Thermal stability of recombinant green fluorescent protein (GFPuv) at various pH values

The thermal stability of the recombinant green fluorescent protein (GFPuv) expressed by Escherichia coli cells and isolated by three-phase partitioning extraction with hydrophobic interaction chromatography was studied. The GFPuv (3.5–9.0 μg of GFPuv/mL) was exposed to various pH conditions (4.91–9.03) and temperatures (75–95°C) in the 10 mM buffers: acetate (pH 5.0–7.0), phosphate (pH 5.5–8.0), and Tris-HCl (pH 7.0–9.0). The extent of protein denaturation (loss of fluorescence intensity) was expressed in decimal reduction time (D-value), the time exposure required to reduce 90% of the initial fluorescence intensity of GFPuv. For pH 7.0 to 8.0, the thermostability of GFPuv was slightly greater in phosphate buffer than in Tris-HCl. At 85°C, the D-values (pH 7.1–7.5) ranged from 7.24 (Tris-HCl) to 13.88 min (phosphate) The stability of GFPuv in Tris-HCl (pH>8.0) was constant at 90 and 95°C, and the D-values were 7.93 (pH 8.38–8.92) and 6.0 min (pH 8.05–8.97), respectively. The thermostability of GFPuv provides the basis for its potential utility as a fluorescent biologic indicator to assay the efficacy of moist-heat treatments at temperatures lower than 100°C.     

Evaluation of the pH- and thermal stability of the recombinant green fluorescent protein (GFP) in the presence of sodium chloride

The thermal stability of recombinant green fluorescent protein (GFP) in sodium chloride (NaCl) solutions at different concentrations, pH, and temperatures was evaluated by assaying the loss of fluorescence intensity as a measure of denaturation. GFP, extracted from Escherichia coli cells by the three-phase partitioning method and purified through a butyl hydrophobic interaction chromatography (HIC) column, was diluted in water for injection (WFI) (pH 6.0-7.0) and in 10 mM buffer solutions (acetate, pH 5.0; phosphate, pH 7.0; and Tris-EDTA, pH 8.0) with 0.9-30% NaCl or without and incubated at 80-95 degrees C. The extent of protein denaturation was expressed as a percentage of the calculated decimal reduction time (D-value). In acetate buffer (pH 4.84+/-0.12), the mean D-values for 90% reduction in GFP fluorescence ranged from 2.3 to 3.6 min, independent of NaCl concentration and temperature. GFP thermal stability diluted in WFI (pH 5.94+/-0.60) was half that observed in phosphate buffer (pH 6.08+/-0.60); but in both systems, D-values decreased linearly with increasing NaCl concentration, with D-values (at 80 degrees C) ranging from 3.44, min (WFI) to 6.1 min (phosphate buffer), both with 30% NaCl. However, D-values in Tris-EDTA (pH 7.65+/-0.17) were directly dependent on the NaCl concentration and 5-10 times higher than D-values for GFP in WFI at 80 degrees C. GFP pH- and thermal stability can be easily monitored by the convenient measure of fluorescence intensity and potentially be used as an indicator to monitor that processing times and temperatures were attained.     

Effects of sodium phosphate buffer on horseradish peroxidase thermal stability

Thermal stability of horseradish peroxidase (HRP) was studied by differential scanning calorimetry, tryptophan fluorescence, the heme absorption and enzymatic activity analysis while the concentrations of sodium phosphate buffer ranged from 2.5 to 50 mM at pH 7.0. The results showed that the denaturation temperature (T _m) values decreased and the intrinsic tryptophan fluorescence intensity of denatured HRP increased as sodium phosphate buffer concentration increased. Furthermore, the heme absorbance at 403 nm and enzymatic activity of HRP decreased with the increasing buffer concentrations. According to data obtained in this experiment, it can be concluded that sodium phosphate accelerated the denaturation process of HRP and reduced the thermal stability of HRP.     

