Change In Fluorescence Quantum Yield Research Articles

BF2-Functionalized Benzothiazole Amyloid Markers: Effect of Donor Substituents on One- and Two-Photon Properties.

Investigation of amyloids with the aid of fluorescence microscopy provides crucial insights into the development of numerous diseases associated with the formation of aggregates. Here, we present a series of BF2-functionalized benzothiazoles with electron-donating methoxy group(s), which are tested as amyloid fluorescent markers. We evaluate how the position of donor functional group(s) influences optical properties (fluorescence lifetime (τ) and fluorescence quantum yield (FQY)) in a solution and upon binding to amyloids. We elucidate the importance of surrounding environmental factors (hydrogen-bonding network, polarity, and viscosity) on the observed changes in FQY and evaluate how the localization of a donor influences radiative and nonradiative decay pathways. We conclude that a donor attached to the benzothiazole ring contributes to the increment of radiative decay pathways upon binding to amyloids (kr), while the donor attached to the flexible part of a molecule (with rotational freedom) contributes to a decrease in nonradiative decay pathways (knr). We find that the donor-acceptor-donor architecture allows us to obtain 58 times higher FQY of the dye upon binding to bovine insulin amyloids. Finally, we measure two-photon absorption (2PA) cross sections (σ2) of the dyes and their change upon binding by the two-photon excited fluorescence (2PEF) technique. Measurements reveal that dyes that exhibit the increase/decrease of σ2 values when transferred from highly polar solvents to CHCl3 present a similar behavior upon amyloid binding. Our 2PA experimental values are supported by quantum mechanics/molecular mechanics (QM/MM) simulations. Despite this trend, the values of σ2 are not the same, which points out the importance of two-photon absorption measurements of amyloid-dye complexes in order to understand the performance of 2P probes upon binding.

Orthogonally-tunable and ER-targeting fluorophores detect avian influenza virus early infection

Cell-based assays can monitor virus infection at a single-cell level with high sensitivity and cost-efficiency. For this purpose, it is crucial to develop molecular probes that respond selectively to physiological changes in live cells. We report stimuli-responsive light-emitters built on a T-shaped benzimidazole platform, and consecutive borylation reactions to produce a library of homologs displaying systematic changes in fluorescence quantum yield and environmental sensitivity. We find that certain fluorophores localize selectively at the endoplasmic reticulum, and interact with proteins involved in the stress signaling pathways. Notably, the mono-borylated compound responds selectively to the stress conditions by enhancing fluorescence, and detects avian influenza virus infection at the single-cell level. Our findings demonstrate the unprecedented practical utility of the stress-responsive molecular probes to differentiate cellular states for early diagnosis.

Acetylene bridged D-(π-A)2 type dyes containing benzophenone moieties: Photophysical properties, and the potential application as photoinitiators

Acetylene bridged acceptor-π-bridge-donor-π-bridge-acceptor (A-π-D-π-A) dyes (DBPs) based on benzophenone were designed and synthesized via Sonogashira coupling reaction utilizing carbazole/phenothiazine/triphenylamine as electron donating groups. The photophysical properties, photochemistry characteristics, as well as the two-photon absorption properties were studied. All dyes exhibited excellent intra-/intermolecular charge transfer properties upon excitation, and large two photon absorption cross sections in the wavelength range of 720~860 nm. Significant differences in fluorescence and photochemistry properties were found depending on the electron donating groups. The rigid carbazole favored hybridization of benzophenone like local excited state and intramolecular charge transfer excited state, which resulted in abnormal changes in fluorescence quantum yield, poor photostability, and good initiating ability both as Norrish Ⅱ initiator and as sensitizer for iodonium salt. However, the butterfly-shaped phenothiazine and propeller-shaped triphenylamine facilitated intramolecular charge transfer excited states, which resulted in typical positive solvatochromism effect, good photostabilities, and lacking of initiating ability as Norrish Ⅱ initiator.

