Fluorescence Polarization Method Research Articles

Lack of association between polymorphisms in the gene encoding protein tyrosine phosphatase 1B (PTPN1) and risk of Type 2 diabetes

Recently, an association of Type 2 diabetes (T2DM) with polymorphisms in PTPN1 located on chromosome 20q was reported. We attempted to replicate this finding in an ethnically homogeneous Polish population. The study groups comprised 474 cases with T2DM and 411 control subjects with normal fasting glucose. All individuals were genotyped for the five previously reported PTPN1 polymorphisms using a fluorescence polarization method. HAPLO.STAT software was used to infer and compare haplotype distributions. The distributions of alleles and genotypes for the five genotyped PTPN1 polymorphisms did not differ between the T2DM cases and control subjects (lowest P = 0.6). Similarly, the frequency of the common haplotype reported to be associated with T2DM did not differ in cases and control subjects. We also failed to find such an association in Whites by performing a meta-analysis of all the available data on the association of those five SNPs with T2DM. This case-control study in a Polish population did not confirm the reported association between polymorphisms in PTPN1 and T2DM.

Molecular Orientation and Optical Anisotropy in Drawn Films of Miscible Blends Composed of Cellulose Acetate and Poly(N-vinylpyrrolidone-co-methyl methacrylate)

Poly(N-vinylpyrrolidone) (PVP) is miscible with cellulose acetate (CA) with a degree of acetyl substitution (DS) of less than ca. 2.75, and a random copolymer of N-vinylpyrrolidone (VP) and methyl methacrylate (MMA) can also form completely miscible blends with CA, when the VP fraction in the copolymer is >30 mol % and the DS of CA is ≤2.5. The molecular orientation and optical anisotropy induced by uniaxial deformation of the miscible blends of the VP-containing vinyl polymers [P(VP-co-MMA)s] with CA were characterized by a fluorescence polarization method and birefringence quantification, respectively. Film samples of more than 10 pairs of CA/P(VP-co-MMA) were cast from mixed polymer solutions in N,N-dimethylformamide containing a slight amount of a stilbene derivative as a fluorescent probe, to assume different blend compositions for each individual polymer pair. Through analysis of polarized fluorescence intensity, it was found that all the drawn films gave a positive orientation function, but the ori...

Crystal Structure of the HRDC Domain of Human Werner Syndrome Protein, WRN

Werner syndrome is a human premature aging disorder characterized by chromosomal instability. The disease is caused by the functional loss of WRN, a member of the RecQ-helicase family that plays an important role in DNA metabolic pathways. WRN contains four structurally folded domains comprising an exonuclease, a helicase, a winged-helix, and a helicase-and-ribonuclease D/C-terminal (HRDC) domain. In contrast to the accumulated knowledge pertaining to the biochemical functions of the three N-terminal domains, the function of C-terminal HRDC remains unknown. In this study, the crystal structure of the human WRN HRDC domain has been determined. The domain forms a bundle of alpha-helices similar to those of Saccharomyces cerevisiae Sgs1 and Escherichia coli RecQ. Surprisingly, the extra ten residues at each of the N and C termini of the domain were found to participate in the domain architecture by forming an extended portion of the first helix alpha1, and a novel looping motif that traverses straight along the domain surface, respectively. The motifs combine to increase the domain surface of WRN HRDC, which is larger than that of Sgs1 and E. coli. In WRN HRDC, neither of the proposed DNA-binding surfaces in Sgs1 or E. coli is conserved, and the domain was shown to lack DNA-binding ability in vitro. Moreover, the domain was shown to be thermostable and resistant to protease digestion, implying independent domain evolution in WRN. Coupled with the unique long linker region in WRN, the WRN HRDC may be adapted to play a distinct function in WRN that involves protein-protein interactions.

