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Biomedical subjects

I Gryczynski

Publications and source records attributed to I Gryczynski.

89 records · Page 5Linked to original sources
PubMed1988

Resolution of the lifetimes and correlation times of the intrinsic tryptophan fluorescence of human hemoglobin solutions using 2 GHz frequency-domain fluorometry.

We used 2 GHz harmonic content frequency-domain fluorescence to measure the intensity and the anisotropy decays from the intrinsic tryptophan fluorescence from human hemoglobin (Hb). The tryptophan intensity decays are dominated by a short-lived component which accounts for 35-60% of the total steady state intensity. The decay time of this short component varies from 9 to 27 ps and this component is sensitive to the ligation state of Hb. Our error analyses indicate the uncertainty is about +/- 3 ps. The intensity decays also show two longer lived components near 0.7 and 8 ns, which are probably due either to impurities or to Hb molecules in conformations which do not permit energy transfer. The anisotropy decays indicate the tryptophan residues in Hb are highly mobile, with apparent correlation times near 55 ps.

Carboxyhemoglobin↗
PubMed1988

Acrylamide quenching of Yt-base fluorescence in aqueous solution.

Acrylamide was found to be an effective quencher of Yt-base (Y-4,9-dihydro-4,6-dimethyl-9-oxo-1H-imidazo-1,2a-purine) in water. In the absence of collisional quenching the decay of Yt-base in water is predominantly a single exponential. The intensity decays become increasingly heterogeneous when quenched by acrylamide. The frequency-domain data were analyzed using the radiation model, which provides estimates of molecular parameters characteristic of the system. The mutual diffusion coefficient at 20 degrees C was found to be 0.5 x 10(5) cm2/s, the Yt-base acrylamide interaction radius was 8 A, and the rate constant for quenching was 100 cm/s. These values indicate that quenching is diffusion-limited, i.e., the encounter complex is deactivated at least 2-fold faster than the rate of diffusive encounters.

Acrylamide↗
PubMed1988

Frequency-domain measurements of the rotational dynamics of the tyrosine groups of calmodulin.

We used frequency-domain fluorometry to determine the intensity and anisotropy decay kinetics of tyrosine residues in calmodulin and its fragments. Excitation was provided by a continuous ultraviolet laser source, a frequency-doubled rhodamine 6G ring dye laser, whose output was externally modulated to 200 MHz. Both the intensity and anisotropy decays were found to be multiexponential and dependent upon temperature and solution conditions. By examination of calmodulin fragments we determined that energy transfer between the two tyrosine residues reduces the steady-state anisotropy values by about 20%. Additionally, the frequency-domain anisotropy decays indicate local torsional motions of the tyrosine residues, as well as significant individual motions of the two domains of calmodulin.

Calmodulin↗
PubMed1988

Distribution of distances in thiopeptides by fluorescence energy transfer and frequency-domain fluorometry.

Frequency-domain fluorescence spectroscopy was employed to examine the decays of tryptophan in Boc-Trp-Met-Asp-Phe-NH2 (donor) and (Formula: see text) (donor-acceptor pair). The efficiency of energy transfer in the thiopeptide amounted to 60%. The measured dispersion of fluorescence decay times was used to recover the donor-acceptor distance distribution. The parameters of the Gaussian distance distribution obtained for this peptide (r, the mean distance (9 A); hw, the halfwidth (25 A)) indicate the lack of a distinct favorable conformation.

Energy Transfer↗
PubMed1988

Lifetime distributions and anisotropy decays of indole fluorescence in cyclohexane/ethanol mixtures by frequency-domain fluorometry.