Understanding the Fluorescence Change in Red Genetically Encoded Calcium Ion Indicators

For over 20 years, genetically encoded Ca2+ indicators have illuminated dynamic Ca2+ signaling activity in living cells and, more recently, whole organisms. We are just now beginning to understand how they work. Various fluorescence colors of these indicators have been developed, including red. Red ones are promising because longer wavelengths of light scatter less in tissue, making it possible to image deeper. They are engineered from a red fluorescent protein that is circularly permuted and fused to a Ca2+-sensing domain. When Ca2+ binds, a conformational change in the sensing domain causes a change in fluorescence. Three factors can contribute to this fluorescence change: 1) a shift in the protonation equilibrium of the chromophore, 2) a change in fluorescence quantum yield, and 3) a change in the extinction coefficient or the two-photon cross section, depending on if it is excited with one or two photons. Here, we conduct a systematic study of the photophysical properties of a range of red Ca2+ indicators to determine which factors are the most important. In total, we analyzed nine indicators, including jRGECO1a, K-GECO1, jRCaMP1a, R-GECO1, R-GECO1.2, CAR-GECO1, O-GECO1, REX-GECO1, and a new variant termed jREX-GECO1. We find that these could be separated into three classes that each rely on a particular set of factors. Furthermore, in some cases, the magnitude of the change in fluorescence was larger with two-photon excitation compared to one-photon because of a change in the two-photon cross section, by up to a factor of two.

Flow-dependent fluorescence of CCVJ

BackgroundThe molecular rotor 9-(2-Carboxy-2-cyanovinyl)julolidine (CCVJ) is presumed to have a sensitivity towards velocity or shear which is supposed to result in a change in fluorescence quantum yield. Furthermore, a previously reported photoisomeric behavior may contribute to the measured fluorescence intensity changes. The goal of this research was to examine the hypothesized behavior theoretically and experimentally from the perspective of fluid dynamics.ResultsA correlation between stirring rate and intensity could not be established in the present experiments with a completely illuminated sample in contrast to previously reported experiments in spectrofluorometers. Experiments and theoretical models of a Poiseuille flow were in good agreement with the photoisomeric behavior but excluded the influence of shear. Further experiments in a flow chamber supported the photoisomery hypothesis as well.ConclusionNo experimental evidence for the influence of velocity on the fluorescence intensity of CCVJ was found. The hypothesis of shear sensitivity was excluded as well. The results are consistent with the photoisomeric behavior of CCVJ.

Tryptophan Fluorescence Yields and Lifetimes as a Probe of Conformational Changes in Human Glucokinase.

Five variants of glucokinase (ATP-D-hexose-6-phosphotransferase, EC 2.7.1.1) including wild type and single Trp mutants with the Trp residue at positions 65, 99, 167 and 257 were prepared. The fluorescence of Trp in all locations studied showed intensity changes when glucose bound, indicating that conformational change occurs globally over the entire protein. While the fluorescence quantum yield changes upon glucose binding, the enzyme's absorption spectra, emission spectra and fluorescence lifetimes change very little. These results are consistent with the existence of a dark complex for excited state Trp. Addition of glycerol, L-glucose, sucrose, or trehalose increases the binding affinity of glucose to the enzyme and increases fluorescence intensity. The effect of these osmolytes is thought to shift the protein conformation to a condensed, high affinity form. Based upon these results, we consider the nature of quenching of the Trp excited state. Amide groups are known to quench indole fluorescence and amides of the polypeptide chain make interact with excited state Trp in the relatively unstructured, glucose-free enzyme. Also, removal of water around the aromatic ring by addition of glucose substrate or osmolyte may reduce the quenching.

Sedimentation of Reversibly Interacting Macromolecules with Changes in Fluorescence Quantum Yield

Sedimentation velocity analytical ultracentrifugation with fluorescence detection has emerged as a powerful method for the study of interacting systems of macromolecules. It combines picomolar sensitivity with high hydrodynamic resolution, and can be carried out with photoswitchable fluorophores for multicomponent discrimination, to determine the stoichiometry, affinity, and shape of macromolecular complexes with dissociation equilibrium constants from picomolar to micromolar. A popular approach for data interpretation is the determination of the binding affinity by isotherms of weight-average sedimentation coefficients sw. A prevailing dogma in sedimentation analysis is that the weight-average sedimentation coefficient from the transport method corresponds to the signal- and population-weighted average of all species. We show that this does not always hold true for systems that exhibit significant signal changes with complex formation—properties that may be readily encountered in practice, e.g., from a change in fluorescence quantum yield. Coupled transport in the reaction boundary of rapidly reversible systems can make significant contributions to the observed migration in a way that cannot be accounted for in the standard population-based average. Effective particle theory provides a simple physical picture for the reaction-coupled migration process. On this basis, we develop a more general binding model that converges to the well-known form of sw with constant signals, but can account simultaneously for hydrodynamic cotransport in the presence of changes in fluorescence quantum yield. We believe this will be useful when studying interacting systems exhibiting fluorescence quenching, enhancement, or Förster resonance energy transfer with transport methods.