Assay Principle for Modulators of Protein−Protein Interactions and Its Application to Non-ATP-Competitive Ligands Targeting Protein Kinase A

Targeting sites that modulate protein-protein interactions represents an ongoing challenge for drug discovery. We have devised an assay principle, named ligand-regulated competition (LiReC), in an effort to find non-ATP competitive small-molecule regulators for type Ialpha cAMP-dependent Protein kinase (PKA-Ialpha), a protein complex that is implicated in disease. Our assay based on the LiReC principle utilizes a competitive fluorescent peptide probe to assess the integrity of the PKA-Ialpha complex upon introduction of an allosteric ligand. The developed fluorescence polarization method screens for small molecules that specifically protect (antagonists) or conversely activate (agonists) this protein complex. In high-throughput format, various cyclic nucleotide-derived agonists and antagonists are successfully detected with high precision. Furthermore, assay performance (Z'-factors above 0.7) far exceeds the minimum requirement for small-molecule screening. To identify compounds that operate through novel modes of action, our method shields the ATP-binding site and purposely excludes ATP-competitive ligands. These proof-of-principle experiments highlight the potential of the LiReC technique and suggest its application to other protein complexes, thereby providing a novel approach to identify and characterize modulators (small molecules, proteins, peptides, or nucleic acids) of protein-protein systems.

Optimization of a Fluorescence Polarization Immunoassay for Rapid Quantification of Deoxynivalenol in Durum Wheat–Based Products

A fluorescence polarization immunoassay previously described for deoxynivalenol (DON) screening in wheat was optimized for the rapid quantification of DON in durum wheat kernels, semolina, and pasta. A background signal was observed in both spiked and naturally contaminated samples, strictly depending on the testing matrix. After subtracting the background DON level for durum wheat (0.27 μg of DON per g), semolina (0.08 μg of DON per g), and pasta (0.04 μg of DON per g), an accurate quantification of DON was possible at levels greater than 0.10 μg/g for all matrices. Average recoveries from spiked samples (0.25 to 1.75 μg/g) were 98, 102, and 101% for wheat, semolina, and pasta, respectively. Comparative analyses of 35 naturally contaminated durum wheat samples, 22 semolina samples, and 26 pasta samples performed by both the fluorescence polarization method and high-pressure liquid chromatography/immunoaffinity cleanup showed a good correlation (r > 0.995). The fluorescence polarization method showed better accuracy and precision with respect to the high-pressure liquid chromatography method and is suitable for the rapid and quantitative determination of DON in durum wheat–based products at levels foreseen by existing or coming international regulations.

A Review of Rapid Methods for the Analysis of Mycotoxins

An overview is presented of the analysis of mycotoxins by rapid methods such as: enzyme linked immunosorbent assay (ELISA); flow through membrane based immunoassay; immunochromatographic assay; fluorometric assay with immunoaffinity clean-up column or with a solid phase extraction clean-up column; and fluorescence polarization method. These methods are currently commercially available and are reliable, rapid methods. This review focuses on the basic principle of each rapid method as well as advantages and limitations of each method. Additionally, we address other emerging technologies of potential application in the analysis of mycotoxins.

High-throughput fluorescence polarization method for identifying ligands of LOX-11

To develop and optimize a competitive fluorescence polarization (FP) method, and use it as a high-throughput screening (HTS) assay for drug discovery. Human lectin-like oxidized low-density lipoprotein receptor-1 (hLOX-1) and oxidized low-density lipoprotein (oxLDL) were used to establish a high-throughput fluorescence polarization assay to screen ligands of human LOX-1. A 96-well plate assay was performed with a fast plate reader. Three fluorescein isothiocyanate-labeled hLOX-1 concentrations (100, 200, and 400 nmol/L) were selected to be titrated by oxLDL (from 0.05 nmol/L to 100 micromol/L) in order to obtain optimal reactive concentrations. The concentration of Me2SO used (0%, 1%, 3%, 5%) and incubation time (15 min, 30 min, 1 h, 2 h) were optimized. The Z' factor was calculated to estimate the quality of FP-based HTS. Concentrations of 200 nmol/L for human LOX-1 and 50 micromol/L for oxLDL were used in the actual assay. Concentrations of 0% to 5% Me2SO and different reaction times did not affect the FP-based HTS. The Z' value was 0.66. By using this detection and screening system, 12 700 compounds were screened and 3 ligands with an IC50 of less than 4.5 micromol/L were found. The established competitive FP-based assay is sensitive, stable, highly reproducible and robust, and suitable for HTS for ligands of the hLOX-1 receptor.

