Biochemical Applications Of Frequency-Domain Fluorometry; Determination Of Time-Resolved Anisotropies And Emission Spectra

Lakowicz, Joseph R.; Gryczynski, I.; Cherek, Henryh; Laczko, Gabor; Joshi, Nanda B.

doi:10.1117/12.966934

Cited by 8 publications

(11 citation statements)

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“…Thus, the rotational correlation time of 1.2–1.4 ns can be explained in terms of the flexibility of the PEC–dye complex, allowing for segmental motion of linked dyes inside the cavities of the PEC particles. This assumption is supported by similar results obtained previously for dyes bound to a protein and tryptophan within the protein …”

Section: Discussionsupporting

confidence: 90%

Synthesis and Characterization of Chitosan‐Based Polyelectrolyte Complexes Doped with Xanthene Dyes

et al. 2015

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Biocompatible chitosan-based polyelectrolyte complexes (PECs) doped with xanthene dyes (fluorescein, eosin Y, erythrosin B, rhodamine 6G) were synthesized and characterized by scanning electron microscopy, dynamic light scattering, zeta potential measurements, and absorption and luminescence (including polarized, time-resolved, and phosphorescence) spectroscopy. The results are discussed in terms of the mechanism and rigidity of dye-PEC binding, the heavy-atom effect in dyes and PEC stability. Eosin Y is found to be the optimal dopant, providing both a high dye content in PECs and a high quantum yield of fluorescence.

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Section: Discussionsupporting

confidence: 90%

Synthesis and Characterization of Chitosan‐Based Polyelectrolyte Complexes Doped with Xanthene Dyes

et al. 2015

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“…As shown in Table II, ACBP showed very fast single rotational correlation time, ϭ 0.76 ns, and a high limiting anisotropy r ϭ 0.320. This high limiting anisotropy was similar to the maximal Trp and Tyr anisotropies measured in propylene glycol glass at Ϫ60°C (34,35). The latter results were consistent with the internal motions of the protein amino acid residues responsible for the observed fluorescence being completely restricted on the nanosecond time scale.…”

Section: Effect Of Oleoyl-coa Binding On Steady State Fluorescence Chsupporting

confidence: 87%

“…The ACBP hydrodynamic radius calculated from the time-resolved anisotropy data was 15.1 Å (Table II). The fact that the measured residual (limiting) anisotropy of ACBP r ϭ 0.156 (Table II) was much less than both the maximal Trp and Tyr anisotropies measured in a propylene glycol glass at Ϫ60°C, r 0 ϭ 0.315 (34), and r 0 ϭ 0.320 (35), respectively, indicates that ACBP aromatic amino acid local segmental motions were relatively fast and not resolvable on the nanosecond time scale. In addition to this fast rotation, the estimated amplitude for such rotational motions, i.e.…”

Section: Effect Of Oleoyl-coa Binding On Steady State Fluorescence Chmentioning

confidence: 87%

Acyl Coenzyme A Binding Protein

Frolov

Schroeder

1998

Journal of Biological Chemistry

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It is now recognized that long chain fatty acyl-CoAs (Ͼ14 carbons) serve at least four essential cellular functions as follows: fatty acid oxidation (1-7), fatty acid esterification (8-16), signal transduction (17-19), and gene expression (20,21). Although cellular long chain fatty acyl levels are normally in the range 5-164 M, these levels increase as much as 4-fold in pathological conditions (reviewed in Ref. 22). Since the critical micellar concentration of long chain fatty acyl-CoAs is 30-60 M and as much as 96-99% of long chain fatty acyl-CoA partitions to membranes, this suggests that high levels of long chain fatty acyl-CoAs may disrupt membrane function as well as cellular signaling/gene regulation (reviewed in Refs. 10 and 22).Because of these considerations, it is important to understand the mechanisms that regulate the distribution of intracellular free long chain fatty acyl-CoAs. One candidate cytosolic protein that may serve this purpose is ACBP 1 which may sequester, store, and/or protect fatty acyl-CoAs from hydrolysis (reviewed in Ref. 22). Acyl-CoA binding protein (ACBP) is a small (10 kDa), highly conserved cytosolic protein broadly distributed among all eukaryotic tissues studied, and elevated expression of ACBP has been observed in malignant tumors and transformed cells (reviewed in . A primary objective of the present investigation was to use a fluorescent fatty acyl-CoA binding assay as well as non-fluorescent oleoyl/acyl-CoA to directly show for the first time that native rat liver ACBP actually bound the most common chain length, C 18 , fatty acyl-CoAs. The second aim was to examine the structural dynamics of native rat liver apo-and holo-ACBP, to characterize the properties of fluorescent fatty acyl-CoAs within the ACBP-binding site, and to examine ACBP conformational dynamics in response to ligand binding. EXPERIMENTAL PROCEDURESMaterials cis-and trans-parinaroyl-CoAs were synthesized as described (9). cisand trans-parinaric acids were from Calbiochem. Coenzyme A (CoASH), acyl-coenzyme A synthase, ATP, p-terphenyl, and 1,4-bis[4-methyl-5-phenyl-2-oxazolyl]benzene, and saturated and unsaturated fatty acylCoAs were from Sigma. All other chemicals were reagent grade or better. MethodsACBP was isolated from rat liver as described previously (24). Protein concentration was determined by the Bradford assay (25) and corrected according to UV spectrophotometric analysis (26). The Bradford assay overestimates ACBP concentration by 1.69-fold. Steady State Fluorescence SpectroscopySteady state fluorescence spectra were measured in a 1-cm quartz cuvette with a PC1 Photon Counting Spectrofluorimeter (ISS Instr., Champaign, IL). Sample temperature was maintained at 25°C (Ϯ0.1°C). Excitation and emission bandwidths were 4 and 8 nm. Sample absorbance at the excitation wavelengths was Յ0.05. Time-resolved Fluorescence SpectroscopyData Acquisition-All measurements of lifetime, differential polarized phase (limiting anisotropy, rotational rate), hydrodynamic radius, and wobbling in a cone angle were perfo...

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“…As shown in Table I intensity decays are very heterogenous and three exponentials are needed to fit the data. This is expected for a protein with several tryptophans and was observed previously for the SR Ca 2ϩ -ATPase (Lakowicz et al, 1986;Gryczynski et al, 1989;Wang et al, 1992). In the presence of isoflurane the lifetimes decrease (compare exponential fits 2 R and ϽϾ in Table I).…”

Section: Ca 2ϩ -Atpases and Volatile Anestheticsmentioning

confidence: 60%