Electrophilic reactivity of tetrabromorhodamine 123 is bromine induced: convergent interpretation through complementary molecular descriptors.

Nucleophilic addition of water and of methanol to 3,6-diamino-2,4,5,7-tetrabromo-9-[2-(methoxycarbonyl) phenyl]-9H-xanthen-9-ylium, 4BrR123, yields respectively 2-(3,6-diamino-2,4,5,7-tetrabromo-9-hydroxy-9H-xanthen-9-yl)xanthyl benzoate, HO4BrR123 and 2-(3,6-diamino-2,4,5,7-tetrabromo-9-methoxy-9H-xanthen-9-yl)xanthyl benzoate, MeO4BrR123. The novel experimental results are addressed theoretically. The linear free energy relationship, LFER, second-order perturbation theory analysis of the natural bond orbital, NBO, and quantum theory of atoms in molecules, QTAIM, lead to the same conclusion: the electron-withdrawing effect of bonded Br atoms in 4BrR123 extremely enhances the molecular electrophilicity, as compared to 3,6-diamino-9-[2-(methoxycarbonyl) phenyl]-9H-xanthen-9-ylium, R123. The reactivity of these diaminoxanthylium cations is discussed in the context of local and global softness in extended conjugated systems.

Monitoring local unfolding of bovine serum albumin during denaturation using steady-state and time-resolved fluorescence spectroscopy.

In a previous report (J. Fluoresc. 16, 153, 2006) we studied the chaotropically induced denaturation of Bovine Serum Albumin (BSA) using the fluorescence decay kinetics at different stages in the denaturation of BSA by guanidinium hydrochloride (GuHCl). In this work, we gain a more detailed insight into the BSA denaturation process by investigating the thermodynamics of the process. Structural changes were monitored spectrophotometrically via the intrinsic protein fluorescence from tryptophan residues, and the extrinsic fluorescence from 1,8-anilinonaphthalene sulphonate (ANS). ANS tends to locate in a variety of binding sites in BSA which are located in different domains, and these can be selectively populated using different, 1:1 and 1:10 molar ratios of BSA to ANS. The data from steady-state and time-resolved fluorescence spectroscopy were analyzed using thermodynamic two-state and three-state models and the lifetime data clearly indicated the presence of an intermediate state during denaturation. A global analysis using non-linear regression gave a DeltaG(H(2)O,D)(0) = 6.7 kcal x mol(-1) for the complete unfolding of the BSA-ANS complexes, and a DeltaG(H(2)O,I) = 0.9 kcal x mol(-1) for the first step to the intermediate. Therefore, the unfolding energy of the intermediate, which appears mostly at intermediate GuHCl concentrations (1.0 to 1.5 M), to the denatured state, is 5.8 kcal x mol(-1). The lifetime analysis of the BSA-ANS complexes also shows clearly that there are differences in stability of the BSA domains, with domain III unfolding first at low GuHCl concentrations (<1.5 M).

Polarity assessment of thermoresponsive poly(NIPAM-co-NtBA) copolymer films using fluorescence methods.

The in-situ, non-contact, and non-destructive measurement of the physicochemical properties such as the polarity of thin, hydrophilic polymer films is desirable in many areas of polymer science. Polarity is a complex factor and encompasses a range of non-covalent interactions including dipolarity/polarizability and hydrogen bonding. A polarity measurement method based on fluorescence would be ideal, but the key challenge is to identify suitable probes which can accurately measure specific polarity related parameters. In this manuscript we assess a variety of fluorophores for measuring the polarity of a series of relatively hydrophilic, thermoresponsive N-isopropylacrylamide/N-tert-butylacrylamide (NIPAM/NtBA) copolymers. The emission properties of both pyrene and 3-Hydroxyflavone (3-HF) based fluorophores were measured in dry polymer films. In the case of pyrene, a relatively weak, linear relationship between polymer composition and the ratio of the first to the third vibronic band of the emission spectrum (I(1)/I(3)) is observed, but pyrene emission is very sensitive to temperature and thus not suitable for robust polarity measurements. The 3-HF fluorophores which can undergo an excited-state intramolecular proton transfer (ESIPT) reaction have a dual band fluorescence emission that exhibits strong solvatochromism. Here we used 4'-diethylamino-3-hydroxyflavone (FE), 5,6-benzo-4'-diethylamino-3-hydroxyflavone (BFE), and 4 -diethylamino-3-hydroxy-7-methoxyflavone (MFE). The log ratio of the dual band fluorescence emission (log (I(N*)/I(T*))) of 3-HF doped, dry, NIPAM-NtBA copolymer films were found to depend linearly on copolymer composition, with increasing hydrophobicity (greater NtBA fraction) leading to a decrease in the value of log (I(N*)/I(T*)). However, the ESIPT process in the polymer matrix was found to be irreversible, non-equilibrated and occurs over a much longer timescale in comparison to the results previously reported for liquid solvents.

