Nanda Joshi

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.

Radiation boundary conditions in collisional quenching of fluorescence; determination by frequency-domain fluorometry

Abstract Quenching of 1,2-benzanthracene (BA) by CBr 4 was studied by frequency-domain fluorometry. The frequency response of the BA emission could not be fit using the Smoluchowski decay law for transient effects in quenching, exp(- t /τ-2 bt 1 2 ). However, the data were consistent with the expression for the radiation boundary conditions. We believe these are the first reported data which support the “radiation boundary condition” model for collisional quenching. Our ability to detect the inadequacy of the t 1 2 model indicates that the frequency-domain method can yield considerable detail about diffusive processes in solution.

Transient effects in quenching detected by harmonic-content frequency-domain fluorometry

Abstract Transient effects in the diffusional quenching of indole fluorescence by acrylamide were detected by frequency-domain fluorometry. The samples are excited with a repetitive train of picosecond pulses, and the frequency response is measured using the harmonic content up to 1 GHz. The decay of unquenched indole in aqueous buffer is dominantly a single exponential. In the presence of acrylamide the partially quenched indole fluorescence became distinctly more complex. Analysis of the frequency response of the fluorescence yields the apparent interaction radius (7.1 A) and mutual diffusion coefficient (1.1 × 10 −5 cm 2 s ).

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.

Frequency-domain fluorescence spectroscopy, a new method for the resolution of complex fluorescence emission

Abstract The fluorescence emission from complex chemical and biological samples can be resolved by measuring the frequency-response of the emission, which is now possible from 1 to 2000 MHz. The frequency-response allows determination of the components in a mixture, construction of time-resolved emission spectra, and measurement of the dynamic and hydrodynamic properties of biological macromolecules. The instrumentation is relatively simple, and data acquisition times can be short. At present, this method may be superior to direct measurements of time-resolved fluorescence emission.

Biophysical Aspects of Ca2+ - Transport Sites in Skeletal and Cardiac Sarcoplasmic Reticulum (Ca2+ + Mg2+)-ATPase

Calcium is involved in a variety of cell functions, such as controlling cell division and growth, muscle contraction, hormone secretion, and in the excitation-contraction coupling mechanism (1). Intracellular calcium is regulated by the plasma membrane and the subcellular organelles. The plasma membrane extrudes calcium via the Ca2+-pump ((Ca2+ + Mg2+) -ATPase) and via the Ca2+ : Na2+ countertransport system (1). The subcellular organelles that contribute to Ca2+ -regulation are mainly the sarcoplasmic reticulum, via the Ca2+-pump, and the mitochondria via the Ca2+ uniport system (2,3). Sarcoplasmic reticulum plays primary role in the regulation of cytoplasmic calcium in skeletal muscle and thus in the contraction-relaxation cycle. In cardiac muscle the important role of sarcoplasmic reticulum in regulating the contraction-relaxation cycle, is more complex since the organelles function is regulated by Catecholamines (4).

Transient Effects In The Acrylamide Quenching Of Single Tryptophan Proteins, Observed Using Frequency-Domain Fluorometry

We used GHz frequency-domain fluorometry to examine the tryptophan intensity decays of NATA (M-acetyl-L-tryptophanamide), gly-trp-gly, and the single tryptophan proteins ACTH, E. nuclease and ribonuclease T, (Rhase 1%). In all cases the intensity decays became more heterogeneous in the presence of quenching, which we attribute to a time-dependent rate constant for quenching (transient effects). The frequency-domain data were analyzed using the Smoluchowski model (exp(-t/t 2b/T)) and the radiation boundary condition (RBC) model. In contrast to the IT model, the RBC model does not assume the fluorophore-quencher pair is immediately deactivated, but rather assumes a rate constant for deactivation of the pair (K) as well as a mutual diffusion coefficient (D). The RBC model provides dramatically improved fits to the data. The values of both D and decreases progressively in the order listed above, which is with decreasing exposure to the aqueous phase. Because the RBC model may not be strictly correct in homogeneous solution, and probably less so in the hindered anisotropic environment of the proteins, the recovered values of D and IC should be regarded as apparent values. The recovered intensity decays can be compared with molecular dynamic calculations of quencher trajectories in proteins.

Anisotropy Decays Of Single Tryptophan Proteins Measured By Ghz Frequency-Domain Fluorometry With Collisional Quenching

We use frequency-domain fluorometry to determine the anisotropy decays of the tryptophan emission from S. nuclease and from the model compound gly-trp-gly. Resolution of the rapid and complex anisotropy decays was enhanced by global analysis of the data measured in the presence of quenching by either oxygen or acrylamide. Data were obtained at four to six quencher concentrations, and the data analyzed globally to recover the anisotropy decay. Because the decay times were decreased by quenching, measurements were possible to a upper frequency limit of 2 GHz. The anisotropy decay of gly-trp-gly revealed 40 ps of the indole ring, which was resolved from the overall 150 ps correlation times of the tripeptide. The anisotropy decay of nuclease displayed a 90/ps component as well as a 10 ns component due to overall rotational diffusion. We believe these highly resolved anisotropy decays are suitably for comparison with molecular dynamic simulations.

Anisotropie Rotation of Yt-Base in n-Propanol at – 20°C Studied by Frequency-Domain Fluorometry and Global Analysis

Abstract The frequency response of the polarized emission of Yt-base in n-propanol at -20°C was measured. Excitation wavelengths of 346, 312 and 285 nm were used for which the fundamental anisotropics were 0.32, 0.19 and 0.04, respectively. Additional data were obtained using CCl4 to decrease the mean decay time from 10 ns to 0.7 ns. These nine sets of data were analyzed globally to recover the anisotropy decay law. Two correlation times, of about 146 ps and 502 ps, were needed for a good fit

Regulation of cardiac sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase

SummaryThe two high affinity calcium binding sites of the cardiac (Ca2+ + Mg2+)-ATPase have been identified with the use of Eu3+. Eu3+ competes for the two high affinity calcium sites on the enzyme. With the use of laser-pulsed fluorescent spectroscopy, the environment of the two sites appear to be heterogeneous and contain different numbers of H2O molecules coordinated to the ion. The ion appears to be occluded even further in the presence of ATP. Using non-radiative energy transfer studies, we were able to estimate the distance between the two Ca2+ sites to be between 9.4 to 10.2 A in the presence of ATP. Finally, from the assumption that the calcium site must contain four carboxylic side chains to provide the 6–8 ligands needed to coordinate calcium, and based on our recently published data, we predict the peptidic backbone of the two sites.