Increase In Fluorescence Polarization Research Articles

Multi-spectroscopic and HPLC Studies of the Interaction Between Estradiol and Cyclophosphamide With Human Serum Albumin: Binary and Ternary Systems

Drug resistance is a phenomenon that frequently impairs a proper treatment of infections and cancer with chemotherapy. Multidrug efflux transporters extrude structurally dissimilar organic compounds often providing resistance to multiple toxic chemotherapeutic agents. The quantitative analysis of drug efflux requires measuring the affinity of ligands. In this work, the interaction between cyclophosphamide (Cyc) and estradiol (ES) with human serum albumin (HSA) was studied by fluorescence polarization, circular dichroism and high-performance liquid chromatography (HPLC) under physiological conditions (pH = 7.4). Gradual addition of HSA led to a marked increase in fluorescence polarization. Our assays indicated that the protein was bound to these drugs with different K d. Also, the Hill coefficient showed a simple drug binding process with no cooperativity. Circular dichroism results revealed the occurrence of conformational changes in HSA molecules by the binding of Cyc and ES. The protein binding of the drug was studied by HPLC. Our results indicated that the drug was bound to the protein and that the presence of a second drug affected the interaction and resistance between the first drug and the protein.

A cyclodextrin-based polymer for sensing diclofenac in water

An assay for the determination of diclofenac concentration, in the micromolar range in water, was developed. It is based on the use of a recently developed cyclodextrin-based polymer that possesses an inherent affinity for the target pharmaceutical. This competitive assay is exploiting the possibility to displace a fluorescent dye, adsorbed in the cyclodextrin-based polymer, by the target drug. This displacement is followed by measuring the increase in fluorescence polarization of the dye released in solution. The assay was successfully tested on a real wastewater sample with a limit of detection of 1μM.

Fluorescence polarization assay to quantify protein-protein interactions: an update.

A fluorescence polarization assay can be used to evaluate the strength of a protein-protein interaction. A green fluorescent protein variant is fused to one of the protein partners. The formation of a complex is then deduced from an increase in fluorescence polarization, and the equilibrium dissociation constant of the complex is determined in a homogeneous aqueous environment. The assay is demonstrated by using the interaction of the S-protein and S-peptide fragments of ribonuclease A as a case study.

Dodecyl and octyl esters of fluorescein as protonophores and uncouplers of oxidative phosphorylation in mitochondria at submicromolar concentrations

In our search for fluorescent uncouplers of oxidative phosphorylation, three esters of fluorescein, n-butyl-, n-octyl-, and n-dodecyl-oxycarbonyl-fluorescein (C4-FL, C8-FL, C12-FL) were synthesized and characterized. With increasing liposomal lipid content, the long-chain alkyl derivatives of fluorescein (C8-FL, C12-FL and commercially available C18-FL), but not C4-FL and unsubstituted fluorescein, exhibited an increase in fluorescence polarization reflecting the dye binding to liposomes. C12-FL induced proton permeability in lipid membranes, while C4-FL was inactive. In contrast to C4-FL and C18-FL, C12-FL and C8-FL increased the respiration rate and decreased the membrane potential of isolated rat liver mitochondria with half-maximal effective concentrations of 700nM and 300nM, respectively. The effect of Cn-FL on the respiration correlated with that on proton permeability of the inner mitochondrial membrane, as measured by induction of mitochondria swelling in the potassium acetate medium. Binding of C8-FL to mitochondria depended on their energization, which was apparently associated with pH gradient generation across the inner mitochondrial membrane in the presence of a respiratory substrate. In wild-type yeast cells, C12-FL localized predominantly in plasma membrane, whereas in AD1-8 mutants lacking MDR pumps, it stained cytoplasmic organelles with some preference for mitochondria. Fluorescent uncouplers can be useful as a tool for determining their localization in a cell or distribution between different tissues in a living animal by fluorescent microscopy.