Trigger factor from the psychrophilic bacterium Psychrobacter frigidicola is a monomeric chaperone.

In eubacteria, trigger factor (TF) is the first chaperone to interact with newly synthesized polypeptides and assist their folding as they emerge from the ribosome. We report the first characterization of a TF from a psychrophilic organism. TF from Psychrobacter frigidicola (TF(Pf)) was cloned, produced in Escherichia coli, and purified. Strikingly, cross-linking and fluorescence anisotropy analyses revealed it to exist in solution as a monomer, unlike the well-characterized, dimeric E. coli TF (TF(Ec)). Moreover, TF(Pf) did not exhibit the downturn in reactivation of unfolded GAPDH (glyceraldehyde-3-phosphate dehydrogenase) that is observed with its E. coli counterpart, even at high TF/GAPDH molar ratios and revealed dramatically reduced retardation of membrane translocation by a model recombinant protein compared to the E. coli chaperone. TF(Pf) was also significantly more effective than TF(Ec) at increasing the yield of soluble and functional recombinant protein in a cell-free protein synthesis system, indicating that it is not dependent on downstream systems for its chaperoning activity. We propose that TF(Pf) differs from TF(Ec) in its quaternary structure and chaperone activity, and we discuss the potential significance of these differences in its native environment.

Investigating tryptophan quenching of fluorescein fluorescence under protolytic equilibrium.

Fluorescein is one of most used fluorescent labels for characterizing biological systems, such as proteins, and is used in fluorescence microscopy. However, if fluorescein is to be used for quantitative measurements involving proteins then one must account for the fact that the fluorescence of fluorescein-labeled protein can be affected by the presence of intrinsic amino acids residues, such as tryptophan (Trp). There is a lack of quantitative information to explain in detail the specific processes that are involved, and this makes it difficult to evaluate quantitatively the photophysics of fluorescein-labeled proteins. To address this, we have explored the fluorescence of fluorescein in buffered solutions, in different acidic and basic conditions, and at varied concentrations of tryptophan derivatives, using steady-state absorption and fluorescence spectroscopy, combined with fluorescence lifetime measurements. Stern-Volmer analyses show the presence of static and dynamic quenching processes between fluorescein and tryptophan derivatives. Nonfluorescent complexes with low association constants (5.0-24.1 M(-1)) are observed at all pH values studied. At low pH values, however, an additional static quenching contribution by a sphere-of-action (SOA) mechanism was found. The possibility of a proton transfer mechanism being involved in the SOA static quenching, at low pH, is discussed based on the presence of the different fluorescein prototropic species. For the dynamic quenching process, the bimolecular rate constants obtained (2.5-5.3 x 10(9) M(-1)s(-1)) were close to the Debye-Smoluchowski diffusion rate constants. In the encounter controlled reaction mechanism, a photoinduced electron transfer process was applied using the reduction potentials and charges of the fluorophore and quencher, in addition to the ionic strength of the environment. The electron transfer rate constants (2.3-6.7 x 10(9) s(-1)) and the electronic coupling values (5.7-25.1 cm(-1)) for fluorescein fluorescence quenching by tryptophan derivatives in the encounter complex were then obtained and analyzed.

Measuring the micro-polarity and hydrogen-bond donor/acceptor ability of thermoresponsive N-isopropylacrylamide/N-tert-butylacrylamide copolymer films using solvatochromic indicators.