We used frequency-domain fluorometry to measure intensity and anisotropy decay of indole fluorescence in cyclohexane/ethanol mixtures at 20 degrees C. In 100% cyclohexane or 100% ethanol the intensity decay of indole appears to be a single exponential with decay times of 7.66 and 4.10 ns, respectively. In cyclohexane containing a small percentage of ethanol (up to 10%), we observed increased heterogeneity in intensity decay, resulting in a 10-fold increase in chi 2R for the single-exponential fit, as compared with the double-exponential model. We obtained comparable or better fits using unimodal Lorentzian and Gaussian lifetime distributions (two floating parameters) than for the two-exponential model (three floating parameters). We believe that the distribution of decay times reflects a range of indole solvation states in the dominately nonpolar solutions. This result suggests that a variety of hydrogen-bonding configurations could be one origin of the distributions of decay times observed for tryptophan emission from proteins. We also measured rotational diffusion of indole in cyclohexane, ethanol and its mixtures at 20 degrees C. The picosecond correlation times required that the mean decay times be decreased by acrylamide quenching (in ethanol) or energy transfer (in cyclohexane). In ethanol we observed nearly isotropic rotation of indole; in cyclohexane we obtained two correlation times of 17 and 73 ps. The shorter correlation time in cyclohexane appears to be due to the slip boundary condition, which was found to be progressively eliminated by small percentages of ethanol. Hence, hydrogen-bonding interactions appear to have a substantial effect on the rotational dynamics of indole.

Cyclohexanes↗
PubMed1988

Time-resolved emission spectra of hemoglobin on the picosecond time scale.

We used front-face illumination to examine the steady-state and time-resolved emission from the intrinsic tryptophan emission of human hemoglobin (Hb). Experimental conditions were identified which eliminated all contributions of scattered light. The sensitivity obtained using front-face optics was adequate to allow measurement of the wavelength-dependent frequency response of the emission to 2 GHz. The intensity decays displayed pico- and nanosecond components in the emission at all wavelengths from 315 to 380 nm. The contribution of the picosecond component decreased from 72 to 37% over this range of wavelengths. Frequency-domain measurements were used to calculate the time-resolved emission spectra and decay-associated emission spectra. These spectra indicate that the picosecond components of the emission display maxima near 320 nm, whereas the nanosecond components are centered at longer wavelengths near 335 nm. The nanosecond components appear to be due to residual impurities which remain even in highly purified samples of Hb. However, we cannot eliminate the possibility that some of these components are due to Hb itself.

Hemoglobins↗
PubMed1988

Gigahertz frequency-domain fluorometry: resolution of complex decays, picosecond processes and future developments.

We describe the principles, instrumentation and applications of frequency-domain fluorescence spectroscopy. This method is useful for the resolution of multi-exponential decays and complex anisotropy decays on the picosecond timescale. The present instrumentation allows measurements to 2 GHz, which has been used to measure rotational correlation times as short as 7 ps. In the future it may be possible to extend the frequency range to 10 GHz, which should allow still faster processes to be quantified. It should be emphasized that resolution of fast processes is not obtained at the expense of losing information on the nanosecond timescale. Additionally, the GHz frequency-domain measurements are performed using low excitation intensities, which do not damage the samples.

Lasers↗
PubMed1988

Resolution of end-to-end distance distributions of flexible molecules using quenching-induced variations of the Forster distance for fluorescence energy transfer.

We describe a new method to recover the distribution of donor-to-acceptor (D-A) distances in flexible molecules using steady-state measurements of the efficiency of fluorescence energy transfer. The method depends upon changes in the Forster distance (Ro) induced by collisional quenching of the donor emission. The Ro-dependent transfer efficiencies are analyzed using nonlinear least squares to recover the mean D-A distance and the width of the distribution. The method was developed and tested using three synthetic D-A pairs, in which the chromophores were separated by alkyl chains of varying lengths. As an example application we also recovered the distribution of distances from the single tryptophan residue in troponin I (trp 158) to acceptor-labeled cysteine 133. The half-width of the distribution increases from 12 A in the native state to 53 A when unfolded by guanidine hydrochloride. For both TnI and the three model compounds the distance distributions recovered from the steady-state transfer efficiencies were in excellent agreement with the distributions recovered using the more sophisticated frequency-domain method (Lakowicz, J.R., M.L. Johnson, W. Wiczk, A. Bhat, and R.F. Steiner. 1987. Chem. Phys. Lett. 138:587-593). The method was found to be reliable and should be generally useful for studies of conformational distributions of macromolecules.