Conformational Dynamics in a Truncated Epidermal Growth Factor Receptor Ectodomain

Structural studies have revealed two forms of the monomeric epidermal growth factor receptor (EGFR) ectodomain: a compact (tethered) form stabilized by interdomain interactions and an extended (untethered) form in the presence of ligand. An important question is whether the ligand induces a conformational transition from a tethered to untethered form or whether there is a preexisting conformational equilibrium between tethered and untethered states. To distinguish between these two possibilities, we investigated a truncated receptor, EGFR501 (spanning residues 1-501), that contains the minimal elements required for high-affinity ligand binding in solution. Conformational transitions and dynamics were inferred by means of fluorescence from five internal tryptophan residues that are located within or close to the ligand-binding domains of EGFR501. A preexisting conformational equilibrium between tethered and untethered states in EGFR501 was deduced from (1) the nonlinear Arrhenius temperature dependence of fluorescence and (2) fluorescence polarization showing independently mobile domains. In contrast, the ligand-EGFR501 complex revealed a linear Arrhenius temperature dependence of fluorescence and increased fluorescence polarization due to a lack of significant interdomain motions. The data suggest that the role of the ligand is to trap the EGFR501 in the untethered state that is transiently formed in solution through a preexisting conformational equilibrium.

O6-Methylguanine-DNA Methyltransferase Gene Promoter Methylation Detection in Glioma Tumors by a Novel Fluorescence Polarization Assay

O⁶-Methylguanine-DNA methyltransferase methylation status has a very good predictive value for benefit from alkylating agent therapy. The stratified therapy assignment of patients according to the O⁶-Methylguanine-DNA methyltransferase methylation status requires a standardized diagnostic test. A novel method detecting the promoter methylation status of O⁶-Methylguanine-DNA methyltransferase in tissue samples by a fluorescence polarization assay was developed. A pair of primers was used to amplify a 266 bp fragment in the promoter region of the O⁶-Methylguanine-DNA methyltransferase gene. Two probes specific for either methylated or unmethylated DNA labeled with different fluorophores hybridized with their target amplicons, and the hybridization increased the fluorescence polarization values. The methylation status was determined by the increased fluorescence polarization values. Ninety-seven glioma tumor samples were analyzed in parallel with the new assay and the nested gel-based methylation-specific polymerase chain reaction assay. The results of the methylation status of the fluorescence polarization assay were in good concordance with the results obtained with the nested gel-based methylation-specific polymerase chain reaction assay. The sensitivity and stability of the fluorescence polarization assay have been measured. The coefficient of variation of the reproducibility for the fluorescence polarization assay was <10%. The minimum detection level established with the fluorescence polarization assay was 20 copies/μL. The fluorescence polarization assay allowed the discrimination of the O⁶-Methylguanine-DNA methyltransferase methylation status at individual CpG sites directly in the solution without the 2-step approach with nested primers.

Analysis of Escherichia coli cell damage induced by HPCD using microscopies and fluorescent staining

Cellular damage of Escherichia coli ( E. coli) induced by high pressure carbon dioxide (HPCD) at 37–57 °C and 10–30 MPa for 5–75 min was investigated using scanning electronic microscopy (SEM), transmission electronic microscopy (TEM), confocal laser scanning microscopy (CLSM), and fluorospectrophotometer (FSM). HPCD-induced alterations in the morphology and the intracellular organization of E. coli cells was more susceptible to HPCD. A vast majority of HPCD-treated E. coli cells with seemingly intact morphology sustained severe damage in their intracellular organization. CLSM suggested that initial disruption of the outer membrane and later permeabilization of the cytoplasmic membrane of HPCD-treated E. coli cells was a consecutive and progressive process. These results were confirmed by FSM with the probes PI and SYTO 9. The membrane fluidity of HPCD-treated E. coli cells decreased as suggested by increased fluorescence polarization using FSM with the probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The temperatures of 37, 42 and 47 °C alone showed no impact on the outer membrane and membrane fluidity of E. coli cells whereas 57 °C alone had greater impact on them. Combined with HPCD, the temperatures of 37, 42 and 47 °C disrupted the outer membrane of E. coli cells without damage to the cytoplasmic membrane and of 57 °C damaged the cytoplasmic membrane, but all these temperatures decreased the membrane fluidity of E. coli cells. Higher temperature increased HPCD-induced outer membrane disruption and the cytoplasmic membrane damage and decreased the membrane fluidity.