Thin polymer films are important in many areas of biomaterials research, biomedical devices, and biological sensors. The accurate in situ measurement of multiple physicochemical properties of thin polymer films is critical in understanding biocompatibility, polymer function, and performance. In this work we demonstrate a facile spectroscopic methodology for accurately measuring the micro-polarity and hydrogen-bond donor/acceptor ability for a series of relatively hydrophilic thermoresponsive copolymers. The micro-polarity of the N-isopropylacrylamide (NIPAM) and N-tert-butylacrylamide (NtBA) co-polymers was evaluated by means of the E(T)(30), alpha, beta, and pi empirical solvatochromic polarity parameters. The data shows that increasing the NtBA fraction in the dry copolymer film reduces polarity and hydrogen-bonding ability. Within the Kamlet-Taft polarity framework, the NIPAM/NtBA copolymer films are strong hydrogen-bond acceptors, strongly dipolar/polarizable, and rather moderate hydrogen-bond donors. This characterization provides a more comprehensive physicochemical description of polymers, which aids the interpretation of film performance. Comparison of the measured E(T)(30) values with literature data for other water-soluble polymers show that dry NIPAM/NtBA copolymers are slightly more polar than poly(ethylene oxide), less polar than polyvinylalcohol, and approximately the same polarity as poly(N-vinyl-2-pyrrolidone). These findings indicate that this spectroscopic method is a facile, rapid, and nondestructive methodology for measuring polymer properties in situ, suitable for most biomaterials research laboratories.

Lipophilic porphyrin microparticles induced by AOT reverse micelles: a fluorescence lifetime imaging study.

Fluorescence Lifetime Imaging Microscopy (FLIM) technique was applied to investigate the fluorescence dynamics and structural features of large colloidal aggregates of meso-tetra(N-dodecyl-4-amino sulfonyl-phenyl)porphyrin (PC12) induced by Sodium 1,4-bis(2-ethyl hexyl)sulfosuccinate (AOT) reverse micelles. The aggregate's particle sizes (down to 1 microm) obtained from the confocal fluorescence images matched with the particle sizes measured in the images obtained from Scanning Electron Microscopy (SEM). The fluorescence decays for those aggregates in the micro spatial domain show triexponential fluorescence lifetimes (tau1 approximately 12 ns, tau2 approximately 3 ns and tau3 approximately 1 ns) which are independent of the aggregate's size.

Photophysical studies of 9,10-phenanthrenequinones.

The characterization of the excited states of 9,10-phenanthrenequinone (PQ) and its derivatives substituted in the 3 and 6 positions with methoxy (PQ1), chloro (PQ2), methyl (PQ3) and fluoro (PQ3) was carried out using steady-state UV-Visible absorption spectroscopy and phosphorescence emission spectroscopy at room temperature and at 77 K. Nanosecond laser flash photolysis was used to obtain the time resolved spectra from the triplet emission decays. The compounds presented phosphorescence in benzene, chlorobenzene and acetonitrile solutions at room temperature and at 77 K. The phosphorescence of the methoxy derivative, however, was observed only at low temperature. The derivatives showed a slightly higher triplet energy than PQ. The Hammett plots were applied to correlate singlet and triplet energies with sigma values that account for resonance and the radical character. It is observed that singlet and triplet energies increase with electron donating groups.

Cell cycle-dependent mobility of Cdc45 determined in vivo by fluorescence correlation spectroscopy.

Eukaryotic DNA replication is a dynamic process requiring the co-operation of specific replication proteins. We measured the mobility of eGFP-Cdc45 by Fluorescence Correlation Spectroscopy (FCS) in vivo in asynchronous cells and in cells synchronized at the G1/S transition and during S phase. Our data show that eGFP-Cdc45 mobility is faster in G1/S transition compared to S phase suggesting that Cdc45 is part of larger protein complex formed in S phase. Furthermore, the size of complexes containing Cdc45 was estimated in asynchronous, G1/S and S phase-synchronized cells using gel filtration chromatography; these findings complemented the in vivo FCS data. Analysis of the mobility of eGFP-Cdc45 and the size of complexes containing Cdc45 and eGFP-Cdc45 after UVC-mediated DNA damage revealed no significant changes in diffusion rates and complex sizes using FCS and gel filtration chromatography analyses. This suggests that after UV-damage, Cdc45 is still present in a large multi-protein complex and that its mobility within living cells is consistently similar following UVC-mediated DNA damage.

