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PubMed2004

Correlation of the thermal stability of phospholipid-based emulsions and the microviscosity measurements using fluorescence polarization.

The fluorescence polarization technique was used to measure the microviscosity of a series of phospholipid-based emulsions. Fourteen different oil-in-water emulsions containing 20% medium chain length triglycerides, various concentrations and types of phospholipids, and 2.2% glycerin were prepared by microfluidization and pH-adjusted to 4.0 or 7.4. Microviscosity was measured by determining the anisotropy of a fluorophore probe (1,6-phenyl 1,3,5-hexatriene) which was found to obey Perrin's equation as has been previously reported for liposomes and membrane bilayers. Moreover the method was validated by comparing viscosities of phospholipid-oil mixtures measured by rheometry and fluorescence polarization. The viscosities determined by fluorescence polarization were within 6% of the values obtained by classic rheometry. Emulsions were also subjected to thermal stress at 121 degrees C. The droplet growth rate was estimated by measuring the time-dependent mean droplet diameter using photon correlation spectroscopy. The logarithm of the droplet growth rate was found to be directly proportional to the interfacial rigidity (i.e., the inverse microviscosity) which suggested that coalescence rather than molecular diffusion is the primary mechanism of droplet growth under these conditions of thermal stress.

Diphenylhexatriene↗
PubMed2001

Conformation of myosin interdomain interactions during contraction: deductions from muscle fibers using polarized fluorescence.

Myosin cross-bridge subfragment 1 (S1) is the ATP catalyzing motor protein in muscle. It consists of three domains that catalyze ATP and bind actin (catalytic), conduct energy transduction (converter), and transport the load (lever arm). Force development during contraction is thought to result from rotary lever arm movement with the cross-bridge attached to actin. To elucidate cross-bridge structure during force development, two crystal structures of S1 were extrapolated to working "in solution" or oriented "in tissue" forms, using structure-sensitive optical spectroscopic signals from two extrinsic probes. The probes were located at two interfaces containing the catalytic, converter, and lever arm domains of S1. Observed signals included circular dichroism (CD) and absorption originating from S1 in solution in the presence and absence of actin and fluorescence polarization from cross-bridges in muscle fibers. Theoretical signals were calculated from S1 crystal structure models perturbed with lever arm movement from swiveling at three conserved glycines, 699, 703, and 710 (chicken skeletal myosin numbering). Best agreement between the computed and observed signals gave structures showing that actin binding to S1 causes movement of the lever arm. A three-state model of S1 conformation during contraction consists of three actin-bound cross-bridge states observed from muscle fibers in isometric contraction, in the presence of MgADP, and in rigor. Structures best representing these states show that most of the lever arm rotation occurs between isometric contraction and the MgADP states, i.e., during phosphate release. Smaller but significant lever arm rotation occurs with ADP dissociation. Structural changes within the S1 interfaces studied are discussed in the accompanying paper [Burghardt et al. (2001) Biochemistry 40, 4834-4843].

Animals↗
PubMed1997

Analysis of the intracellular processing of proteins: application of fluorescence polarization and a novel fluorescent probe.

Previous studies indicated that fluorescein derivatized bovine serum albumin was an ideal probe to monitor the time-dependent kinetics of antigen processing in the murine macrophage cell line J774. Whereas previous work focused on fluorescence intensity measurements, the present study relied on fluorescence polarization to dissect the local environment of the fluorescent hapten-protein within the endocytic system of the cell. A steady increase in both fluorescence intensity and fluorescence polarization of the cell population was detected for the first 100 min. However, at 100 min, a plateau in both fluorescence intensity and polarization was observed and was followed by a decrease in fluorescence polarization and a corresponding increase in fluorescence intensity. Western blot analyses revealed that the decrease in fluorescence polarization was due to proteolytic degradation of the probe within the cell. Using a combination of in vitro experiments and an additional fluorescent probe, it was determined that the initial increase in fluorescence polarization was due to movement of the probe through a pH gradient within the cell, suggestive of transport through the endocytic system. By combining fluorescence polarization, flow cytometry, and a unique fluorescent enhancement substrate, these studies represented a novel approach for monitoring intracellular trafficking and processing of proteins within macrophages.