The unusual solvatochromism and solvatofluorochromism of longwave absorbing and emitting barbiturate merocyanine dyes

Spectral-fluorescent properties of a series of merocyanine dyes comprising the barbituric acid residue as the electron-accepting terminal group are investigated in comparison with those of their N,N-methylated analogues in media of various polarity. It is revealed that in polar aprotic solvents the electronic absorption spectra of the studied compounds are influenced dramatically by the formation of hydrogen bonds between the NH-groups of barbituric residue and solvent molecules. An effect of such nucleophilic solvation on the electronic structure of the studied dyes is analysed using both the spectral data obtained and the DFT/B3LYP quantum chemical simulation. It is found also, that solvation has comparatively weak influence on the shape and position of the fluorescence bands of the studied merocyanines while the fluorescence quantum yield changes substantially in solvents of various polarity.

Modeling of the redox state dynamics in photosystem II of Chlorella pyrenoidosa Chick cells and leaves of spinach and Arabidopsis thaliana from single flash-induced fluorescence quantum yield changes on the 100ns-10s time scale.

The time courses of the photosystem II (PSII) redox states were analyzed with a model scheme supposing a fraction of 11-25% semiquinone (with reduced [Formula: see text]) RCs in the dark. Patterns of single flash-induced transient fluorescence yield (SFITFY) measured for leaves (spinach and Arabidopsis (A.) thaliana) and the thermophilic alga Chlorella (C.) pyrenoidosa Chick (Steffen et al. Biochemistry 44:3123-3132, 2005; Belyaeva et al. Photosynth Res 98:105-119, 2008, Plant Physiol Biochem 77:49-59, 2014) were fitted with the PSII model. The simulations show that at high-light conditions the flash generated triplet carotenoid (3)Car(t) population is the main NPQ regulator decaying in the time interval of 6-8μs. So the SFITFY increase up to the maximum level [Formula: see text]/F 0 (at ~50μs) depends mainly on the flash energy. Transient electron redistributions on the RC redox cofactors were displayed to explain the SFITFY measured by weak light pulses during the PSII relaxation by electron transfer (ET) steps and coupled proton transfer on both the donor and the acceptor side of the PSII. The contribution of non-radiative charge recombination was taken into account. Analytical expressions for the laser flash, the (3)Car(t) decay and the work of the water-oxidizing complex (WOC) were used to improve the modeled P680(+) reduction by YZ in the state S 1 of the WOC. All parameter values were compared between spinach, A. thaliana leaves and C. pyrenoidosa alga cells and at different laser flash energies. ET from [Formula: see text] slower in alga as compared to leaf samples was elucidated by the dynamics of [Formula: see text] fractions to fit SFITFY data. Low membrane energization after the 10ns single turnover flash was modeled: the ∆Ψ(t) amplitude (20mV) is found to be about 5-fold smaller than under the continuous light induction; the time-independent lumen pHL, stroma pHS are fitted close to dark estimates. Depending on the flash energy used at 1.4, 4, 100% the pHS in stroma is fitted to 7.3, 7.4, and 7.7, respectively. The biggest ∆pH difference between stroma and lumen was found to be 1.2, thus pH- dependent NPQ was not considered.

Fluorescent probe 7-(prop-2-yn-1-yloxy)-2H-chromen-2-one: Experimental and DFT based approach to photophysical properties

Compound 7-(prop-2-yn-1-yloxy)-2H-chromen-2-one was synthesized by Pechmann condensation reaction and characterized by various spectroscopic techniques. The structure of title compound was confirmed by single crystal X-ray diffraction. The compound crystallized in the orthorhombic system with P 21 21 21 space group and the corresponding lattice parameters were found to be a=4.0138 (11)Å, α=90°; b=23.536 (6)Å, β=90°; c=10.93 (2)Å, γ=90°. The crystal packing of molecule showed that intermolecular hydrogen bonds C3–H3⋯O3 [D=3.53Å], C-13–H13⋯O2 [D=3.67Å] and intermolecular short interaction between C1–H1⋯C1–H1 [2.68 Å] forms a dimeric unit which finally stabilizes the crystal packing in three dimensional network in the molecule. Absorption and emission spectra shows that compound is fluorescent with good Stoke shift values ranging between 57 and 62nm. Thermal analysis further supports by TGA, DTA. The photophysical results show that the compound exhibits change in fluorescence quantum yield with change in solvent polarity. The structural parameters and the vibrational wave numbers obtained from the optimized geometry of the compound from DFT-B3LYP calculations employing 6-311G (d,p) basis set are in good agreement with the experimental data. UV–Vis spectrum calculated by employing time dependent density functional theory (TD-DFT) is also in very good agreement with the experiment for all solvents.

