Loss Of Fluorescence Intensity Research Articles

Thermal characteristics of recombinant green fluorescent protein (GFPuv) extracted from Escherichia coli.

The thermal stability of isolated and extracted recombinant green fluorescent protein (GFPuv) was evaluated by analysing the loss of fluorescence intensity. GFPuv was expressed by Escherichia coli, extracted by the three-phase partitioning method and purified by elution through an hydrophobic interaction column. The collected fractions were further diluted in Tris-HCl-EDTA (pH 8.0) and subjected to continuous heating at set temperatures (45-95 degrees C). From a standard curve relating fluorescence intensity to GFPuv concentration, the loss of fluorescence intensity was converted to denatured GFPuv concentration (microg ml-1). To determine the extent of the thermal stability of GFPuv, decimal reduction times (D-values), z-value and energy of activation (Ea) were calculated. For temperatures between 45 and 70 degrees C, extracted native GFPuv activity decreased from 11 to 75% relative to initial native protein concentration above 70 degrees C, the average decrease in GFPuv fluorescence was between 72 to 83%. The thermal stability of GFPuv provides the basis for its potential utility as a fluorescent biological indicator to assess the efficacy of the treatment of liquids and materials exposed to steam.

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 microg of GFPuv/mL) was exposed to various pH conditions (4.91-9.03) and temperatures (75-95 degrees 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 degrees 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 degrees 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 degrees C.

Effect of Charge Reversal Mutations on the Ligand- and Membrane-binding Properties of Liver Fatty Acid-binding Protein

Liver fatty acid-binding protein (FABP) is able to bind to anionic phospholipid vesicles under conditions of low ionic strength. This binding results in the release of ligand, the fluorescent fatty acid analogue 11-dansylaminoundecanoic acid (DAUDA), with loss of fluorescence intensity (Davies, J. K., Thumser, A. E. A., and Wilton, D. C. (1999) Biochemistry 38, 16932-16940). Using a strategy of charge reversal mutagenesis, the potential role of specific cationic residues in promoting interfacial binding of FABP to anionic phospholipid vesicles has been investigated. Cationic residues chosen included those within the alpha-helical region (Lys-20, Lys-31, and Lys-33) and those that make a significant contribution to the positive surface potential of the protein (Lys-31, Lys-36, Lys-47, Lys-57, and Arg-126). Only three cationic residues make a significant contribution to interfacial binding, and these residues (Lys-31, Lys-36, and Lys-57) are all located within the ligand portal region, where the protein may be predicted to exhibit maximum disorder. The binding of tryptophan mutants, F3W, F18W, and C69W, to dioleoylphosphatidylglycerol vesicles, containing 5 mol% of the fluorescent phospholipid dansyldihexadecanoylphosphatidylethanolamine, was monitored by fluorescence resonance energy transfer (FRET). All three mutants showed enhanced dansyl fluorescence due to FRET on addition of phospholipid to protein; however, this fluorescence was considerably greater with the F3W mutant, consistent with the N-terminal region of the protein coming in close proximity to the phospholipid interface. These results were confirmed by succinimide quenching studies. Overall, the results indicate that the portal region of liver FABP and specifically Lys-31, Lys-36, and Lys-57 are involved in the interaction with the interface of anionic vesicles and that the N-terminal region of the protein undergoes a conformational change, resulting in DAUDA release.

Kinetics of fluorescence expression in nonhuman primates transplanted with GFP retrovirus-modified CD34 cells.

Downregulation and loss of proviral expression have been demonstrated to occur in a variety of in vitro studies and in mouse models. Here we evaluated the kinetics of proviral expression after transplantation in a competitive repopulating model in the baboon. Transgene persistence and green fluorescent protein (GFP) expression in peripheral blood leukocytes (PBL) were analyzed in four animals by semiquantitative PCR and flow cytometry for up to 80 weeks (range 17-80). All animals were transplanted with cells transduced with EGFP or EYFP reporters driven by Moloney murine leukemia virus (MoMuLV) or a modified promoter/enhancer, (MND) respectively. Simultaneous dual-color analysis of fluorescence levels in granulocyte and lymphocyte subsets following hematopoietic reconstitution demonstrated progressive loss of fluorescence intensity occurring predominantly early after transplant in cells transduced with both retrovirus backbones and at serial time points. In addition, we carried out PCR analysis of DNA extracted from sorted EGFP(-)/EYFP(-) cells and confirmed the presence of cells genetically marked by either vector in this population, indicating the persistence of cells that have downregulated or lost retroviral gene expression. In comparison to mouse studies, however, we did not detect substantial differences between MND and MoMuLV backbones.