Animals↗
PubMed2002

Quantitative analysis of c-myc-tagged protein in crude cell extracts using fluorescence polarization.

A fluorescence polarization (FP) assay was developed to determine the concentration of a c-myc-tagged recombinant protein in a crude cell extract. The basis of the assay was a competition between a c-myc-tagged protein and a fluorescein-labeled c-myc peptide for a c-myc antibody Fab. Fluorescein-labeled c-myc peptide produced a high-fluorescence polarization signal upon binding to the c-myc antibody, which can be inhibited in the presence of a c-myc-tagged protein. Quantitation of a c-myc-tagged protein was realized by measuring the decrease in fluorescence polarization. The observed IC(50) values in the competition FP assay were similar among all monomeric c-myc-tagged proteins tested, indicating that the interaction of the c-myc tag with the antibody was independent of the fusion protein sequence. The c-myc-tagged protein concentrations measured by FP were found to correlate well with values derived from a spike experiment and with values obtained by quantitative immunoblot. This assay was not perturbed by the presence of crude cell lysate, dithiothreitol or detergents, and worked with both native and denatured samples from several expression systems, including Escherichia coli, Pichia, insect cells, and mammalian cells. The assay under the current condition can detect as low as 0.05% expression level of c-myc-tagged protein with regards to total proteins, depending on the expression system. This assay is both quantitative and rapid (less than 15min) and is therefore suitable for the optimization of recombinant protein expression conditions as well as for the monitoring of protein purification procedures.

Binding, Competitive↗
PubMed1983

The effect of fetal pulmonary surfactant production on the apparent microviscosity of amniotic fluid measured by fluorescence polarization.

The fluorescence properties of 1,6 diphenyl 1,3,5-hexatriene (DPH) in human amniotic fluid from the 10--20th week and from the 36--40th week of gestation are investigated. The probe environment of two stages of fetal development are studied by measuring the effect of temperature on the fluorescence polarization of DPH. The temperature profiles of fluorescence polarization of DPH in amniotic fluid and in liposomes prepared from chloroform/methanol extracts of the fluid are compared to the temperature profiles of liposomes prepares from phospholipids of known composition. The DPH binding structures of human amniotic fluid are further characterized by fractionation of lipid bodies by centrifugation on a Ficoll density gradient. The major part of fluorescence in third trimester amniotic fluid is emitted by DPH bound to pulmonary lamellar lipid bodies. The DPH environment in early gestational amniotic fluid is a distinctly different structure, which is more rigid than both sphingomyelin and dipalmityl lecithin at temperatures above 40 degrees C, and which is not chloroform/methanol extractable.

Amniotic Fluid↗
PubMed1976

Theory of fluorescence polarization of oriented pigment molecules in spherical arrays.

Theoretical results are presented which are appropriate for the analysis of the static polarized fluorescence experiment with oriented pigment molecules in spherical arrays (vesicles). Though the global orientation mediated over the whole sphere is isotropic, the fluorescent molecules may have preferred local orientation with respect to the local plane. As in a former paper, concerning fluroescence polarization in planar arrays, three basic (local) orientation distributions of the electronic transition moments are investigated, which may be expected to describe a wide class of real cases with sufficient accuracy. Analytic expressions for the degree of polarization are derived. One important result is that the degree of polarization may be extremely dependent on the local orientation of transition moments. Hence the usual method of determination of microviscosities from experiments with vesicles with the use of the theory of fluorescence polarization for macromolecules in solutions should be regarded with great caution.

Electromagnetic Phenomena↗
PubMed1981

Principles of fluorescence polarization measurements.

Fluorescence polarization measurements using high-speed single-cell flow cytometry have found increasing use in cellular biology. In most flow systems, the detection axis generally is aligned orthogonally to the direction of flow and the excitation axis, and asymmetric apertures along the excitation and emission axes by cylindrical lenses and/or nonplanar transition between optical indices complicate the numerical correction of systematic depolarization effects. In addition, recent studies on fluorescence emission of structured particles have shown remarkable anisotropies of polarized fluorescence emission dependent on the direction of the detection axis.

