anisotropy (r infinity) of the membrane probe diphenylhexatriene (DPH) in solvents and lipid vesicles exposed to hydrostatic pressures ranging from 1 bar to 2 kbar. These measurements reveal the effect of pressure on the phase-transition temperatures of the phosphatidylcholine vesicles, and the effects of pressure on order parameter of the acyl side-chain region of the membranes, the latter as indicated by r infinity. In addition to the well-known elevation of the transition temperature (Tc) with pressure, our results demonstrate that increased pressure restores the order of the bilayers to that representative of temperatures below the transition temperature. We also found that solvents which allowed free isotropic rotation of DPH at 1 bar no longer allowed free rotation when sufficiently compressed; moreover, the apparent DPH rotational rate increased with r infinity. Pressure studies using both DPH and the charged DPH analogue, trimethylammonium DPH (TMA-DPH) indicated that the Tc of dipalmitoylphosphatidylcholine vesicles increased 23 K/kbar and an apparent volume change of 0.036 ml/mol lipid at the phase transition. Assuming, as has been proposed, that TMA-DPH is localized near the glycerol backbone region of the bilayers, these results indicate a similar temperature- and pressure-dependent phase transition in this region and the acyl side-chain region of the membrane.

Cell Membrane↗
PubMed1983

Binding of an indole derivative to micelles as quantified by phase-sensitive detection of fluorescence.

Phase-sensitive detection of fluorescence was used to measure the fractional binding of an amphipathic derivative of indole to detergent micelles. By selection of the detector phase angle, the emissions from the free and bound indole could be individually suppressed. The phase-sensitive intensity was then proportional to the concentrations of the bound and free indole, respectively. Also, the ratio of the phase-sensitive intensities, recorded at the phase angles for suppression, revealed the ratio of the fluorophore present in each environment. As the detergent concentration was increased, the measurements revealed a cooperative formation of detergent micelles and binding of the indole derivative to these micelles. The theory and procedures for this specific analysis can be applied to the analysis of any association reaction in which one of the species is fluorescent, and in which the fluorescence lifetime is altered by the binding process. An advantage of this procedure is that binding can be quantified without physical separation of the individual species.

Colloids↗
PubMed1983

Oxygen quenching and fluorescence depolarization of tyrosine residues in proteins.

We examined the dynamics of oxygen quenching and depolarization of tyrosine fluorescence in small peptides and proteins lacking tryptophan. The oxygen-quenching constants and the apparent correlation times for fluorescence depolarization were found to be sensitive to the conformational state of the proteins. For small peptides and random coil proteins, the oxygen bimolecular quenching constants indicated complete accessibility of the tyrosine residues to collisions with oxygen. For folded proteins, the quenching constants were about 2-fold smaller, indicating only limited shielding of the tyrosine residues from oxygen by the protein matrix. We also used the steady state anisotropies, measured under conditions of oxygen quenching, to estimate the motional freedom of the tyrosine residues. For random coil proteins, such as a tyrosine copolymer and histones at low pH, the data clearly indicated that depolarization occurs due to subnanosecond segmental motions of the tyrosine residues which are independent of overall protein rotation. For some folded proteins, including bovine pancreatic trypsin inhibitor, the data are consistent with, but do not unambiguously demonstrate, motional freedom of the residues. In these cases, energy transfer among tyrosine residues may also contribute to the observed depolarization. Overall, these results indicate that the rate and extent of tyrosine rotation in proteins depend upon the conformation of the protein and the specific protein under observation.

Chemical Phenomena↗
PubMed1983

Rotational freedom of tryptophan residues in proteins and peptides.

We studied the rotational motions of tryptophan residues in proteins and peptides by measurement of steady-state fluorescence anisotropies under conditions of oxygen quenching. By fluorescence quenching we can shorten the fluorescence lifetime and thereby decrease the average time for rotational diffusion prior to fluorescence emission. This method allowed measurement of rotational correlation times ranging from 0.03 to 50 ns, when the unquenched fuorescence lifetimes are near 4 ns. A wide range of proteins and peptides were investigated with molecular weights ranging from 200 to 80 000. Many of the chosen substances possessed a single tryptophan residue to minimize the uncertainties arising from a heterogeneous population of fluorophores. In addition, we also studied a number of multi-tryptophan proteins. Proteins were studied at various temperatures, under conditions of self-association, and in the presence of denaturants. A wide variety of rotational correlation times were found. As examples we note that the single tryptophan residue of myelin basic protein was highly mobile relative to overall protein rotation whereas tryptophan residues in human serum albumin, RNase T1, aldolase, and horse liver alcohol dehydrogenase were found to be immobile relative to the protein matrix. These results indicate that one cannot generalize about the extent of segmental mobility of the tryptophan residues in proteins. This physical property of proteins is highly variable between proteins and probably between different regions of the same protein.