Stability of green fluorescent protein using luminescence spectroscopy: is GFP applicable to field analysis of contaminants?

Green fluorescent protein (GFP) was first isolated in the early 1970s for experimental use from coelenterates or the Pacific jellyfish, Aequorea victoria (Morin and Hastings, 1971). GFP has since become a favored biomarker in the photophysical analysis of molecular and cell biology because of its strong intrinsic visible fluorescence and the feasibility of fusing it to other proteins without affecting their normal functions (Creemers et al., 2000). Here we report using Bacillus subtilis expressing GFP to evaluate the influence of different environmental pH conditions on GFP fluorescence. Emission acquisitions were configured to excite at 471 nm and detect at an emission from 490 to 650 nm at 1-nm increments. Fluorescence intensity was significantly better at pH 7 (4.2×105 cps; P-value<0.01) than at acid or alkaline conditions. GFP is a good biomarker for environments near netural conditions: however, GFP may be unsuitable where soils or waters are below or above pH 7 because of loss in fluorescence intensity. Alternative fluorescent markers and delivery systems must be examined in different environments to optimize responses from bioreporter molecules.

Fluorescence Emission Spectral Shift Measurements of Membrane Potential in Single Cells

Previous measurements of transmembrane potential using the electrochromic probe di-8-ANEPPS have used the excitation spectral shift response by alternating excitation between two wavelengths centered at voltage-sensitive portions of the excitation spectrum and recording at a single wavelength near the peak of the emission spectrum. Recently, the emission spectral shift associated with the change in transmembrane potential has been used for continuous membrane potential monitoring. To characterize this form of the electrochromic response from di-8-ANEPPS, we have obtained fluorescence signals from single cells in response to step changes in transmembrane potentials set with a patch electrode, using single wavelength excitation near the peak of the dye absorption spectrum. Fluorescence changes at two wavelengths near voltage-sensitive portions of the emission spectrum and shifts in the complete emission spectrum were determined for emission from plasma membrane and internal membrane. We found that the fluorescence ratio from either dual-wavelength recordings, or from opposite sides of the emission spectrum, varied linearly with the amplitude of the transmembrane potential step between −80 and +60 mV. Voltage dependence of difference spectra exhibit a crossover point near the peak of the emission spectra with approximately equal gain and loss of fluorescence intensity on each side of the spectrum and equal response amplitude for depolarization and hyperpolarization. These results are consistent with an electrochromic mechanism of action and demonstrate how the emission spectral shift response can be used to measure the transmembrane potential in single cells.

Transbilayer lipid redistribution accompanies poly(ethylene glycol) treatment of model membranes but is not induced by fusion.

Small, unilamellar vesicles (SUV) or large, unilamellar vesicles (LUV) containing a small amount of N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) or the corresponding phosphatidylserine (NBD-PS) were made asymmetric in labeled lipid by reduction of outer leaflet probe with externally added sodium dithionite. Following removal of dithionite, transbilayer lipid redistribution (presumably due to lipid flip-flop) was indicated by a loss of fluorescence intensity upon readdition of dithionite. Vesicle rupture and fusion in the presence of PEG were measured by changes in the fluorescence of trapped Tb3+ complexed with dipicolinic acid (DPA) or by the increase of fluorescence from 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) coencapsulated with a quenching agent. NBD-PE redistributed slowly (approximately 2%/h) in all symmetrically labeled vesicles examined, while NBD-PS did not. NBD-PE redistribution was not accelerated by treatment of vesicles with PEG below concentrations that induced vesicle rupture or fusion, but was enhanced at or above these PEG concentrations. SUV prepared from hen egg yolk phosphatidylcholine (egg PC) or from dioleoylphosphatidylcholine (DOPC)/dilinolenoylphosphatidylcholine (diC(18:3)PC) (85/15) mixtures were shown to fuse without rupturing in the presence of appropriate concentrations of PEG. Matching the osmolalities inside and outside the vesicle mitigated against rupture but did not prevent fusion. Under these conditions, NBD-PE flip-flop was proportional to the amount of fusion, but with different proportionality constants for the two lipid systems, while NBD-PS flip-flop did not occur. Redistribution of total mass from the outer to the inner leaflet during the fusion process could be detected in terms of a change in the ratio of dithionite-reducible probe to total probe. Both probes detected inwardly directed redistribution of lipid mass under conditions that induced fusion of SUV. We conclude that inward mass redistribution must accompany PEG-mediated SUV fusion, but that lipid flip-flop is not mechanistically related to the fusion process.