Chlorophyta↗
PubMed2001

Polarized fluorescence microscopy of individual and many kinesin motors bound to axonemal microtubules.

Kinesin is a molecular motor that interacts with microtubules and uses the energy of ATP hydrolysis to produce force and movement in cells. To investigate the conformational changes associated with this mechanochemical energy conversion, we developed a fluorescence polarization microscope that allows us to obtain information on the orientation of single as well as many fluorophores. We attached either monofunctional or bifunctional fluorescent probes to the kinesin motor domain. Both types of labeled kinesins show anisotropic fluorescence signals when bound to axonemal microtubules, but the bifunctional probe is less mobile resulting in higher anisotropy. From the polarization experiments with the bifunctional probe, we determined the orientation of kinesin bound to microtubules in the presence of AMP-PNP and found close agreement with previous models derived from cryo-electron microscopy. We also compared the polarization anisotropy of monomeric and dimeric kinesin constructs bound to microtubules in the presence of AMP-PNP. Our results support models of mechanochemistry that require a state in which both motor domains of a kinesin dimer bind simultaneously with similar orientation with respect to the microtubule.

Actin Cytoskeleton↗
PubMed1986

Change of intracellular fluidity during keratinocyte differentiation measured by fluorescence polarization.

The fluorescence polarization method was applied to measure the intracellular fluidity of fractionated guinea pig keratinocytes. Guinea pig epidermal cell suspension was obtained by treatment with EDTA and trypsin, and was separated into high, intermediate, and low density fractions using Percoll density gradient centrifugation. Morphological observation and cytofluorometric analysis of DNA content in the fractionated epidermal cells showed that the high, intermediate, and low density fractions were basal, spinous, and granular cell-rich fractions, respectively. Intracellular fluorescence polarization of each fraction was determined by a polarization spectrofluorometer (Hitachi MPF-4, prototype) with fluorescein diacetate. The P-values were calculated for high, intermediate, and low density fractions as 0.192 +/- 0.021, 0.172 +/- 0.019, and 0.147 +/- 0.012, respectively. Since low P-values indicate a high degree of fluidity, the results indicate that intracellular fluidity of keratinocytes is lower in basal cells and higher in granular cells. Dye-binding experiments showed that fluorescein-binding proteins were not detected in the soluble fraction of the epidermal cells. The present findings suggest that intracellular fluidity of the guinea pig keratinocyte increases during the process of its differentiation.

Animals↗
PubMed1997

Measurement of specific protease activity utilizing fluorescence polarization.

A fluorescence polarization assay was designed to measure proteolytic cleavage of a specific peptide substrate for human cytomegalovirus protease. The peptide substrate was derivatized by biotinylation of a gamma-aminobutyric acid-modified amino-terminus and labeled with 5-(4,6-dichlorotriazinyl)aminofluorescein at the carboxy-terminus. Incubation of this substrate with recombinant human cytomegalovirus protease and subsequent addition of egg white avidin produced a polarization signal that was proportional to the relative amounts of cleaved and uncleaved substrate. The uncleaved substrate produced a high polarization value upon binding to avidin, whereas the cleaved, low-molecular-weight fluorescently tagged peptide that cannot bind to avidin produced a low polarization value. The inhibitory activity of a 3,4-dichloroisocoumarin against the protease was evaluated by comparing the change in polarization with a noninhibited control. The fluorescence polarization protease assay does not suffer from interference due to the presence of absorptive interferants making this a convenient, homogenous assay for high throughput screening.

Amino Acid Sequence↗
PubMed1979

Assay of angiotensin I by fluorescence polarization method.