Effect of pH on thermal- and chemical-induced denaturation of GFP

Green fluorescent protein (GFP) is an unusually stable autofluorescent protein that is increasingly being exploited for many applications. In this report, we have used fluorescence spectroscopy to study the effect of pH on the denaturation of GFP with sodium dodecyl sulfate (SDS), urea, and heat. Surprisingly, SDS (up to 0.5%) did not have any significant effect on the fluorescence of GFP at pH 7.5 or 8.5 buffers; however, at pH 6.5, the protein lost all fluorescence within 1 min of incubation. Similarly, incubation of GFP with 8 M urea at 50°C resulted in time dependent denaturation of GFP, but only in pH 6.5 buffer. At higher pH values (pH 7.5 and pH 8.5), the GFP was quite stable in 8 M urea at 50°C, showing only a slight decrease in fluorescence. Heat denaturation of GFP was found to be pH dependent as well, with the denaturation being fastest at pH 6.5 as compared to pH 7.5 or pH 8.5. Like the denaturation studies, renaturation of heat-denatured GFP was most efficient at pH 8.5, followed by pH 7.5, and then pH 6.5. These results suggests that GFP undergoes a structural/stability shift between pH 6.5 and pH 7.5, with the GFP structure at pH 6.5 being very sensitive to denaturation by SDS, urea, and heat.     

Characterization of a Neutral and Thermostable Glucoamylase from the Thermophilic Mold Thermomucor indicae-seudaticae: Activity, Stability, and Structural Correlation

Glucoamylase from the thermophilic mold Thermomucor indicae-seudaticae was purified by anion exchange and gel filtration chromatographic techniques using a fast protein liquid chromatographic system. The structure and thermal stability of this unique ‘thermostable and neutral glucoamylase’ were analyzed by circular dichroism (CD). T. indicae-seudaticae glucoamylase (TGA) contained typical aromatic amino acid (tryptophan/tyrosine) fingerprints in its tertiary structure. Analysis of the far-UV CD spectrum at pH 7.0 and 25 °C revealed the presence of 45% α-helix, 43% β-sheet, and 12% remaining structures. The α-helix content was highest at pH 7.0, where glucoamylase is optimally active. This observation points towards the possible (α/α)₆ barrel catalytic domain in TGA, as reported in microbial glucoamylases. Thermal denaturation curves of the pure protein at different pH values revealed maximum stability at pH 7.0, where no change in the secondary structure was observed upon heating in the temperature range between 20 °C and 60 °C. The observed midpoint of thermal denaturation (T _m) of glucoamylase at pH 7.0 was 67.1 °C, which decreased on either sides of this pH. Thermostability of TGA enhanced in the presence of starch (0.1%) as no transition curve was obtained in the temperature range between 20 °C and 85 °C. The only product of TGA action on starch was glucose, and it did not exhibit transglycosylation activity even at 40% glucose that can also be considered as an advantage during starch saccharification.     

Determination of the different oxidation states of As and Sb by a new electrochemical hydride generator coupled with atomic fluorescence spectrometry

A novel disk electrochemical hydride generator has been developed for the determination of As and Sb. Compared with the traditional thin-layer cell, the disk cell combined the advantages of quick assembly and easy operation. This electrochemical system for hydride generation in neutral buffer solutions has been studied for analytical usefulness in coupling with atomic fluorescence spectrometry. It was found that the use of neutral phosphate buffer solution could markedly increase the fluorescence intensity of As(III) and Sb(III) and reduce the impact of cathode erosion on the stability of signal intensity. At the same time, the fluorescence intensity of As(V) and Sb(V) were almost suppressed totally. The detection limits (3σ) of 0.031 μg L⁻¹ As(III) and 0.026 μg L⁻¹ Sb(III) in aqueous solutions were obtained, respectively. The precisions (n = 11) for 20 μg L⁻¹ As(III) and Sb(III) were 2.0% and 2.7%, respectively. The method was successfully applied for determination of different oxidation states of As and Sb in environmental samples.     

Evidence for a Molten Globule State in Cicer α-Galactosidase Induced by pH, Temperature, and Guanidine Hydrochloride

Physiologically as well as industrially, α-galactosidases are very important enzymes, but very little is known about the stability and folding aspect of enzyme. In the present study, we have investigated the temperature, pH, and guanidine hydrochloride (GuHCl) induced unfolding of Cicer α-galactosidase using circular dichroism and fluorescence spectroscopy. Strong negative ellipticities at 208, 215, and 222 nm indicate the presence of both α and β structures in Cicer α-galactosidase and showed that its secondary structure belongs to α?+?β class of proteins with 31 % α-helicity. For Cicer α-galactosidase the emission maximum was found to be 345 nm which suggests that tryptophan residues are less exposed to solvent. However, at pH?2.0, protein showed blue-shift. This state of protein lacked activity but it retained significant secondary structure. Enhanced binding of ANS at pH?2.0 indicated significant unfolding and exposure of hydrophobic regions. The unfolded state of Cicer α-galactosidase showed a red-shift of 15 nm with a concomitant decrease in the fluorescence intensity. The enzyme maintained its native structure and full activity up to 40 °C; however, above this temperature, denaturation was observed.     

