Denisio M. Togashi

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.

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.

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.

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.

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 (I1/I3) 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 (IN*/IT*)) 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 (IN*/IT*). 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.

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 Forster 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.

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 ET(30), α, β, and π* 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 ET(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 polyvinyl-alcohol, 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.

Fluorescence study of Bovine Serum Albumin and Ti and Sn Oxide Nanoparticles Interactions.

Nanochemistry offers stimulating opportunities for a wide variety of applications in the biosciences. Understanding of the interaction of nanoparticles with biomolecules such as proteins is very important as it can help better design and fabricate nanocomposites for applications in diagnostics, drug delivery, and cell monitoring. In this work, the interaction of Bovine Serum Albumin (BSA) and two types of metal oxide nanoparticles (titanium and tin) have been studied using the intrinsic fluorescence of tryptophan residue from the proteins measured by steady state and time resolved fluorescence techniques. The nanoparticles which were fabricated using a novel synthetic process have average sizes of ∼2 nm (SnO 2 ) and ∼6 nm (estimated for TiO 2 ) and have very high solubilities in a variety of solvents. The Stem-Volmer plots indicate an effective quenching process by TiO 2 nanoparticles whereas SnO 2 nanoparticles have a lower quenching efficiency for BSA fluorescence. Static quenching is the major contribution in the overall process which may indicate a high degree of association between protein and nanoparticles. The difference in BSA fluorescence quenching efficiency between the two types of nanoparticles can be explained by the non-covalent interaction differences and the thermal stability of protein-nanoparticle associated species for both materials.