Fluorescence polarization technique was applied for the assay of angiotensin I (Al) in human plasma. In this assay system, fluorescein labeled Al (F-Al), which retained the original antigenicity, and antibody to Al was allowed to interact in a cuvette in the instruments yielding an increase in the fluorescence polarization (P) value. Non-labeled Al in the sample blocked the binding of F-Al to the antibody resulting lower P value. Log of antigen concentration and P value was found to exhibit reverse linear proportionality between 0.05 ng to 2 ng/ml of antigen (Al) concentration. The present method was compared with standard radioimmunoassay method and the result showed that data were compatible with each other. The calculation of P value is automated and three cavity filter and optics of the instrument gave reliable results. The method is fast (less than 2 min), sensitive (less than 10 picomole/ml) and simple (no separation step before readout of the results).

Angiotensin I↗
PubMed2002

Fluorescence polarization of green fluorescence protein.

We report here the striking anisotropy of fluorescence exhibited by crystals of native green fluorescence protein (GFP). The crystals were generated by water dialysis of highly purified GFP obtained from the jellyfish Aequorea. We find that the fluorescence becomes six times brighter when the excitation, or emission, beam is polarized parallel (compared with perpendicular) to the crystal long axis. Thus, the major dipoles of the fluorophores must be oriented very nearly parallel to the crystal long axis. Observed in a polarizing microscope between parallel polars instead of either a polarizer or analyzer alone, the fluorescence polarization ratio rises to an unexpectedly high value of about 30:1, nearly the product of the fluorescence excitation and emission ratios, suggesting a sensitive method for measuring fluorophore orientations, even of a single fluorophore molecule. We have derived equations that accurately describe the relative fluorescence intensities of crystals oriented in various directions, with the polarizer and analyzer arranged in different configurations. The equations yield relative absorption and fluorescence coefficients for the four transition dipoles involved. Finally, we propose a model in which the elongated crystal is made of GFP molecules that are tilted 60 degrees to align the fluorophores parallel to the crystal long axis. The unit layer in the model may well correspond to the arrangement of functional GFP molecules, to which resonant energy is efficiently transmitted from Ca2+-activated aequorin, in the jellyfish photophores.

Animals↗
PubMed1981

Fluorescence polarization immunoassay. II. Analyzer for rapid, precise measurement of fluorescence polarization with use of disposable cuvettes.

This instrument, developed for quantitating fluorescence polarization immunoassays, automatically measures both polarization components and computes a polarization value corrected for background and optical bias. An electronically switched liquid crystal provides a nonmechanical means of rotating the plane of polarization in the excitation optics. The electronic design features digital integration and microprocessor controlled functions. Readings are made in disposable 12 X 75 mm round culture tubes. in less than 10 s. Results are precise to 0.001 polarization unit, linear from 10(-7) to 10(-10) mol of fluorescein per liter, and correlate well with those obtained with other "high-performance" instrumentation.

Autoanalysis↗
PubMed2002

Fluorescence polarization discriminates green fluorescent protein from interfering autofluorescence in a microplate assay for genotoxicity.

An unconventional use for the polarization optics, associated with a variety of commercially available fluorescence microplate readers, is reported. This novel application has allowed the discrimination of green fluorescent protein (GFP) fluorescence in genetically modified yeast cells from interfering autofluorescent species. The method exploits the unusually high fluorescence anisotropy of GFP compared to smaller fluorophores and autofluorescent species. The principle was successfully applied to resolve the induced GFP signal from that of autofluorescent test compounds, in an assay for genotoxic species. The use of fluorescence polarization enabled both proflavin and methapyrilene to be identified as genotoxic agents in the yeast assay. This would not have been possible using conventional fluorescence alone since these compounds were found to be intensely autofluorescent at the same wavelength as GFP and thus effectively mask the GFP signal.

Anisotropy↗
PubMed1988

Measurement of angiotensinogen in human serum by fluorescence polarization immunoassay.

Fluorescence polarization immunoassay (F.P.I.A.) has rarely been used to measure components of the renin system. Using an Abbott TDX polarimeter and fluorescein - labelled angiotensin I as a tracer we measured angiotensin I by F.P.I.A. Combining this procedure with a renin incubation step enabled measurement of angiotensinogen in human serum. Using sera from male patients and from pregnant females, a good correlation between radioimmunoassay (R.I.A.) and F.P.I.A. was found. Two procedures were developed; one involving taking samples from the renin incubation mixture, the other involving measurement of generated angiotensin I at intervals without interrupting the renin incubation procedure. F.P.I.A. is less expensive and somewhat simpler than R.I.A. but, with the instrument used, it was less sensitive. An unexpected observation was that human renin increases polarization in solutions containing fluorescein labelled angiotensin I, indicating possible binding of renin to angiotensin I.