Cholera DFA: An improved direct fluorescent monoclonal antibody staining kit for rapid detection and enumeration of Vibrio cholerae O1

An improved fluorescent monoclonal antibody staining kit, Cholera DFA, for direct detection and enumeration of Vibrio cholerae O1 has been developed, employing a highly specific anti-A antigen monoclonal antibody, COLTA, labeled with fluorescein isothiocyanate (FITC). An optimized quantity of anti-photobleaching agent is used in a glycerol mounting medium to retard the rapid fading of immunofluorescent stained cells during fluorescent microscopy, thus enabling prolonged inspection of individual fields, as well asimproved photographic recording of results without loss of fluorescence intensity. When tested for specificity, all 30 strains of V. cholerae O1 reacted with Cholera DFA, whereas 100 heterologous species examined did not, yielding 100% specificity for all strains examined in this study. A field trial was conducted in Bangladesh, employing Cholera DFA and the results were compared with those obtained by conventional culture methods. Of 44 diarrheal stool specimens tested, Cholera DFA was positive for V. cholerae O1 in all culture-positive stool specimens and negative for all culture-negative stool specimens. The procedure is sensitive and highly specific, as well as simple, i.e., less complex than the indirect fluorescent assay, requiring only one reagent and less than 30 min to complete the staining process, while retarding rapid fading that often occurs with fluorescent microscopy.

Cholera DFA: An improved direct fluorescent monoclonal antibody staining kit for rapid detection and enumeration ofVibrio choleraeO1

An improved fluorescent monoclonal antibody staining kit, Cholera DFA, for direct detection and enumeration of Vibrio cholerae O1 has been developed, employing a highly specific anti-A antigen monoclonal antibody, COLTA, labeled with fluorescein isothiocyanate (FITC). An optimized quantity of anti-photobleaching agent is used in a glycerol mounting medium to retard the rapid fading of immunofluorescent stained cells during fluorescent microscopy, thus enabling prolonged inspection of individual fields, as well as improved photographic recording of results without loss of fluorescence intensity. When tested for specificity, all 30 strains of V. cholerae O1 reacted with Cholera DFA, whereas 100 heterologous species examined did not, yielding 100% specificity for all strains examined in this study. A field trial was conducted in Bangladesh, employing Cholera DFA and the results were compared with those obtained by conventional culture methods. Of 44 diarrheal stool specimens tested, Cholera DFA was positive for V. cholerae O1 in all culture-positive stool specimens and negative for all culture-negative stool specimens. The procedure is sensitive and highly specific, as well as simple, i.e., less complex than the indirect fluorescent assay, requiring only one reagent and less than 30 min to complete the staining process, while retarding rapid fading that often occurs with fluorescent microscopy.

The kinetic aspects of intracellular fluorescence labeling with TMA-DPH support the maturation model for endocytosis in L929 cells.

TMA-DPH (1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene), a hydrophobic fluorescent membrane probe, interacts with living cells by instantaneous incorporation into the plasma membrane, where it becomes fluorescent. It then follows the intracellular constitutive membrane traffic and acts as a bulk membrane marker of the endocytic pathway (Illinger, D., P. Poindron, P. Fonteneau, M. Modolell, and J. G. Kuhry. 1990. Biochim. Biophys. Acta. 1030:73-81; Illinger, D., P. Poindron, and J. G. Kuhry. 1991. Biol. Cell. 73:131-138). As such, TMA-DPH displays particular properties mainly due to partition between membranes and aqueous media. From these properties, original arguments can be inferred in favor of the maturation model for the endocytic pathway, against that of pre-existing compartments, in L929 cultured mouse fibroblasts. (a) TMA-DPH labeling is seen to progress from the cell periphery to perinuclear regions during endocytosis without any noticeable loss in fluorescence intensity; with a vesicle shuttle model this evolution would be accompanied by probe dilution with a decrease in the overall intracellular fluorescence intensity, and the labeling of the inner (late) compartments could in no way become more intense than that of the peripheral (early) ones. (b) From TMA-DPH fluorescence anisotropy assays, it is concluded that membrane fluidity is the same in the successive endocytic compartments as in the plasma membrane, which probably denotes a similar phospholipidic membrane composition, as might be expected in the maturation model. (c) TMA-DPH internalization and release kinetics are more easily described with the maturation model.