Effect of nucleophilic agents and organized media on the fluorimetric determination of histamine with o-phthalic aldehyde

The addition of nucleophilic agents, 2-mercaptoethanol, 3-mercaptopropionic acid (3-MPA), and N-acetyl cysteine, to the system histamine-o-phthalic aldehyde (OPA) resulted in the following effects: the exclusion of the acidification step, the 1.5-2-fold increase in the time of attaining the optimum fluorescence, the shift of the pH of fluorophore formation of a less alkaline region by 2 units and the widening of its range by two to three times, and the twofold increase in the fluorophore stability (in time in the case of 3-MPA). The addition of surfactant micelles reduced the time of attaining the optimum fluorescence intensity by two times, but its intensity increased (by a factor of 1.3–1.5) only in the presence of nonionic surfactant block copolymers. The widest plateau range (2 pH units), the highest stability of an analytical signal in time (3 h), and the best reaction sensitivity were obtained after the addition of 2-hydroxypropyl-β-cyclodextrin to the histamine-OPA-3-MPA system. Procedures were proposed for the determination of histamine in fish products and wine using nucleophilic agents and different organized media.     

Energy dispersive X-ray fluorescence determination of cadmium in uranium matrix using Cd Kα line excited by continuum

An energy dispersive X-ray fluorescence method for determination of cadmium (Cd) in uranium (U) matrix using continuum source of excitation was developed. Calibration and sample solutions of cadmium, with and without uranium were prepared by mixing different volumes of standard solutions of cadmium and uranyl nitrate, both prepared in suprapure nitric acid. The concentration of Cd in calibration solutions and samples was in the range of 6 to 90 µg/mL whereas the concentration of Cd with respect to U ranged from 90 to 700 µg/g of U. From the calibration solutions and samples containing uranium, the major matrix uranium was selectively extracted using 30% tri-n-butyl phosphate in dodecane. Fixed volumes (1.5 mL) of aqueous phases thus obtained were taken directly in specially designed in-house fabricated leak proof Perspex sample cells for the energy dispersive X-ray fluorescence measurements and calibration plots were made by plotting Cd Kα intensity against respective Cd concentration. For the calibration solutions not having uranium, the energy dispersive X-ray fluorescence spectra were measured without any extraction and Cd calibration plots were made accordingly. The results obtained showed a precision of 2% (1σ) and the results deviated from the expected values by < 4% on average.     

Isolation of quercetin and kaempferol from Rubus ulmifolius and their fluorometric assay

A fluorometric method for the determination of the flavonols quercetin and kaempferol in the leaves of Rubus ulmifolius was developed.A chromatographic technique for the isolation and separation of the two compounds was described. Working curves with standard solutions of quercetin and kaempferol at pH 2.5 were constructed. The effect of pH on the fluorescence intensity was studied with fluorescence measurements in acidic solutions of the compounds. The wavelengths for all fluorescence measurements were selected from fluorescence spectra of the two compounds.     

Glucose oxidase electrodes of a terpolymer poly(aniline-co-o-anisidine-co-o-toluidine) as biosensors

A simple technique is described for constructing a glucose sensor by the entrapment of glucose oxidase (GOD) in a poly (aniline-co-o-anisidine-co-o-toluidine) [P(A-co-o-A-co-o-T)] thin films, which were electrochemically deposited on a platinum plate in phosphate and acetate buffer. The maximum current response was observed for the said electrodes at pH 5.5 and potential 0.60 V (versus Ag/AgCl). The phosphate buffer gives high stability and fast response as compared to acetate buffer in amperometric measurements.     

A new strategy for optimizing sensitivity, speed, and resolution in capillary electrophoretic separation of DNA