Angiotensin I↗
PubMedDate not supplied

Efficient and expedient two-step pyranose-retaining fluorescein conjugation of complex reducing oligosaccharides: galectin oligosaccharide specificity studies in a fluorescence polarization assay.

Fluorescence labeling of naturally occurring saccharides provides a tool for studying lectins. A practical and efficient two-step protocol for fluorescence labeling of reducing sugars without disrupting their pyranose structure has been developed, consisting of generation of the amino sugar using NH(4)HCO(3)(s)/NH(3)(aq, concentrated) followed by BOP-mediated acylation with derivatives of 5- or 6-carboxyfluorescein. The acylated conjugates were subsequently run against galectins-1, -3, and -8, beta-galactoside recognizing lectins of current interest, in a fluorescence polarization binding assay. Upon analyzing a collection of isomerically pure 5- and 6-carboxyfluorescein derivatives with different tether lengths, we found that conjugates based on 5-carboxyfluorescein gave significantly better results than the ones based on 6-carboxyfluorescein and that galectins-1 and -8 favored conjugates with different tether lengths than did galectin-3. The results show that fluorescence labeling can be chemically tuned to find optimal probes for individual galectins but also probes interacting well with many galectins.

Binding Sites↗
PubMed1996

Instability of standard calibrators may be involved in overestimating vancomycin concentrations determined by fluorescence polarization immunoassay.

Fluorescence polarization immunoassay (FPIA) is widely used to determine serum vancomycin concentrations, and it has been shown to over-estimate vancomycin concentrations in sera from renally impaired patients. This phenomenon has generally been thought to result from interference by vancomycin crystalline degradation products (CDP-1). In this study, we confirmed that serum vancomycin concentrations in various patients determined by FPIA were higher than those determined by high-performance liquid chromatography (HPLC) or enzyme multiplied immunoassay (EMIT). However, the quantitative differences in the serum vancomycin concentrations determined by FPIA versus HPLC were higher than the CDP-1 concentrations, even when the cross-reactivity of FPIA to CDP-1 is assumed to be 100%. When the vancomycin calibrators for FPIA were stored at 4 degrees C for 30 days, their concentrations determined by FPIA and HPLC decreased by 14 and 20%, respectively, and CDP-1 corresponding to 20% of primary vancomycin was formed. When stored at 25 degrees C, the degradation of vancomycin was more marked. We concluded that not only the cross-reactivity of FPIA to CDP-1 but also the instability of calibrators may cause the overestimation of serum vancomycin concentrations determined by FPIA.

Aged↗
PubMed2002

Field trial of the brucellosis fluorescence polarization assay.

Fluorescence polarization assay (FPA) is a homogeneous technique which was applied to the serological diagnosis of bovine brucellosis. Because of its simplicity and because it may be performed very rapidly, it was an ideal test to adapt to field use. The FPA was used to test cattle on six dairy farms in Baja California, Mexico. Anticoagulated blood, serum, and milk were collected from each animal. The anticoagulated blood was tested immediately on the farm while serum and milk were tested subsequently in the laboratory. Cattle on one farm (n = 140) were thought not to be infected with Brucella abortus and the other farms were thought to have high prevalence of the infection. The whole blood FPA (FPA(bld)) did not detect antibody in any of the cattle on the first premise. This finding was confirmed using a number of other serological tests, including the buffered antigen plate agglutination test, the complement fixation test, the indirect and competitive enzyme immunoassays, and the FPA using serum and milk. Cattle on the other premises (n = 1122) were tested in a similar fashion. The sensitivity of the FPA(bld), relative to the serum FPA (considered the definitive test), was 99.1% and the relative specificity of the FPA(bld) was 99.6%. These results compared favourably with those obtained using the other serological tests.

Animals↗