Orientation of Adsorbed Dyes in the Interlayer Space of Clays. 2 Fluorescence Polarization of Rhodamine 6G in Laponite Films

A new fluorescence polarization method is applied to evaluate the preferential orientation of rhodamine 6G (R6G) adsorbed into laponite (Lap) clay mineral in supported films. For this purpose, the fluorescence spectra for the horizontal and vertical directions of the emission polarizer were recorded for a common horizontally polarized excitation light as a function of the twist angle δ of the film with respect to the excitation (and emission) beam. The orientation of the fluorescent species adsorbed in the clay films can be evaluated from the linear relationship between the fluorescence dichroic ratio and the twist angle δ recently established.1,2 For low dye-loading R6G, molecules are adsorbed into the Lap surface as monomeric units with a tilt ψ angle of 28° with respect to the clay layer, in agreement with results obtained by visible absorption with polarized light. Fluorescent coplanar J type dimers of R6G are also observed in Lap films for moderate dye loadings with a similar orientation angle around 30°. The presence of coplanar R6G H type dimers and high-order aggregates in moderate- and high-loading R6G/Lap films causes a depolarization for the fluorescence anisotropy of the J dimer, ascribed to an excitation energy-transfer process and reducing the applicability of this fluorescence method. However, present experimental results confirm a more-perpendicular disposition of these H aggregates with respect to the Lap layer. The new fluorescence polarization method can be extrapolated to determine the orientation of any adsorbed fluorophore in rigid and ordered 2D host materials.

Application of Fluorescence with Polarized Light to Evaluate the Orientation of Dyes Adsorbed in Layered Materials

A new method of fluorescence polarization is applied to evaluate the angle of the preferential orientation of Rhodamine 6G (R6G) dye adsorbed in supported thin films of Laponite (Lap) clay. The method is based in the determination of the fluorescence dichroic ratio, obtained from the recorded fluorescence spectra with the detection polarizer horizontally and vertically oriented, as a function of the twisted angle of the film around its vertical axis, keeping the excitation polarizer in a fixed direction. The validity of the method is checked by comparing the experimental results obtained with both vertically and horizontally polarized excitations to that previously provided by absorption spectroscopy with linearly polarized light. A preferential orientation angle with respect to the normal to the clay layer of 62 degrees is derived for R6G monomers adsorbed in Lap films.

New fluorescent polarization method to evaluate the orientation of adsorbed molecules in uniaxial 2D layered materials

A new fluorescence polarization method is developed as a powerful tool to evaluate the preferential orientation of Rhodamine 6G (R6G) molecules adsorbed in the two-dimensional (2D) interlayer space of Laponite (Lap) clay films. The fluorescence anisotropy of a very low R6G loading film, around the 0.1% of the total cation exchange capacity (% CEC) of Lap, reveals that the dye is adsorbed as monomeric units with a preferential orientation angle of 62° with respect to the normal to the clay layers. This tilted angle agrees with that previously obtained by absorption spectroscopy with linearly polarized light [V. Martínez Martínez, F. López Arbeloa, J. Bañuelos Prieto, I. López Arbeloa, Chem. Mater. 17 (2005) 4134–4141], justifying the validity of the present emission technique. The fluorescent method can be applied to evaluate the orientation of any fluorescent molecules preferentially adsorbed along the normal of the plane of any rigid 2D host arrays.