DNA separations were performed in poly(ethylene oxide) (PEO) solutions prepared in 100 mM Tris-boric acid (TB) buffers using a capillary filled with TB buffers with concentrations up to 2.5 M, pH 10.0. The electroosmotic flow (EOF) increased with increasing the concentration of TB buffers till 1.5 M as a result of decreasing PEO adsorption on the capillary wall. At high TB concentrations (> 1.5 M), the peaks corresponding to small DNA fragments (11 and 8 base pairs) became sharper and were detected. Relative standard deviations of the EOF coefficient and the migration times of the DNA fragments were all less than 1% using a capillary filled with TB buffers at concentrations higher than 1.5 M. When separations were performed at different pH values of PEO solutions and TB buffers, better results in terms of sensitivity, speed, and resolution were generally achieved. The fluorescence intensity of the 2176 bp fragment obtained at pH values of TB buffers/PEO solutions 10.0/8.2 was 27-fold of that at pH values 8.2/8.2. The enhancement was related to effects of pH and borate on fluorescence intensity, DNA conformation, stacking, and interactions with the capillary wall. Using a capillary filled with 400 mM TB buffers, pH 10.0, the separation of DNA (pBR 322/HaeIII digest, pBR 328/Bg/I digest and pBR 328/HinfI digest) in 1.5% PEO solutions prepared in 100 mM TB buffers, pH 9.0, at 375 V/cm was accomplished in less than 18 min.     

Second Derivative Spectrophotometric Determination of the Binding Constant Between Codeine Phosphate and Bovine Serum Albumin

Second derivative spectrophotometry was applied to determine the binding constant (K) between codeine phosphate (COD) and bovine serum albumin (BSA) at simulated physiological conditions (37.00?°C and pH = 7.4). The second derivative spectra of COD in buffer solutions containing various amounts of BSA showed derivative isosbestic points. The residual background signals derived from incomplete suppression of BSA signals can be entirely eliminated in the second derivative spectra indicating that BSA has spectrophotometrically one kind of binding site for COD. The fractions of COD bound to BSA were calculated from the derivative intensity differences (??D values) of COD before and after the addition of BSA. Scatchard plot calculation suggested that the binding of COD to BSA can be explained by a partition-like non-specific binding model. The binding constant (K) was calculated from ??D values according to the non-specific binding model by a nonlinear least-squares method. K values were almost constant for all of the COD concentrations studied with good reproducibility. The fractions predicted by the K values were in good agreement with the observed values. The results indicate the usefulness of the derivative method in drug?Calbumin binding studies without the need for prior separation procedures which may disturb the equilibrium states of the samples solutions.     

Rhenium bipyridine complexes for the recognition of glucose

Bipyridine ligands containing pendant methyl, amino, and amino-boronic acid groups were synthesized. Coordination complexes of these ligands with rhenium were prepared straightforwardly and in good yield. The fluorescence behavior of the Re complexes was studied as a function of pH and exposure to various concentrations of glucose. The methyl bipyridine complex showed no change in fluorescence with pH, the amino derivative showed a rapid decrease from low pH to neutral, and the amino-boronate derivative showed little change from pH 4 to 10. Fluorescence quenching was observed at high pH as expected on the basis of a photoinduced electron transfer (PET) signaling mechanism. This behavior can be explained on the basis of the first oxidation and reduction potentials of these complexes. Glucose testing showed a significant dependence on the solvent system used. In pure methanol, the rhenium boronate complex exhibited a 55% fluorescence intensity increase upon increasing glucose concentration from 0 to 400 mg/dL. However, in 50 vol % methanol/phosphate buffered saline, none of the complexes showed significant response in the glucose range of physiological interest.     

Extended detection range for an optical enzymatic glucose sensor coupling with a novel data-processing method

A new data-processing method was established and applied for optical enzymatic glucose sensing, in which oxygen and glucose were simultaneously consumed. The oxygen level remaining in the detection system, which was equal to the difference between the initial and consumed oxygen concentrations, could be measured using fluorescent oxygen indicators immobilized in the sensing layer. It was deduced that the ratio of I ₀ and I was inversely proportional to glucose concentration, where I ₀ is the maximum fluorescence intensity in various glucose solutions, and I is the fluorescence intensity at various concentrations of glucose. Using the new data-processing method, the detection range of the calibration curve method was extended from 0 to 1.2 mmol L^?1, which was enlarged about 2–3 folds over that in ordinary approaches. The prepared glucose sensor could be directly applied to detect high concentrations of glucose.     

Resorufin as an electron acceptor in glucose oxidase-catalyzed oxidation of glucose

Resorufin (1) has been found to act as an electron acceptor in glucose oxidase (GOD)-catalyzed oxidation of glucose. When a 1: 1: 1 mixture of solutions of 1 (5.0 microM), glucose, and GOD (4.0 mg/ml) in phosphate buffer (pH 7.4, 0.1 M) was incubated at 36 degrees C under aerobic conditions and the reaction was followed by a measurement of changes in fluorescence intensity due to 1, only two types of fluorometric traces were observed: (1) when a glucose solution of less than 0.7 mM was subjected to the enzymatic reaction, no consumption of 1 was observed; (2) the reaction with glucose at more than 1.0 mM always consumed 1, affording a regression fluorometric curve, and yet the obtained fluorometric traces could be almost superimposed on one another with no dependence on the glucose concentration. The reasons for the observed phenomena are discussed.     