Comparison of antifading agents used in immunofluorescence

Various chemicals including p-phenylenediamine, n-propyl gallate, 1,4-diazobicyclo[2,2,2]-octane and Citifluor were tested to determine their effectiveness in retarding the fading of fluorescein-conjugated antibody bound to eosinophil granule major basic protein in tissues. Immunofluorescence fading and intensity were measured quantitatively using the Zeiss Zonax microphotometer system. All of these agents effectively retarded fluorescence fading; p-phenylenediamine and n-propyl gallate were the most effective. Comparison of glycerol-based and polyvinyl alcohol-based media containing n-propyl gallate or p-phenylenediamine showed that the glycerol-based medium yields higher mean fluorescence intensities than the polyvinyl alcohol-based medium. Slides mounted with p-phenylenediamine-glycerol could be stored for up to 2 weeks without loss of fluorescence intensity. Although p-phenylenediamine has many undesirable chemical properties, we conclude that it is the agent of choice for retarding the fading of tissue preparations stained with fluorescein isothiocyanate conjugates when used in a glycerol-based medium.

A sodium-selective optrode

An ion selective optrode (ISO) for continuous determination of sodium is presented. It is based on the optical measurement of the membrane potential between an aqueous sample solution and a lipid membrane phase with the help of a potential-sensitive dye. The lipid membrane is composed of rhodamine B C-18 ester together with the sodium selective ionophore ETH 157 and either arachidic acid or 1-octadecanol, and is prepared by using the Langmuir-Blodgett (LB) technique. The fluorescence intensity of the dye reversibly responds to the sodium ion concentration. Fluorescence decreases with increasing sodium concentraction. Interferences by potassium or calcium can be compensated for by using a reference optrode. The sodium sensor works in the 1 to 100 mM sodium ion concentration range with a maximal signal change of −4.2% of the full scale signal for 100 mM. The relationship between the relative signal change (ΔI/I) and the negative logarithm of the sodium concentration is linear over two orders of magnitude. The selectivity coefficients over potassium or calcium are better than 1 × 105 when use is made of a reference optrode. The sensors can be stored under air for more than half a year without loss in fluorescence intensity or changes in response function.

Apparent loss of 2H4+ T cells in peripheral blood lymphocytes of normal donors: A 2H4-specific artefact unique to T cells

The antigenic cluster designated CD45R is recognized by a family of monoclonal antibodies. However, one of those most frequently used because of its commercial availability is 2H4. In a series of experiments we show that 2H4 immunofluorescence is almost completely lost from CD4+ or CD8+ T cells of human origin if they are fixed after staining with 2H4. Loss of fluorescence intensity of the total population of 2H4+ lymphocytes and nearly complete quantitative loss of CD45R+ T cells is observed after fixation. This apparent loss of CD45R as detected by 2H4 is not seen if other CD45R-specific monoclonal antibodies are used. Fixation of B cells previously stained with 2H4 gives rise to slightly diminished fluorescence intensity, but no reduction in number of 2H4+ B cells. This appears to be due to a lower antigen density on T cells are compared to B cells. The effect is unique to 2H4, as other monoclonal antibodies recognizing CD45R, while exhibiting a decrease in fluorenscence intensity after fixation, still uniquivocally detect CD45R+ CD4+ or CD8+T cells. The effect is restricted to human T cells, as primate lymphocytes, fixed after staining with 2H4, show no loss in fluorescence intensity. We conclude that if it is necessary to fix human T cells after fluorescent staining, the use of CD45R-specific antibodies other than 2H4 is mandatory.

Investigation of membrane structure using fluorescence quenching by spin-labels. A review of recent studies.

Fluorescence quenching is the loss of fluorescence intensity which is observed when a fluorescent molecule or group interacts with another molecule or group, called the quencher. By use of tryptophan residues of proteins, together with specific probe molecules, quenching can be applied to problems of biological and model membrane structure. Quenching interactions are short range (less than 50 A) so that structure on the scale of molecular dimensions can be examined. This review summarizes the recent applications of fluorescence quenching by spin (nitroxide)-labeled molecules to problems of membrane structure, including determination of the distance of membrane-bound molecules from the membrane surface, the strength of lipid-protein interactions and the strength of protein-protein interactions within membranes. The unique advantages and the limitations of this powerful method are examined.

Effect of extracellular Ca 2+, K + and OH − on erythrocyte membrane potential as monitored by the fluorescent probe 3,3′-dipropylthiodicarbocyanine

Changes in fluorescence intensity of thiodicarbocyanine, DiS-C 3(5), were correlated with direct microelectrode potential measurements in red blood cells from Amphiuma means and applied qualitatively to evaluate the effects of extracellular Ca 2+, K + and pH on the membrane potential of human red cells. Increasing extracellular [Ca 2+] from 1.8 to 15 mM causes a K +-dependent hyperpolarization and decrease in fluorescence intensity in Amphiuma red cells. Both the hyperpolarization and fluorescence change disappear when the temperature is raised from 17 to 37°C. No change in fluorescence intensity is observed in human red cells with comparable increase in extracellular Ca 2+ in the temperature range 5–37°C. Increasing the extracellular pH, however, causes human red cells to respond to an increase in extracellular Ca 2+ with a significant but temporary loss in fluorescence intensity. This effect is blocked by EGTA, quinine or by increasing extracellular [K +], indicating that at elevated extracellular pH, human erythrocytes respond to an increase in extracellular Ca 2+ with an opening of K + channels and associated hyperpolarization of the plasma membrane.