Energy Transfer↗
PubMed1987

Diffusion coefficients of quenchers in proteins from transient effects in the intensity decays.

We used 2-GHz frequency-domain fluorometry to examine the intensity decays of N-acetyl-L-tryptophamide (NATA) and the protein staphylococcal nuclease in the presence and absence of quenching by oxygen or acrylamide. When analyzed with a multiexponential model, the decays of NATA and nuclease both become more heterogeneous in the presence of quenching. We attribute the increased complexity to transient effects in quenching or equivalently a time-dependent rate constant for quenching. The frequency-domain data were analyzed using the Smoluchowski model (exp(-t/tau-2b square root t)) and the radiation model, which is known to correct some flaws in the more approximate Smoluchowski model. The radiation model provides improved fits to the data, as evidenced by average 10-fold decreases in chi R2. The radiation model also provides an estimate of the sum of the diffusion coefficients and the specific rate constant for quenching. The apparent diffusion coefficients for acrylamide and oxygen in nuclease, as seen by its single tryptophan (residue 140) are 15- and 11-fold lower than in water, respectively. The apparent values of the oxygen diffusion coefficient in water, as seen by NATA, are 2- to 3-fold larger than expected from earlier steady-state measurements. The ability to recover the detailed form of the intensity decays by the frequency-domain method should allow comparison of experimental results with calculated trajectories of quenchers in proteins.

Acrylamide↗
PubMed1987

Correction for contaminant fluorescence in frequency-domain fluorometry.

We describe a general method to correct for contaminant fluorescence when using the technique of frequency-domain fluorometry. The method can be applied regardless of the origin of the background signal, from scattered light, impurity fluorescence, or both. The procedure requires measurement of the frequency-dependent phase and modulation of the background at enough frequencies to approximate the decay law of the background. We also describe a general method to propagate the uncertainties in the measured phase and modulation values into the corrected values. This propagation is necessary to ensure proper weighting of the frequency-dependent data in the least-squares fitting algorithms. The practical usefulness of this correction method is demonstrated using frequency-domain data for one and two component mixtures which were deliberately contaminated with scattered light and/or other fluorophores.

Anthracenes↗
PubMed1987

Picosecond resolution of tyrosine fluorescence and anisotropy decays by 2-GHz frequency-domain fluorometry.

We extended the technique of frequency-domain fluorometry to an upper frequency limit of 2000 MHz. This was accomplished by using the harmonic content of a laser pulse train (3.76 MHz, 5 ps) from a synchronously pumped and cavity-dumped dye laser. We used a microchannel plate photomultiplier as the detector to obtain the 2-GHz bandwidth. This new instrument was used to examine tyrosine intensity and anisotropy decays from peptides and proteins. These initial data sets demonstrate that triply exponential tyrosine intensity decays are easily recoverable, even if the mean decay time is less than 1 ns. Importantly, the extended frequency range provides good resolution of rapid and/or multiexponential tyrosine anisotropy decays. Correlation times as short as 15 ps have been recovered for indole, with an uncertainty of +/- 3 ps. We recovered a doubly exponential anisotropy decay of oxytoxin (29 and 454 ps), which probably reflects torsional motions of the phenol ring and overall rotational diffusion, respectively. Also, a 40-ps component was found in the anisotropy decay of bovine pancreatic trypsin inhibitor, which may be due to rapid torsional motions of the tyrosine residues and/or energy transfer among these residues. The rapid component has an amplitude of 0.05, which is about 16% of the total anisotropy. The availability of 2-GHz frequency-domain data extends the measurable time scale for fluorescence to overlap with that of molecular dynamics calculations.

Kinetics↗
PubMed1987

Analysis of fluorescence decay kinetics measured in the frequency domain using distributions of decay times.