Detection of the four major human herpesviruses simultaneously in whole blood and cerebrospinal fluid samples by the fluorescence polarization assay

Herpes simplex virus type 1/2 (HSV-1/-2), cytomegalovirus (CMV), and Epstein-Barr virus (EBV) correlate strongly with infections of the central nervous system. The objective of this study was to develop a method for the simultaneous detection of HSV-1/-2, CMV, and EBV DNA by the fluorescence polarization assay based on asymmetric polymerase chain reaction (PCR) and hybridization. DNA of HSV-1/-2, CMV, and EBV was amplified in an asymmetric PCR by a universal primer system. The amplicons were then detected by the fluorescence polarization assay. In this method, the probes for HSV-1/-2, CMV, and EBV hybridized with their respective target amplicons, and the hybridization resulted in an increase in the fluorescence polarization values. Infections of HSV-1/-2, CMV, and EBV were determined by the increased fluorescence polarization values. The DNA extracted from whole blood and cerebrospinal fluid samples was subjected to fluorescence polarization and a previously published multiplex PCR assay in parallel. Compared to the multiplex PCR assay, no significant difference in the numbers of samples positive for the human herpesviruses was identified by the fluorescence polarization assay. The fluorescence polarization assay presented in this study is a reliable, convenient, and cost-effective diagnostic tool that allows the detection of the four major human herpesviruses.

Detection of hepatitis B virus genotypes A to D by the fluorescence polarization assay based on asymmetric PCR

The hepatitis B virus genotypes A and D have global distributions, while the hepatitis B genotypes B and C are predominant in Asia. Individuals infected with genotype C or D have a lower response rate to therapy than individuals infected with genotype A or B. The conventional detections of hepatitis B genotypes A to D, however, have not been used routinely due to technical difficulties and high costs. A simple and cost-effective method for simultaneous detection of the hepatitis B genotypes A to D has been developed. A pair of general primers in the preS region of the hepatitis B were used in an asymmetric PCR. Four probes specific for the hepatitis B genotypes A to D labeled with different fluorophores hybridized, respectively with their target amplicons, and the hybridization increased the fluorescence polarization (FP) values. The genotypes were determined by the increased fluorescence polarization values. DNA extracted from 1398 samples was detected by the FP and the type-specific PCR assay in parallel. No significance difference was found between the two methods.

The damage of outer membrane of Escherichia coli in the presence of TiO 2 combined with UV light

The biological consequences of exposure to TiO 2, UV light, and their combined effect were studied on the Escherichia coli ( E. coli) cells. The damage of outer membrane was observed for the cells after treatment of TiO 2 or UV light. TiO 2 alone can break down lipopolysacchride (LPS), the outermost layer of the E. coli cells, but was not able to destroy peptidoglycan underneath. The same phenomenon was observed for E. coli under 500 W UV light treatment alone. However, the outer membrane of E. coli could be removed completely in the presence of both TiO 2 and UV light, and the cells became elliptical or round without a mechanically strong network. From the analysis of the concentrations for Ca 2+ and Mg 2+, a large amount of Ca 2+ and Mg 2+ were detected in the solution of the treated cells by photo-catalysis, and this was attributed to the damage of LPS dispatches. After TiO 2 or UV light treatment, a significant decrease in membrane fluidity of E. coli was found from an increase in fluorescence polarization by a fluorescence probe. The permeability of the treated cells increased to some degree that can be confirmed by quantum dots labeling technique.

Design, Synthesis, and Pharmacology of Fluorescently Labeled Analogs of Serotonin: Application to Screening of the 5-HT2C Receptor

Novel fluorescent derivatives of serotonin have been synthesized and used as tracers for the development of a 5-HT2C fluorescence polarization assay. Serotonin analogs that feature a fluorescent probe attached through an ether linkage at the tryptamine 5-position have high affinity for the 5-HT2C receptor, and affinity is dependent on both linker length and pendent dye. These variables have been optimized to generate Cy3B derivative 5a, which has 10-fold higher 5-HT2C affinity relative to serotonin (Kd=0.23 nM). In receptor activation experiments, 5a acts as a full agonist of 5-HT2C. Upon binding to 5-HT2C cell membranes, 5a shows a robust increase in fluorescence polarization (FP) signal. In an FP binding assay using 5a as a tracer ligand, Ki values for known 5-HT2C agonists and antagonists showed excellent agreement with Ki values from radioligand binding (r2=0.93). The FP ligand assay is suitable for high-throughput drug screening applications with respect to speed of analysis, displaceable signal, precision, and sensitivity to various reagents. A 384-well-based high-throughput assay that is rapid, economical, and predictive of test compounds' ability to bind to the 5-HT2C receptor has been compiled and validated.