An Image Processing Method for Quantifying Fiber Formation in Meat Analogs Under High Moisture Extrusion

ABSTRACTHigh moisture extrusion using twin‐screw extruders shows great promise for producing meat analog products with vegetable proteins. The resulting products have well‐defined fiber formations and resemble real meat in both visual appearance and taste sensation. Developing reliable nondestructive techniques to quantify the textural properties of extrudates is important for quality control in the manufacturing process. In this study, we developed an image‐processing technique to automatically characterize sample fiber formation using digital imaging. The algorithm is based on statistical analysis of Hough transform. This objective method can be used as a standard method for evaluating other noninvasive methods. We have compared the fiber formation indices measured using this technique and a noninvasive fluorescence polarization method and obtained a high correlation.

Inhibition of mitochondrial respiration by nitric oxide is independent of membrane fluidity modulation or oxidation of sulfhydryl groups

Nitric oxide (NO) modulates the fluidity of a variety of membranes. Thus, the aim of the present work was to study if the inhibitory effect of NO on mitochondrial respiration is associated with its effects on membrane fluidity. Liver mitochondria and an inner mitochondrial membrane fraction (IMMF) were isolated from male Wistar rats by differential centrifugation. Oxygen consumption was measured polarographically and fluidity by the fluorescence polarization method. S-nitroso-N-acetylpenicillamine (SNAP) was used as a NO donor. It was observed that NO decreased IMMF fluidity and oxygen consumption in a concentration dependent fashion. However, SAM a fluidizing agent that prevented the decrement in fluidity produced by SNAP, failed to preserve oxygen consumption. Protection of sulfhydryl groups with dithiotreitol was utilized to evaluate the role of oxidation of these groups on IMMF respiration. Incubation with dithiotreitol did not preserve IMMF oxygen consumption. The data shown herein suggest that NO inhibits the respiratory chain by a mechanism not involving the modulation of membrane fluidity or the oxidation of sulfhydryl groups. Thus, it seems that the mechanism by which NO modulates mitochondrial respiration is by cytochrome oxidase inhibition, because (as reported by others) low concentrations of NO specifically inhibit reversibly cytochrome oxidase in competition with oxygen.

Programmed cell death as a prognostic indicator for radiation therapy in cervical carcinoma patients: A pilot study

In clinical practice, radiation therapy often fails in cervical carcinoma stage IIIB and there is a need to develop a predictive assay for prognosis of radiation treatment outcome in cancer patient. We have attempted to evaluate the relevance of changes in Membrane Fluidity (MF) and associated apoptotic cell death in cervical cancer cells after first fractionated dose of radiation therapy to treatment outcome of stage IIIB cervical carcinoma patients. Biopsies of 15 patients with histologically proven cervix cancer were collected from the patients before and 24 h after first fractionated radiation dose of 2 grays (Gy). Cell suspension made in Dulbecco's Modified Eagle's Medium (DMEM) were used for further investigations and cell suspension of cervix cancer patient were used to measure MF by fluorescence polarization method and apoptotic index (AI) was determined by Tdt dUTP Nucleotide End Labeling (TUNEL) assay. A substantial increase in MF and AI was observed in cervical cancer cells irradiated ex vivo . A significant correlation ( P P > 0.1) was detected between changes in MF and treatment outcome of patients. Preliminary results showed significant change in MF and a marked increase in percentage apoptosis of cervix cancer cells irradiated ex vivo . The changes in AI after first fractionated dose of radiotherapy in cervical carcinoma patients may provide a predictor of prognosis for radiotherapy in uterine cervical carcinoma patients.

Protein self-association in crowded protein solutions: a time-resolved fluorescence polarization study.

The self-association equilibrium of a tracer protein, apomyoglobin (apoMb), in highly concentrated crowded solutions of ribonuclease A (RNase A) and human serum albumin (HSA), has been studied as a model system of protein interactions that occur in crowded macromolecular environments. The rotational diffusion of the tracer protein labeled with two different fluorescent dyes, 8-anilinonaphthalene-1-sulfonate and fluorescein isothiocyanate, was successfully recorded as a function of the two crowder concentrations in the 50-200 mg/mL range, using picosecond-resolved fluorescence anisotropy methods. It was found that apoMb molecules self-associate at high RNase A concentration to yield a flexible dimer. The apparent dimerization constant, which increases with RNase A concentration, could also be estimated from the fractional contribution of monomeric and dimeric species to the total fluorescence anisotropy of the samples. In contrast, an equivalent mass concentration of HSA does not result in tracer dimerization. This different effect of RNase A and HSA is much larger than that predicted from simple models based only on the free volume available to apoMb, indicating that additional, nonspecific interactions between tracer and crowder should come into play. The time-resolved fluorescence polarization methods described here are expected to be of general applicability to the detection and quantification of crowding effects in a variety of macromolecules of biological relevance.