We describe the theoretical and practical aspects of analyzing complex fluorescence decay kinetics using continuous distributions of decay times. Our analysis uses frequency-domain data, provides for global analysis of multiple data sets and includes the possibility of excited-state processes. Simulated data were used to estimate the types of distributions which can be reasonably recovered from the measurements. Additionally, we describe a variety of distributions recovered from experimental data. For mixtures of one, two or three exponentially decaying fluorophores we recovered narrow lifetime distributions, which are essentially identical to a multiexponential decay. Similarly, a two-state excited-state reaction also yielded a narrow distribution with negative preexponential factors. The presence of time-dependent spectral relaxation of labeled lipids results in a wide distribution of decay times, which becomes narrower for faster relaxation rates at higher temperatures. Hence, the decay-time distributions appear to be sensitive to the dynamics of the environment surrounding the fluorophore. Additionally, distributions of decay times were observed to result from transient effects in collisional quenching, from energy transfer in the presence of a range of donor-to-acceptor distances, and for several single-tryptophan proteins.

Energy Transfer↗
PubMed1987

Enhanced resolution of fluorescence anisotropy decays by simultaneous analysis of progressively quenched samples. Applications to anisotropic rotations and to protein dynamics.

Enhanced resolution of rapid and complex anisotropy decays was obtained by measurement and analysis of data from progressively quenched samples. Collisional quenching by acrylamide was used to vary the mean decay time of indole or of the tryptophan fluorescence from melittin. Anisotropy decays were obtained from the frequency-response of the polarized emission at frequencies from 4 to 2,000 MHz. Quenching increases the fraction of the total emission, which occurs on the subnanosecond timescale, and thereby provides increased information on picosecond rotational motions or local motions in proteins. For monoexponential subnanosecond anisotropy decays, enhanced resolution is obtained by measurement of the most highly quenched samples. For complex anisotropy decays, such as those due to both local motions and overall protein rotational diffusion, superior resolution is obtained by simultaneous analysis of data from quenched and unquenched samples. We demonstrate that measurement of quenched samples greatly reduces the uncertainty of the 50-ps correlation time of indole in water at 20 degrees C, and allows resolution of the anisotropic rotation of indole with correlation times of 140 and 720 ps. The method was applied to melittin in the monomeric and tetrameric forms. With increased quenching, the anisotropy data showed decreasing contributions from overall protein rotation and increased contribution from picosecond tryptophan motions. The tryptophan residues in both the monomeric and the tetrameric forms of melittin displayed substantial local motions with correlation times near 0.16 and 0.06 ns, respectively. The amplitude of the local motion is twofold less in the tetramer. These highly resolved anisotropy decays should be valuable for comparison with molecular dynamics simulations of melittin.

Acrylamide↗
PubMed1986

Measurement of subnanosecond anisotropy decays of protein fluorescence using frequency-domain fluorometry.

We report the first anisotropy decays of protein fluorescence obtained using a frequency-domain fluorometer. The ultraviolet light source (300 nm) was a ring dye laser equipped with an intracavity frequency doubler, pumped by an argon ion laser. The data, measured at modulation frequencies from 2 to 200 MHz, reveal the presence of subnanosecond motions (0.1-0.2 ns) of the single tryptophan residues in melittin and monellin. For melittin the data also indicate the presence of slower motions near 1 ns, which may be the result of concerted motions of several peptide units. Smaller amplitude motions, on a similar timescale, were observed for the single tryptophan residue in staphylococcal nuclease. We demonstrate using N-acetyl-L-tryptophanamide in water that the method of frequency-domain fluorometry is capable of measuring correlation times as short as 50 ps. This method can provide data for the direct comparison of measured anisotropy decays with those predicted from molecular dynamics calculations.

Fluorescence↗
PubMed1986

Picosecond resolution of oxytocin tyrosyl fluorescence by 2 GHz frequency-domain fluorometry.

The technique of frequency-domain fluorometry has been extended to 2000 MHz using the harmonic content of a picosecond laser source and a microchannel plate photomultiplier tube. This new instrument was used to resolve complex subnanosecond intensity and anisotropy decays of the tyrosyl emission of oxytocin. The intensity decay was found to contain at least three exponential components, 80, 359 and 927 ps. The anisotropy analysis revealed a 29 ps torsional motion of the tyrosine residue as well as a 454 ps overall rotational correlation time. The time resolution of this method should permit the comparison of experimental results with theoretical models for motions of proteins.

Kinetics↗