Lectin Biosensing Using Digital Analysis of Ru(II)-Glycodendrimers

A novel, digital, single-operation analytical method to study glycodendrimer-lectin interactions is described. Robust, highly fluorescent derivatives of tris(bipyridine)ruthenieum(II) ([Ru(bipy)(3)](2+)) bearing 2, 4, 6, or 18 mannose or galactose units were designed to perform molecular logic operations. Inputs for these systems were pH, N,N'-4,4'-bis(benzyl-2-boronic acid)bipyridinium dibromide, and different lectins (concanavalin A, Galantus nivalis agglutinin, and asialoglycoprotein). The relative change in fluorescence quantum yield of the Ru(II)-glycodendrimers served as output. Together, the fluorescent emission readout, the logic analysis of the photoinduced electron transfer, and the optical behavior provide a single-step method to quickly screen a glycodendrimer library and select the best dendrimer model for studying carbohydrate-lectin interactions.

Fluorescence of 5-Arylvinyl-5′-Methyl-2,2′-Bipyridyl Ligands and Their Zinc Complexes

The photophysical properties of 5-arylvinyl-5'-methyl-2,2'-bipyridyls (AVMBs, 1-9, 11) and their zinc complexes were studied. Similar 2,2'-bipyridyl-based ligands have been applied as optical sensors for metal ions and sensitizers for solar energy conversion. The goal of this investigation is to reveal the factors that determine the emission band shift and fluorescence quantum yield change of the title ligand system upon zinc binding. The outcome of this study will not only advance the fundamental understanding of the coordination-driven photophysical processes embodied in the AVMB platform but facilitate the rational design of fluorescent probes for metal ions, particularly zinc. The AVMB ligands were synthesized using the Horner-Wadsworth-Emmons reaction. AVMBs containing electron-donating aryl groups show absorption and emission in the visible region, which can be assigned to charge-transfer transitions as supported by solvent-dependency and computational studies. The binding between AVMB ligands and zinc ion in acetonitrile was studied using isothermal titration calorimetry (ITC). A multicomponent equilibrium model is suggested that explains the multiple transitions evidenced in fluorescence titration isotherms. Coordination to zinc ion stabilizes the charge-transfer excited state of an AVMB ligand with an electron-donating aryl substituent, consequently results in bathochromic shifts in both absorption and emission. However, unlike the emission band shift, the fluorescence quantum yield change upon zinc complex formation does not have an intuitive correlation with the electronic nature of the aryl group. Lifetime measurements using the Time-Correlated Single Photon Counting method enabled the determination of nonradiative and radiative decay rate constants. Both rates of an AVMB ligand decrease upon zinc binding. The collective effect gives rise to the change in fluorescence quantum yield with the apparent lack of correlation with the electronic property of the aryl group.

Occurrence and characterization of carbon nanoparticles below the soot laden zone of a partially premixed flame

An experimental study has been performed to detect the occurrence of nanosized carbon particulates below the soot laden zone of a co-flowing partially premixed flame. Samples have been extracted from different points across the flame and passed through DI water. Absorption and fluorescence spectroscopies have been performed with the collected water suspensions. The occurrence of carbon nanoparticles is evident across the inner flame front. In addition, evidence of naphthalene has also been found inside the inner rich premixed flame. The concentration of naphthalene decreases while that of the carbon nanoparticles increases as the inner flame front is reached. The stability of the nanoparticles in the sample has been ensured by observing that the change in fluorescence quantum yield from the sample over a long duration is small. The band gap energy has been evaluated using the absorption data to characterize the likely structures of the particles in the collected suspension. Two kinds of particles having different zones of band gap energy are found in the flame. Dynamic light scattering measurements show that the particle size grows with the increase in height in the lower part of the flame. While, at 3 and 6 mm elevations the particles are observed to be below 2.5 nm in diameter, the particles at 10 mm elevation are found in the size range of 2.5–5.5 nm.