Kinetics↗
PubMed1983

Evidence for normal fibroblast cell membranes from individuals with Huntington's disease. A fluorescence probe study.

We used fluorescence spectroscopy in an attempt to identify differences in the fibroblast cell membranes from individuals with Huntington's disease (HD), compared to those from normal individuals. Eight pairs of age- and sex-matched fibroblast cultures were examined, each using four different fluorescent probes. To maximize the possibility of finding a difference between the normal and HD cells fluorescence probes were selected which localize in different regions of the membrane. Also, these probes differed in their sensitivity to the surrounding environment. Procedures were developed for labeling of the fibroblasts while they were still attached to glass coverslips. All fluorescence measurements were done on attached cells, which were all found to be viable by staining with trypan blue. Both low (5-7) and high (11-13) passage cultures were examined since earlier reports indicated that normal and HD fibroblasts could be distinguished at low passage using a fluorescence method. Using these 16 cultures, and multiple determinations of the fluorescence emission spectra, anisotropies, and lifetimes, we found no significant difference between normal and HD fibroblasts. If such a difference exists, it appears to be too small for use as a diagnostic indicator for Huntington's disease.

Cell Membrane↗
PubMed1982

Interactions of chlorinated hydrocarbon insecticides with membranes.

Chlorinated hydrocarbon insecticides quench the fluorescence of N-alkyl derivatives of carbazole. We used phospholipids with covalently attached carbazole as probes for the interactions of chlorinated hydrocarbon insecticides with lipid bilayers, the object being to understand better the toxicities of chlorinated hydro-carbons. Fluorescence quenching measurements revealed the lipid-water partition coefficients of the chlorinated hydrocarbons, their diffusion coefficients in the membranes, and the binding capacities of the membranes for the chlorinated hydrocarbons. Active insecticides were compared with inactive analogues to test whether activities correlated with chlorinated hydrocarbon-membrane interactions. Thus DDT and methoxychlor were compared with inactive DDE, and insecticidal gamma-lindane was compared with three less active stereoisomers. The partition coefficients, diffusion coefficients and membrane saturation capacities did not correlate with insecticidal potency. The partition coefficients of these chlorinated hydrocarbons were larger in bilayers containing unsaturated fatty acyl chains as compared to bilayers containing saturated fatty acyl chains. Interestingly, neural membranes are known to contain a large percentage of unsaturated lipids. Our results indicate that the activities of chlorinated hydrocarbons are not a result of specific interactions of these compounds with the lipids of membranes. However, the neurotoxicity of chlorinated hydrocarbons may be amplified by selective partitioning in the unsaturated neural membranes.

Diffusion↗
PubMed1982

Theory of phase-modulation fluorescence spectroscopy for excited-state processes.

Theory is presented for the analysis of excited-state reactions by fluorescence phase shift and demodulation methods. Initially, a two-state model with spectral overlap is considered to illustrate most simply the effects of excited-state reactions on the expected phase and modulation values. Secondly, a multistate model is described to illustrate the probable effects of a fluorophore interacting with several solvent molecules. We note the following unique features of phase-modulation data expected from a fluorophore whose emission spectrum shifts during the lifetime of the excited state: (1) The modulation frequency dependence of the apparent phase (tau p) and modulation (tau m lifetimes of the reacted species is opposite to that of a heterogeneous population of fluorophores. (2) For the reacted species tau p greater than tau m. For a heterogeneous sample tau p less than tau m. (3) The phase angle of the reacted species can exceed 90 degrees. For a heterogeneous sample phase angles are always less than 90 degrees. Thus, phase and modulation measurements can distinguish between time-dependent processes and spectral heterogeneity by observation of any feature described above. Additionally: (4) The lifetime of the product species can be measured directly. (5) Reverse relaxation can be identified, and the reverse relaxation rates calculated. (6) The wavelength-dependent phase and modulation data can be used to resolve the individual spectra from a two-state reaction. (7) And finally, under favorable conditions, a two-state excited-state process can be distinguished from a continuous multiple-state process. In each instance, model calculations are presented to illustrate the unique potentials of phase-modulation fluorometry for investigations of excited-state processes.

Kinetics↗
PubMed1982

Analysis of excited-state processes by phase-modulation fluorescence spectroscopy.