Effect of Genistein on Antioxidative Defence System and Membrane Fluidity in Chick Skeletal Muscle Cells

; This study was conducted to investigate the protective effect of genistein on the antioxidative defence system and membrane fluidity in chick skeletal muscle cells after supplementation with 0, 20, 40, and 80 μmol/L genistein in 50 μmol/L FeSO 4 / H 2 O 2 treated cells for 24 h. Genistein supplementation recovered the decreased activity of total superoxide dismutase induced by FeSO4/ H 2 O 2 , significantly increased glutathione peroxidase activity (p<0.05) and decreased malondialdehyde production (p<0.05). The treatment of 80 μmol/L genistein in FeSO 4 /H 2 O 2 treated cells decreased the secretion of creatine kinase (p<0.05). Fluorescence polarization values and microviscosities observed with FeSO 4 /H 2 O 2 treated cells were significantly higher than those observed with no FeSO 4 /H 2 O 2 treated cells. The addition of 80 μmol/L genistein improved the increased fluorescence polarization value (p<0.05) caused by FeSO 4 /H 2 O 2 treatment. The microviscosity value was significantly decreased by adding genistein (p<0.05). In conclusion, genistein protected skeletal muscle cells from oxidative damage by improving antioxidative status and membrane fluidity.

Molecular Chaperones and the Assembly of the Prion Ure2p in Vitro

The protein Ure2 from Saccharomyces cerevisiae possesses prion properties at the origin of the [URE3] trait. In vivo, a high molecular weight form of inactive Ure2p is associated to [URE3]. The faithful and continued propagation of [URE3]is dependent on the expression levels of molecular chaperones from the Hsp100, -70, and -40 families; however, so far, their role is not fully documented. Here we investigate the effects of molecular chaperones from the Hsp40, Hsp70, Hsp90, and Hsp100 families and the chaperonin CCT/Tric on the assembly of full-length Ure2p. We show that Hsp104p greatly stimulates Ure2p aggregation, whereas Ssa1p, Ydj1p, Sis1p, and Hsp82p inhibit aggregation to different extents. The nature of the high molecular weight Ure2p species that forms in the presence of the different molecular chaperones and their nucleotide dependence is described. We show that Hsp104p favors the aggregation of Ure2p into non-fibrillar high molecular weight particles, whereas Ssa1p, Ydj1p, Sis1p, and Hsp82p sequester Ure2p in spherical oligomers. Using fluorescently labeled full-length Ure2p and Ure2p-(94-354) and fluorescence polarization, we show that Ssa1p binding to Ure2p is ATP-dependent, whereas that of Hsp104p is not. We also show that Ssa1p preferentially interacts with the N-terminal domain of Ure2p that is critical for prion propagation, whereas Ydj1p preferentially interacts with the C-terminal domain of the protein, and we discuss the significance of this observation. Finally, the affinities of Ssa1p, Ydj1p, and Hsp104p for Ure2p are determined. Our in vitro observations bring new insight into the mechanism by which molecular chaperones influence the propagation of [URE3].

Spectroscopic studies on the interaction between CdTe nanoparticles and lysozyme

Nanoparticles of cadmium telluride (CdTe) coated with thioglycolic acid (TGA) were prepared in the water phase. The interaction between CdTe nanoparticles (NPs) and lysozyme (Lyz) was investigated by fluorescence and circular dichroism (CD) spectroscopy at pH 7.40. It was proved that the fluorescence quenching of Lyz by CdTe NPs was mainly a result of the formation of CdTe–Lyz complex. By the fluorescence quenching results, the Stern–Volmer quenching constant ( K SV), binding constant ( K a) and binding sites ( n) were calculated. The binding distance ( r) between Lyz (the donor) and CdTe NPs (the acceptor) was obtained according to fluorescence resonance energy transfer (FRET). Gradual addition of CdTe NPs to the solution of Lyz led to a marked increase in fluorescence polarization ( P) of Lyz, which indicated that CdTe NPs were located in a restricted environment of Lyz. The effect of CdTe NPs on the conformation of Lyz has been analyzed by means of synchronous fluorescence spectra and CD spectra, which provided the evidence that the secondary structure of Lyz has been changed by the interaction of CdTe NPs with Lyz.