Fluorescence anisotropy as a probe to study tracer proteins in crowded solutions.

Fluorescence anisotropy has been widely used to study the dynamics and interactions of biomolecules in diluted solutions. Comparable studies on single tracer macromolecules at the cellular level are now feasible because of the recent development of non-invasive fluorescence markers, like the growing family of the green fluorescence proteins (GFPs), and the advances in time-resolved fluorescence microscopy instrumentation. The interpretation of fluorescence polarization data in terms of dynamics and biological function of the macromolecular complexes in these physiological environments requires a deep understanding of the tracer rotational diffusion in such complex media. In this work we have studied the rotational diffusion of a tracer protein, apomyoglobin labeled with 1-anilino-8-naphthalene sulfonate, in crowded solutions of an unrelated protein, ribonuclease A. We have evaluated the deviation of the different tracer rotational motions from the Stokes-Einstein-Debye diffusion behavior, and its relation to the properties of the transient molecular cavities where the tracer is rotating in the fluorescence lifetime window. Finally, we have analyzed the application of fluorescence polarization methods to determine the apparent equilibrium constants of homo and hetero-associations of macromolecules in crowded conditions.

Diagnosis of Militarily Relevant Diseases Using Oral Fluid and Saliva Antibodies: Fluorescence Polarization Immunoassay

This laboratory is developing fluorescence polarization (FP) methods as diagnostic tools to assay antibodies in saliva and other oral fluids. FP provides quantitation of molecular interaction, such as antigen-antibody binding, of a single, small-volume sample in real time and without prior separation of components such as blood cells. There is potential for wide-spread use of these homogeneous assays as noninvasive tests, especially as more compact, simplified fluorescence polarimeters become available. FP tests can be designed that are applicable to a wide spectrum of microorganisms and may be used in a clinic or far-forward deployed setting to aid in diagnosis of disease or verification of vaccination. Rapid salivary diagnostics, including FP, have been identified by the Office of Naval Research as requirements for future naval capabilities in basic and applied medical research for warfighter protection in casualty prevention. The applications of FP salivary diagnostics for determination of tuberculosis exposure and of anthrax immunization status are discussed as examples.

High-throughput methods to detect dimerization of Bcl-2 family proteins

Members of the Bcl-2 family are critical regulators of apoptosis. Antiapoptotic family proteins such as Bcl-2 and Bcl-x L function, at least in part, by binding proapoptotic members such as Bax and Bak and thereby preventing release of apoptotic proteins, including cytochrome c, from the mitochondria. “BH3-only” members of the family disrupt this interaction by binding, via their BH3 domain, to a hydrophobic pocket on the surface of the antiapoptotic members. Disruption of heterodimerizations by small-molecule inhibitors could be used to modulate cell death in both cancer (to increase apoptosis) and degenerative disorders (to decrease apoptosis), and assays are necessary to screen compound libraries. Fluorescence polarization and enzyme-linked immunosorbent assay-based methods to detect Bcl-2 protein interactions have been described. Here, two further methods that are rapid, “mix and read,” homogeneous reactions, insensitive to compound autofluorescence, and amenable to high-throughput screening, are described: a scintillation proximity assay and a time-resolved fluorescence resonance energy transfer assay (HTRF). The assays are designed using tags such that different Bcl-2 family members or BH3 domain peptides can be readily applied to either format, as exemplified by the use here of histidine-tagged Bcl-x L and biotinylated BH3 peptides.