Fluorescence phase shift and demodulation methods were used in the analysis of excited-state reactions and to investigate solvent relaxation around fluorophores in viscous solvents. The chosen samples illustrate the results expected for fluorophores bound to biological macromolecules. These moderately simple samples served to test the theoretical predictions described in the preceding paper (J.R. Lakowicz and A.B. Balter, Biophys. Chem. 16 (1982) 99.) and to illustrate the characteristic features of phase-modulation data expected from samples which display time-dependent spectral shifts. The excited-state protonation of acridine and exciplex formation between anthracene and diethylaniline provided examples of one-step reactions in which the lifetimes of the initially excited and the reacted species were independent of emission wavelength. Using these samples we demonstrated the following: (1) Wavelength-dependent phase shift and demodulation values can be used to prove the occurrence of an excited-state process. Proof is obtained by observation of phase angles (ø) larger than 90 degrees or by measurement of ratios of m/cos ø greater than 1, where m is the modulation of the emission relative to that of the excitation. (2) For a two-state process the individual emission spectra of each state can be calculated from the wavelength-dependent phase and modulation data. (3) The phase difference or demodulation factor between the initially excited and the reacted states reveals directly the fluorescence lifetime of the product of the reaction. (4) Phase-sensitive detection of fluorescence can be used to prove that the lifetimes of both the initially excited and the reacted states are independent of emission wavelength. (5) If steady-state spectra of the individual species are known, then phase-sensitive emission spectra can be used to measure the lifetimes of the individual components irrespective of the extent of spectral overlap. (6) Spectral regions of constant lifetime can be identified by the ratios of phase-sensitive emission spectra. In addition, we examined 6-propionyl-2-dimethylaminonaphthalene (PRODAN) and N-acetyl-L-tryptophanamide (NATA) in viscous solvents where the solvent relaxation times were comparable to the fluorescence lifetimes. Using PRODAN in n-butanol we used m/cos ø measurements, relative to the blue edge of the emission, to demonstrate that solvent relaxation requires more than a single step. For NATA in propylene glycol we used phase-sensitive detection of fluorescence to directly record the emission spectra of the initially excited and the solvent relaxed states. These measurements can be easily extended to fluorophores which are bound to proteins and membranes and are likely to be useful in studies of the dynamic properties of biopolymers.

Biopolymers↗
PubMed1982

Differential-wavelength deconvolution of time-resolved fluorescence intensities. A new method for the analysis of excited-state processes.

We describe a new procedure for the analysis of time-resolved decays of fluorescence intensity observed for excited-state reactions. This procedure is particularly valuable because it simplifies both determination of the rate constants of excited-state reactions and calculation of the emission spectra of the unreacted and the reacted species. These advantages are obtained without additional data acquisition, by the further analysis of data which would already be collected. As is usual for an excited-state process, time-resolved decays of fluorescence intensity are collected at wavelengths across the emission spectrum. Generally, one derives analytical expressions for the impulse response by deconvolution using the time profile of the exciting pulse. We suggest that in addition to the procedure just described, the response observed at longer wavelengths be deconvolved using the response observed on the blue side of the emission. Typically, this emission is from the initially excited state of the fluorophore (F). Then, the derived decay time is the decay rate of the reacted species (R). In addition, spectral overlap of the F and R states is revealed quantitatively as an apparent zero-decay-time component in the derived impulse response function. This is because the population of the initially excited state is considered to be the excitation function. This spectral overlap component is easily quantified, and allows the emission spectra of the F and R states to be calculated. This deconvolution procedure requires one favorable circumstance, which is the ability to choose a wavelength at which only the initially excited state is emitting. We tested our procedure on the excited state protonation of acridine and the excited-state deprotonation of 2-naphthol. The former reaction is essentially irreversible, whereas depending upon pH the dissociation of 2-naphthol can be reversible. We demonstrated that this procedure, which we call differential-wavelength deconvolution, revealed the individual spectra and simplified determination of the kinetic constants from the time-resolved decays. Without differential-wavelength deconvolution considerably more complex methods are required. We expect this procedure to greatly facilitate the use of pulse fluorometry methods in the analysis of excited-state processes.

Acridines↗
PubMed1982

Fluorescence studies of red blood cell membranes from individuals with Huntington's disease.

Fluorescence spectroscopic methods were used to investigate and compare the properties of erythrocyte membranes from individuals with Huntington's Disease (HD) and from normal individuals. Erythrocyte ghosts were labeled with four different fluorescent probes: 1,6-diphenylhexatriene (DPH); 6-lauroyl-2-(dimethylamino)-naphthalene (Laurdan); 2-(4-maleimide anilino)-naphthalene-6-sulfonic acid (MIANS) and 5-(iodoacetamidoethyl)aminoaphthalene-1-sulfonic acid (IAEDANS). DPH is sensitive to the microviscosity of the membranes. Laurdan is highly sensitive to the polarity and relaxation time of its environment. MIANS and IAEDANS both react covalently with sulfhydryl groups in membrane proteins. Erythrocyte membranes were labeled separately with each of these four probes, and we measured the centers of gravity of the fluorescence emission, the fluorescence anisotropies, and the fluorescence lifetimes. In 10 separate experiments, including a total of 24 patients and 14 control individuals, we found no significant differences between the two groups for any of the probes or spectral parameters. These results do not support the existence of a "generalized membrane defect" in individuals with HD.

Erythrocyte Membrane↗