Assessing protein-surface interactions with a series of multi-labeled BSA using fluorescence lifetime microscopy and Förster Energy Resonance Transfer.

Reliably measuring the physicochemical properties of protein thin layers deposited on surfaces is critical to understanding the surface chemistry, biocompatibility, and performance of implanted biomaterials. Here we apply a series of multi-fluorophore labeled Bovine Serum Albumin (BSA) proteins as model probes to investigate surface-induced conformational changes of BSA by the use of a confocal Fluorescence Lifetime Imaging Microscopy and Förster Resonance Energy Transfer (FLIM-FRET) method. In this FLIM-FRET approach we study six different constructs where the BSA is covalently linked to one (BSA-F1) or five (BSA-F5) fluorescein molecules, one (BSA-T1) or seven (BSA-T7) rhodamine molecules, and hetero labeled with both (BSA-F4-T2 and BSA-F6-T1). The fluorescence intensity and decays were simultaneously measured at two different emission regions (green and red channels) of the labeled BSA deposited on substrates of different hydrophilicity and hydrophobicity. To generate reliable data, several different regions (10(4)µm(2) in each case) of the surfaces were scanned for each measurement. The amplitude-weighted lifetimes, obtained from the fluorescence decay parameters, are discussed based on the average distance between the conjugated fluorophores acting as a donor and acceptor pair in the Energy Transfer framework. The number of probes conjugated has significant effects on the fluorescence emission intensity and lifetimes in solution and on surfaces. The BSA-F4-T2 constructs showed a significant ability to differentiate using lifetime the hydrophilicity and hydrophobicity of the surfaces, by detecting local expansion and contraction of protein structure in the deposited layers. Using these multiple labeled BSA probes in conjunction with FLIM-FRET can provide a way to assess structural changes in proteins induced by variations in surface chemistry of biomaterials.

Quantifying adsorbed protein on surfaces using confocal fluorescence microscopy.

The quantification and analysis of protein adsorption on solid surfaces are of significant importance in many areas of biosensors, biomaterials, and biomedical devices research. The accurate, in situ, measurement of multiple physicochemical properties from the thin protein layers which adsorb on surfaces is critical to understanding biocompatibility, surface chemistry factors, and the performance of implanted medical devices. To implement such studies, new tools and simple protocols based on instrumentation available in typical bioscience laboratories are desirable. In this work, we have developed an approach using confocal fluorescence microscopy to quantify the amount of bovine serum albumin (BSA) adsorbed onto a flat hydrophilic glass surface, under different pH conditions. This approach which can be implemented using most confocal fluorescence microscopes is described in detail and its limitations are discussed. This quantitative method coupled with the Langmuir model allowed for the determination of adsorption parameters at pH 2.0, 4.0, 7.4, and 9.2. The adsorption parameters were validated by comparison with literature values obtained from different techniques for a similar protein-surface system. The Derjaguin-Landau-Verwey-Overbeek (DLVO) theory was then used for a detailed analysis of these parameters, to understand in general terms how pH affected the surface adsorption interactions.

A fluorescence analysis of ANS bound to bovine serum albumin: binding properties revisited by using energy transfer.

Determination of binding parameters such as the number of ligands and the respective binding constants require a considerable number of experiments to be performed. These involve accurate determination of either free and/or bound ligand concentration irrespective of the measurement technique applied. Then, an appropriate theoretical model is used to fit the experimental data, and to extract the binding parameters. In this work, the interaction between bovine serum albumin (BSA) and 1-anilino-8-naphthalene sulphonate (ANS) is revisited. Using steady state fluorescence spectroscopy, the binding isotherm of BSA/ANS was obtained applying the Halfman-Nishida approach. The binding parameters, site number, and binding site association constants, were determined from the stoichiometric Adair model and Job's plot. The binding parameters obtained were then correlated to the distance of the respective binding site to the tryptophan residues using the energy transfer technique. This approach, that uses both tryptophans independently from each other, is presented as a tool to help understand the binding mechanism of the albumin fluorescent complex. The results show that ANS molecules bind to BSA in up to five different binding sites. Energy transfer from the tryptophan residues to the BSA/ANS complex shows that the four highest affinity binding sites (>10(4) M(-1)) are located at a reasonably close distance (18-27 A) to at least one of two tryptophan residues, while the lowest affinity binding site (approximately 10(4) M(-1)) is located over 34 A away from the both tryptophans.