Smart optical probes for near-infrared fluorescence imaging of Alzheimer’s disease pathology

Near-infrared fluorescent probes for amyloid-beta (Abeta) are an exciting option for molecular imaging in Alzheimer's disease research and may translate to clinical diagnostics. However, Abeta-targeted optical probes often suffer from poor specificity and slow clearance from the brain. We are designing smart optical probes that emit characteristic fluorescence signal only when bound to Abeta. We synthesized a family of dyes and tested Abeta-binding sensitivity with fluorescence spectroscopy and tissue-staining. Select compounds exhibited Abeta-dependent changes in fluorescence quantum yield, lifetime, and emission spectra that may be imaged microscopically or in vivo using new lifetime and spectral fluorescence imaging techniques. Smart optical probes that turn on when bound to Abeta will improve amyloid detection and may enable quantitative molecular imaging in vivo.

High Affinity of the Cell-Penetrating Peptide HIV-1 Tat-PTD for DNA

During cellular uptake of fluorescently labeled cell-penetrating peptides (CPPs), intense fluorescent signals are commonly observed in the nucleus of the cell, suggesting intracellular CPP relocation and potential binding to the genome of the host. We therefore investigated the interaction of the CPP HIV-1 Tat(47-57) with double-stranded DNA, and we also tested whether the fluorescence intensity of the labeled CPP allows for linear predictions of its intracellular concentration. Using isothermal titration calorimetry, we observe that the CPP has a high affinity for salmon sperm DNA as characterized by a microscopic dissociation constant of 126 nM. The binding is exothermic, with a reaction enthalpy of -4.63 kcal/mol CPP (28 degrees C). The dissociation constant and reaction enthalpy decrease further at higher temperatures. The affinity of the CPP for DNA is thus 1-2 magnitudes higher than for extracellular heparan sulfate, the likely mediator of the CPP uptake. Accordingly, the high affinity for DNA confers stability to extracellular transport complexes of CPP and DNA but potentially affects the regulation and molecular organization of the host's genome after nuclear uptake. Moreover, the CPP leads to the condensation of DNA as evidenced by the pronounced increase in light-scattering intensity. The fluorescence quantum yield of the FITC-labeled CPP decreases considerably at concentrations > 5 micromol/L, at pH < 7, and upon binding to DNA and glycosaminoglycans. This change in fluorescence quantum yield impedes the microscopic identification of uptake routes and the comparison of uptake efficiency of different CPPs, especially if the accumulation in subcellular compartments (self-quenching and pH difference) and transitory binding partners (quenching and condensation) is unknown.

A Monte Carlo simulation of photon propagation in fluorescent poly(ethylene glycol) hydrogel microsensors

A Monte Carlo simulation of photon propagation through fluorophore-containing poly(ethylene glycol) hydrogel networks is described. Poly(ethylene glycol) (PEG) hydrogel microspheres are being studied for fluorescent biosensing of small molecular weight analytes such as glucose and paraoxon. A computational approach to assess the fluorescent output from these sensors was developed, in particular investigating the impact of sphere characteristics such as size, scattering, and fluorophore concentrations on sensor response. Monte Carlo simulations of both individual spheres and packed sphere beds were developed to model photon propagation through homogeneous, water-swollen PEG hydrogel microspheres with encapsulated fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) dyes. Experimental studies using an integrating sphere were conducted to determine the hydrogel optical properties used in the simulation and spectrofluorimetry studies performed to verify assumptions made in the model regarding quantum yield. Changes in fluorescence quantum yield, hydrogel scattering and absorption coefficients ( μ s and μ a), hydrogel scattering anisotropy ( g), and depth of the sphere bed were used to predict optimum sphere size, gel chemistry, and collection geometry.