Displacement of protein-bound aptamers with small molecules screened by fluorescence polarization

Small molecule inhibitors of proteins are invaluable tools in research and as starting points for drug development. However, their screening can be tedious, as most screening methods have to be tailored to the corresponding drug target. Here, we describe a detailed protocol for a modular and generally applicable assay for the identification of small organic compounds that displace an aptamer complexed to its target protein. The method relies on fluorescence-labeled aptamers and the increase of fluorescence polarization upon their binding to the target protein. The assay has high Z'-factors, making it compatible with high-throughput screening. It allows easy automation, making fluorescence readout the time-limiting step. As aptamers can be generated for virtually any protein target, the assay allows identification of small molecule inhibitors for targets or individual protein domains for which no functional screen is available. We provide the step-by-step protocol to screen for antagonists of the cytohesin class of small guanosine exchange factors.

Fluorescence polarization immunoassays and related methods for simple, high-throughput screening of small molecules

Fluorescence polarization immunoassay (FPIA) is a homogeneous (without separation) competitive immunoassay method based on the increase in fluorescence polarization (FP) of fluorescent-labeled small antigens when bound by specific antibody. The minimum detectable quantity of FPIAs with fluorescein label (about 0.1 ng analyte) is comparable with chromatography and ELISA methods, although this may be limited by sample matrix interference. Because of its simplicity and speed, FPIA is readily automated and therefore suitable for high-throughput screening (HTS) in a variety of application areas. Systems that involve binding of ligands to receptor proteins are also susceptible to analysis by analogous FP methods employing fluorescent-labeled ligand and HTS applications have been developed, notably for use in candidate drug screening.

Fluorescently Labeled Analogues of Dofetilide as High-Affinity Fluorescence Polarization Ligands for the Human Ether-a-go-go-Related Gene (hERG) Channel

Novel fluorescent derivatives of dofetilide (1) have been synthesized. Analogues that feature a fluorescent probe attached through an aliphatic spacer to the central tertiary nitrogen of 1 have high affinity for the hERG channel, and affinity is dependent on both linker length and pendent dye. These variables have been optimized to generate Cy3B derivative 10e, which has hERG channel affinity equivalent to that of dofetilide. When bound to cell membranes expressing the hERG channel, 10e shows a robust increase in fluorescence polarization (FP) signal. In a FP binding assay using 10e as tracer ligand, Ki values for several known hERG channel blockers were measured and excellent agreement with the literature Ki values was observed over an affinity range of 2 nM to 3 muM. 10e blocks hERG channel current in electrophysiological patch clamp experiments, and computational docking experiments predict that the dofetilide core of 10e binds hERG channel in a conformation similar to that previously predicted for 1. These analogues enable high-throughput hERG channel binding assays that are rapid, economical, and predictive of test compounds' potential for prolonged QT liabilities.

In Vitro Fluorescence Anisotropy Analysis of the Interaction of Full-length SRC1a with Estrogen Receptors α and β Supports an Active Displacement Model for Coregulator Utilization