苯乙烯嘧啶化合物的合成及其作为pH探针的光谱特点

合成并表征了化合物4,6-二[（4′-二乙基胺基）苯乙烯基]嘧啶-2-醇。 通过研究该化合物在不同pH值的磷酸盐缓冲溶液中紫外-可见吸收光谱和荧光光谱的变化,发现λmax约为500 nm的吸收峰在溶液pH=6.78中吸收强度最大。 荧光发射峰随着pH值的升高逐渐减弱,直至淬灭（pH=5.51）。     

Specificity of Glucose Oxidase from Penicillium funiculosum 46.1 Towards Some Redox Mediators

Glucose oxidase (GOx) from Penicillium funiculosum 46.1 was purified using step-by-step ultrafiltration and it was characterized by spectrophotometric and spectrofluorometric methods. It was shown that spectra of GOx produced by P. funiculosum are typical for flavoproteins. Absorption spectrum has distinct peaks at 380 and 457 nm, excitation spectrum at 373 and 447 nm, and emission spectrum at 530 and 562 nm. The pH correlation of enzyme activity and catalytic characteristics in various buffer systems (phosphate (pH 5.0–9.0), citrate (pH 3.0–5.0), citrate-phosphate (pH 3.0–9.0), and universal (pH 3.0–9.0)) were registered. It was determined that the GOx is the most efficiently interacting with substrate (glucose) in phosphate buffer at pH 7.0 with k _cat/K _m?=?21,825 M^?1 s^?1. Interaction of several different redox mediators (9,10-phenantroline-5,6-dione, 9,10-phenanthrenequinone, N-methylphenazonium methyl sulfate, ferrocene, ferrocenecarboxylic acid, α-methylferrocenemethanol, ferrocenecarboxaldehyde) with GOx from P. funiculosum was investigated by evaluation of the difference in fluorescence emission intensity of FAD_(oxidized) and FADH_2(reduced) forms. It was found that 9,10-phenantroline-5,6-dione and 9,10-phenanthrenequinone are the best redox mediators for this type of GOx.

Influence of micellar media on the fluorescence of various benzo- and methyl-quinolizinium salts

The bebaviour of several different micelar systems (anionic, cationic and non-ionic) on the fluorescence of quinolizinium salts is studied. Important factors, such as pH and ionic strength that influence fluorescence parameters, are discussed. Fourteen quinolizinium salts (benzo and methyl derivatives) are examined as fluorescent probes in micellar media. All of them showed a marked increase of fluorescence intensity when sodium dodecyl sulfate solutions of critical micelle concentration (CMC) are added. The presence of non-ionic surfactants did not change the fluorescent emission of the probes. The emission intensity is much decreased when N-cetyl-N,N,N- trimethylammonium bromide concentrations are above the CMC. Changes in pH ido not significantly affect the fluorescence intensity of the benzo derivatives. Increasing the ionic strength decreases the fluorescence. For 9-cyanobenzo[a]phenanthro [9,10-g] quinolizinium chloride, the spectrum changes when the surfactant concentration is high than the CMC thereforre this compound is considered to be a good fluorescent probe in icell     

Einfluß von Lösungsmittel und Halogenessigsäure auf die Fluorescenz von N-Aryl-anthranilsäuren und verwandten Verbindungen

The influence of a pretreatment of flufenamic acid and other N-aryl-anthranilic acids by alkaline saponification and oxidation, as well as the influence of the solvent and the added halogenoacetic acid on fluorescence intensity has been investigated. Derivatives of the flufenamic acid (e.g. esters or amides) must first be hydrolyzed. To render the solution fluorescent solvents with a DK = 0 are required. The halogeno-acetic acids (e.g. mono-, di-, trichloro-, -bromo- and -fluoroacetic acids) with a pK_a-value <1 are suitable. Acids with a pK_a-value >1 are completely unsuitable. Arylsubstituation influences the position of the fluorescence maxima but not the intensity. Anthranilic acid itself shows fluorescence in chloroform-trichloroacetic acid while their m- and p-analogues as well as diphenylamine do not.