The Dependence of Benzo-15-Crown-5 Ether-Containing Oligo Paraphenylene Vinylene (CE-OPV) Emission Upon Complexation with Metal Ions in Solution

The effects of metal salts (NaCl, CaCl2·2H2O, EuCl3·6H2O and Eu(OAc)3) on the steady state and time resolved fluorescence behaviour of poly(para phenylene vinylene) oligomers containing benzo-15-crown-5 ether units (CE-OPV) have been investigated. The presence of EuCl3 causes a significant (8–9 fold) increase in the fluorescence emission intensity of the OPV segments, as compared to pure CE-OPV, in 99:1 methanol/chloroform solution and a small (∼9 nm) red shift in the emission maximum. The presence of Na+ or Ca2+ results in less marked increases in fluorescence intensity compared to Eu3+. In the presence of Eu3+ and Na+, the fluorescence intensity increases approximately linearly with metal ion concentration up to a metal ion/CE-OPV molar ratio of ∼10. The emission enhancement is not related to a simple 1:1 (CE-OPV:metal ion) complex formation process. In contrast, in acetonitrile, CE-OPV shows complex fluorescence quenching behaviour as a function of EuCl3 concentration. This solvent dependence suggests that the emission changes with metal concentration are related to the formation of charge-transferred complexes. The marked changes in fluorescence quantum yield of the PPV backbone due to complexation with metal ions makes CE-OPV a sensitive fluorescent probe for metal ions, or may be exploited for improving the quantum yield of PPV-based devices.

Excitation Dependent Fluorescence Quantum Yield in Hydrocarbon Fuels Containing Polycyclic Aromatic Compounds

Fluorescence emission spectra and quantum yields have been measured for neat sample of six different hydrocarbon fuels over different possible excitation wavelengths. Hydrocarbon fuels show different excitation fluorescence cutoff depending on their commercial use and applications in the UV ∼ visible region. The fluorescence emission maximum of these fuels shows a red shift with excitation wavelength. Resonance energy transfer and self-quenching through solvent collision of fluorophores play an important role on the fluorescence characteristics of multifluorophoric mixture like petroleum fuels at higher concentration. The fluorescence quantum yield of these fuels is a function of the excitation wavelength and is different for different fuels. The change in quantum yield with excitation wavelength for petroleum fuels is different from that of crude oils. The change in fluorescence quantum yield at lower excitation wavelength provides a promising tool to estimate contamination of diesel and petrol by kerosene.

Time-resolved monitoring of flash-induced changes of fluorescence quantum yield and decay of delayed light emission in oxygen-evolving photosynthetic organisms.

The present contribution describes a new experimental setup that permits time-resolved monitoring of the rise kinetics of the relative fluorescence yield, Phi(rel)(t), and simultaneously of the decay of delayed light emission, L(t), induced by strong actinic laser flashes. The results obtained by excitation of dark-adapted samples with a train of eight flashes reveal (a) in suspensions of spinach thylakoids, Phi(rel)(t) exhibits a typical period four oscillation that is characteristic for a dependence on the redox states S(i)() of the water oxidizing complex (WOC), (b) the relative extent of the unresolved "instantaneous" rise to the level (100 ns) at 100 ns and the maximum values of Phi(rel)(t) attained at about 45 s after each actinic flash, (45 s) synchronously oscillate and exhibit the largest values at flash nos. 1 and 5 and minima after flash nos. 2 and 3, (c) opposite effects are observed for the normalized extent of the rise kinetics in the 100 ns to 5 s time domain of relative fluorescence yield, Phi(rel)(5 s) - Phi(rel)(100 ns), i.e., both parameters attain minimum and maximum values after the first/fifth and second/third flash, respectively, and (d) analogous features for the "fast" and "slow" ns-kinetics of the fluorescence rise were observed in suspensions of Chlamydomas reinhardtii cells. A slight phase shift by one flash is ascribed to physiological differences. The applicability of this noninvasive technique to study reactions of photosystem II, especially the reduction kinetics of P680(*)(+) and their dependence on the redox state S(i)() of the WOC, is discussed.

Probing the Origins of Spectroscopic Responses to Analyte-Induced Conformational Changes in Fluorescently-Labeled Cod III Parvalbumin

An emerging strategy for the development of reagentless biosensors is the coupling of fluorescence responses to analyte-induced conformational changes within fluorescently-labeled proteins. In this work, we have examined the absorbance and the steady-state and time-resolved fluorescence responses to Ca2+-induced conformational changes within cod III parvalbumin (C3P), which was labeled at cysteine-18 with the fluorescent probes fluorescein, acrylodan or nitrobenzoxadiazole (NBD). The basis of the analyte-induced responses was further characterized by examining reporter group accessibility and rotational reorientation dynamics in the presence and absence of analyte. We show that the fluorescence responses are often based on a combination of direct and indirect effects, and that changes in fluorescence quantum yield can be reinforced or opposed by simultaneous changes in absorbance, dramatically affecting the sensitivity of the observed signal to analyte concentration. In the case of NBD, the response is fu...