Binding of full-length P160 coactivators to hormone response element-steroid receptor complexes has been difficult to investigate in vitro. Here, we report a new application of our recently described fluorescence anisotropy microplate assay to investigate binding and dissociation of full-length steroid receptor coactivator-1a (SRC1a) from full-length estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta) bound to a fluorescein-labeled (fl) estrogen response element (ERE). SRC1a exhibited slightly higher affinity binding to flERE.ERbeta than to flERE.ERalpha. Binding of SRC1a to flERE.ERalpha and to flERE.ERbeta was 17beta-estradiol (E2)-dependent and was nearly absent when ICI 182,780, raloxifene, or 4-hydroxytamoxifen were bound to the ERs. SRC1a binds to flERE.E2-ERalpha and flERE.E2-ERbeta complexes with a t1/2 of 15-20 s. Short LXXLL-containing nuclear receptor (NR) box peptides from P160 coactivators competed much better for SRC1a binding to flERE.E2-ER than an NR box peptide from TRAP220. However, approximately 40-250-fold molar excess of the P160 NR box peptides was required to inhibit SRC1a binding by 50%. This suggests that whereas the NR box region is a primary site of interaction between SRC1a and ERE.E2-ER, additional contacts between the coactivator and the ligand-receptor-DNA complex make substantial contributions to overall affinity. Increasing amounts of NR box peptides greatly enhanced the rate of dissociation of SRC1a from preformed flERE.E2-ER complexes. The data support a model in which coactivator exchange is facilitated by active displacement and is not simply the result of passive dissociation and replacement. It also shows that an isolated coactivator exhibits an inherent capacity for rapid exchange.

Isolation of a High-Affinity Functional Protein Complex between OmcA and MtrC: Two Outer Membrane Decaheme c -Type Cytochromes of Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a facultatively anaerobic bacterium capable of using soluble and insoluble forms of manganese [Mn(III/IV)] and iron [Fe(III)] as terminal electron acceptors during anaerobic respiration. To assess the structural association of two outer membrane-associated c-type decaheme cytochromes (i.e., OmcA [SO1779] and MtrC [SO1778]) and their ability to reduce soluble Fe(III)-nitrilotriacetic acid (NTA), we expressed these proteins with a C-terminal tag in wild-type S. oneidensis and a mutant deficient in these genes (i.e., Delta omcA mtrC). Endogenous MtrC copurified with tagged OmcA in wild-type Shewanella, suggesting a direct association. To further evaluate their possible interaction, both proteins were purified to near homogeneity following the independent expression of OmcA and MtrC in the Delta omcA mtrC mutant. Each purified cytochrome was confirmed to contain 10 hemes and exhibited Fe(III)-NTA reductase activity. To measure binding, MtrC was labeled with the multiuse affinity probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (1,2-ethanedithiol)2, which specifically associates with a tetracysteine motif engineered at the C terminus of MtrC. Upon titration with OmcA, there was a marked increase in fluorescence polarization indicating the formation of a high-affinity protein complex (Kd < 500 nM) between MtrC and OmcA whose binding was sensitive to changes in ionic strength. Following association, the OmcA-MtrC complex was observed to have enhanced Fe(III)-NTA reductase specific activity relative to either protein alone, demonstrating that OmcA and MtrC can interact directly with each other to form a stable complex that is consistent with their role in the electron transport pathway of S. oneidensis MR-1.

Comparison of the effects of selected chalcones, dihydrochalcones and some cyclic flavonoids on mitochondrial outer membrane determined by fluorescence spectroscopy

The effect on mitochondrial outer membrane of 4-hydroxychalcone ( 1), the cyclic chalcone analogues E-2-(4′-hydroxybenzylidene)-1-indanone ( 2a) and E-2-(4′-hydroxybenzylidene)-1-tetralone ( 2b), the dihydrochalcones phloretin ( 3a) and phloridzin ( 3b), the flavanones naringenin ( 4a) and naringin ( 4b), and the flavonol quercetin ( 5) was investigated by fluorescence spectroscopy. Excitation and emission fluorescence spectra of each flavonoid and synthetic analogue were recorded in respiration medium containing 1 mM succinate. Initial interaction of the compounds with the outer mitochondrial membrane was investigated by recording their fluorescence polarization in the presence of rat liver mitochondria. Most of the compounds displayed an elevated fluorescence polarization on mixing with mitochondria at the zero time point. During the investigated 20 min period the initial fluorescence polarization values remained constant ( 1, 2a), or a gradual depression of the measured polarization values could be observed ( 2b, 3a, 4b, 5). In the case of naringenin ( 4a), however, similar to the previously investigated seven-membered cyclic chalcone analogue E-2-(4´-methoxybenzylidene)-1-benzosuberone, a slight, continuous increase of fluorescence polarization could be detected during the 20 min experiment. Phloridzin ( 3b) showed an increased fluorescence polarization in first 10 min, which was slightly depressed by the 20 min time point.