Fluorescence lifetime imaging study of a thin protein layer on solid surfaces.

Understanding the fundamental interactions between proteins and solid surfaces is essential in the area of implantable medical devices. Fluorescence methods offer the sensitivity required to study the formation of the initial thin protein layers that mediate biocompatibility of materials. Thin protein layers (bovine serum albumin labelled with 1-anilino-8-naphthalenesulfonate, BSA-ANS) deposited on several surfaces (glass, silicon, stainless steel, polystyrene, and silver island film) were studied using confocal frequency domain Fluorescence Lifetime Imaging Microscopy (FLIM) and single-point multifrequency lifetime analysis techniques. FLIM provides spatial information about both fluorophores located on the surface and physicochemical parameters of the surface microenvironment. The average fluorescence lifetimes (tau(av)) of the adsorbed BSA-ANS generated by the contact between a protein solution and the material surface were measured by the multifrequency modulation and phase shift. Results indicate that tau(av) values of the albumin complexes on the surfaces (approximately 12 ns) are, in general, shorter than tau(av) found in the bulk solution (approximately 14 ns). For some surfaces, like polystyrene and silver island film the differences in tau(av) of the adsorbed BSA-ANS were found to be much greater. The differences in fluorescence lifetimes may indicate structural changes in the BSA protein induced by contact with the surface.

Mobility and distribution of replication protein A in living cells using fluorescence correlation spectroscopy.

Replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA) binding protein, is essential for all pathways of DNA metabolism. To study the function of RPA in living cells the second largest RPA subunit and an N-terminal deletion mutant thereof were fused to green fluorescent protein (GFP; GFP-RPA2 and GFP-RPA2deltaN, respectively) in a controlled, molecular biological way. These proteins were expressed in HeLa cells under the control of the inducible tetracycline expression system. GFP-RPA2 and GFP-RPA2deltaN are predominately nuclear proteins as determined by confocal laser scanning microscopy. Low basal expression of GFP-RPA2deltaN allowed the measurement of kinetic parameters of RPA. Using fluorescence correlation spectroscopy (FCS) two populations--a fast and a slow moving species--were detected in the nucleus and the cytosol of human cells. The translational diffusion rates of these two RPA populations were approximately 15 microm2/s and 1.8 microm2/s. This new finding reveals the existence of different multiprotein and ssDNA-protein complexes of RPA in both cellular compartments and opens the possibility for their analyses.

Time-resolved fluorescence studies on bovine serum albumin denaturation process.

The denaturation of Bovine Serum Albumin (BSA) by a chaotropic agent, guanidinium hydrochloride (GuH+Cl-) was studied by fluorescence lifetime analysis. The BSA was labelled with 1-anilino-8-naphthalene sulfonate (ANS) at two different molar ratios (1:1) and (1:10). The non-exponential fluorescence kinetics of the BSA-ANS complex at different stages of denaturation is analysed using three different models: a discrete tri-exponential sum, stretched exponential, and Gaussian lifetime distribution. In all cases, the fluorescence decay times decreased with protein denaturation. The results from the models show that there are at least two different binding sites located in the BSA protein with different water accessibility.

Self-organization of a sulfonamido-porphyrin in Langmuir monolayers and Langmuir-Blodgett films.

Langmuir monolayers (LM) and Langmuir-Blodgett (LB) films of pure lipophilic meso-tetra(4-dodecylaminosulfophenyl)porphyrin (PC12) and mixed with the anionic surfactant sodium hexadecylsulfate (SHS) were studied. The molecular packing and structure of PC12 and PC12-4SHS with variable surface pressure were investigated by surface pressure-area measurements, steady-state absorption, fluorescence emission and anisotropy, as well as by fluorescence lifetime imaging microscopy (FLIM). At low surface pressure, the porphyrin molecules are organized with the rings tilted on the water surface whereas at high surface pressure the porphyrin rings achieve a more perpendicular arrangement. Using the FLIM images a gradual change of aggregates into large "islands" is observed. Different patterns are observed in the pure PC12 multilayer films (n = 3 and 5) with ordered patches superimposed which are not observed in the PC12-4SHS multilayer LB films.