Physicochemical characterization of branched chain polymeric polypeptide carriers based on a poly-lysine backbone.

A systematic study is reported on the physicochemical characteristics of two branched chain polymers (based on a poly-L-lysine backbone) with a general formula poly[Lys-(DL-Alam-Xi)], where X = Orn (OAK) or N-acetyl-Glu (Ac-EAK) and m approximately equal to 3, using surface pressure and fluorescence polarization methods. These data are compared with those of the linear poly(L-Lys) from which OAK and Ac-EAK are derived. These two polymers show a moderate surface activity, able to form stable monomolecular layers at the air-water interface. Poly(L-Lys), the most hydrophilic, has the lowest surface activity. The interaction of these polymers with phospholipid bilayers either neutral or negatively charged was studied with vesicles labeled with two fluorescent probes: ANS and DPH. Results indicate that these polymers are able to accommodate in their internal structure, mainly through electrostatic interactions, a certain amount of ANS marker molecules, but fluorescence increases of the ANS-polypeptide complexes were so low that its influence in further polarization measurements could be discarded. After interaction with liposomes, these polymers induce an increase in the polarization of the probes, thus indicating a rigidification of the bilayers. Electrostatic forces seem to be very important in this interaction; cationic polymers are clearly more active, with PG-containing liposomes, than Ac-EAK. Moreover, in these assays poly(L-Lys) behaves as the more active compound. This fact is probably due to its major ability to form alpha-helical structures that could insert easily in the bilayers. These results indicate that the polymeric structures studied can be used as carriers for biologically active molecules, because their interactions with bilayers remain soft and have a positive effect on the stability of the membranes.

Surface Orientation of Fibrils in Cotton Fiber Measured by the Fluorescence Polarization Method

Surface Orientation of Fibrils in Cotton Fiber Measured by the Fluorescence Polarization Method

Cyclosporine and its metabolites before and 2 h post-dose: comparative measurements of a monoclonal and a polyclonal immunoassay.

The aim of the study was to investigate the better accuracy of the 2-h post-dose (C2) levels of cyclosporine (CyA), compared with the pre-dose (C0) levels and to evaluate the results measured by a monoclonal or a polyclonal immunoassay. The parent compound of CyA in C2 (monoclonal2) was measured in 53 kidney transplant patients by the monoclonal fluorescence polarization method, as well as the parent compound plus metabolites (polyclonal2) by the polyclonal fluorescence polarization method. Also, the parent compound was measured in 21 of the patients for the C0 (monoclonal0), whereas the parent compound plus metabolites in 36, for the C0 (polyclonal0). As level of metabolites was considered the difference between polyclonal and monoclonal values (polyclonal-monoclonal), either in C0 (metabolites0) or in C2 (metabolites2). The ratio polyclonal2/monoclonal2 gave a mean value of 1.7+/-0.2 (mean+/-SD), whereas the mean value of the ratio polyclonal0/monoclonal0 was 2.3+/-0.6, with almost double variation. The mean value of the ratio metabolites2/monoclonal2 was 0.7+/-0.2 and of the ratio metabolites0/monoclonal0 was 1.3+/-0.6. The difference between the two ratios is very significant (p = 0.000001) and they are not correlated with each other (r = 0.18, p = 0.44). The measurements of monoclonal0 and polyclonal0 or monoclonal2 and polyclonal2 are very significantly correlated (r = 0.94, p = 0.000001 and r = 0.97, p = 0.000001, respectively). In C0 the proportion of metabolites is higher than in C2, with a double variation, as the degree of metabolism is diverse. Consecutively, in monoclonal methods, as cross-reactions occur with metabolites, it is more accurate to use the C2 measurement for the evaluation of CyA. The application of both methods, the polyclonal and the monoclonal, could be a useful tool as it gives an estimation of metabolites whose degree of contribution to the immunosuppressive result is difficult to ascertain. Finally, if for reasons of clinical experience, the polyclonal method is used, then the mean therapeutic levels of polyclonal2 are 1.5-1.7 compared with monoclonal2.