A model for the effect of lipid oxidation on diphenylhexatriene fluorescence in phospholipid vesicles

We have determined by means of a standard spectrophotometric assay that lipid oxidation occurred at a significant rate in large, multilamellar vesicles containing egg phosphatidylcholine under normal experimental conditions. We have also observed that the fluorescence intensity of the vesicle-associated probe, 1,6-diphenyl-1,3,5-hexatriene, decreased with time in vesicles containing such oxidizing lipids. The spectrophotometric data utilized to monitor lipid oxidation were found to fit an apparent first-order kinetic model. The loss of diphenylhexatriene fluorescence intensity in oxidizing liposomes was analyzed in terms of a first-order event superimposed (and thus presumably dependent) upon the ongoing formation of oxidized lipid. These and other data were used to conclude that the oxidation-induced loss of diphenylhexatriene fluorescence intensity was due to chemical modification of the fluorophore rather than to excited-state quenching or ground-state complex formation. Finally, the loss of fluorescence intensity in oxidizable membranes was found to alter drastically the ‘microviscosity’ parameter as derived from diphenylhexatriene fluorescence anisotropy and relative intensity measurements.

Mechanism of Action of Nitroimidazole Antimicrobial and Antitumour Radiosensitizing Drugs

Electrolytic reduction of the hypoxic tumour cell radiosensitizing drug misonidazole was carried out at a controlled potential under anaerobic conditions in the presence of Escherichia coli DNA. During the reduction process the DNA was examined by viscometry, thermal hyperchromicity, melting and renaturation profiles, hydroxyapatite chromatography, agarose gel electrophoresis and alkaline sucrose density gradient centrifugation. The reduced drug decreases the viscosity, hyperchromicity and renaturation of DNA. These effects are consistent with strand breakage of the molecule which was corroborated by finding an increase in the single-strand content of DNA, increased migration and loss of fluorescence intensity on agarose gels and sedimentation to a less dense region in alkaline sucrose density gradients. The results are discussed in relation to postulated mechanisms of the selective toxicity of the drug towards anaerobes and cytotoxicity of electron affinic radiosensitizers of hypoxic tumour cells.

A possible mutation of a fluorescence polymorphism.

The segregation of the Q-band polymorphisms in 32 families have been studied. From 90 matings in these families, there were a total of 208 offspring. In one of these offspring it could be shown that there had been a change of a fluorescent polymorphism, resulting in the loss of fluorescent intensity in the satellite of a chromosome 21. The origin of such a 'mutation' and a consideration of mutation rates is discussed.

The Effect of Ultraviolet Irradiation on Tyrosinase

The ultraviolet irradiation of mushroom tyrosinase exhibited gradual loss in catalytic activity as a function of radiation dose. Destruction of tryptophanyl residues in the enzyme was studied chemically (determined using pDAB reagent) as well as by fluorescence measurement. The rate constant for the loss in fluorescence intensity and the destruction of tryptophanyl residue was found to be $10.3\pm 0.6\times 10^{-2} {\rm min}^{-1}$ and $4.54\pm 0.45\times 10^{-2} {\rm min}^{-1}$ , respectively. It was observed that the Km for dihydroxy-phenylalanine (dopa), catechol, and tyrosine was affected to varying degrees on irradiation. The $D_{37}$ and kinetic data of native and irradiated tyrosinase support the existence of two active centers on the enzyme molecule. It is proposed that the loss in catalytic activity of tyrosinase may be due to conformational changes.

A Spectrophotometric Procedure, Using Carboxypeptidase A, for the Quantitative Measurement of Ochratoxin A

In the method described, ochratoxin A is eleaved into ochratoxin alpha (free isocoumarin chromophore) and phenylaline, using carboxypeptidase. Detection is based on the difference in fluorescence excitation spectra of ochratoxin A (380 NM, maximum) and ochratoxin alpha (340 nm, maximum). The quantitation of ochratoxin A is based on the loss of fluorescence intensity at 380 nm. The method has been used for the quantitative determination of as little as 4 mug ochratoxin A/kg barley and barley meal but it could be extended to other products.