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.     

Fluorescence polarization immunoassay of ractopamine

A technique was developed for fluorescence polarization immunoassay (FPIA) of ractopamine, a toxic low molecular weight nonsteroidal growth regulator belonging to the most controlled contaminants of food products of animal origin. The assay is based on the competition between a sample containing ractopamine and ractopamine–fluorophore conjugate for binding to antibodies. The competition is monitored via changes in the degree of fluorescence polarization for plane-polarized excitation light, which differs for the free and antibody-bound forms of the conjugate. The optimal assay conditions were established, ensuring a high accuracy and minimal detection limit. The developed assay demonstrated a detection limit of 1 ng/mL and a range of detectable concentrations of 2.3–50 ng/mL, which met the requirements of sanitary control. The duration of the analysis was 10 min. The possible application of the developed FPIA was demonstrated with testing of turkey meat. The speed and simplicity of the proposed assay define its efficiency as a screening tool for safety of foods.     

Fluorescence polarization: a novel indicator of cardiomyocyte contraction

The changes measured in intracellular fluorescein fluorescence polarization (IFFP) are used as a new tool for tracing cytoplasmic effects during contractile cycles of cardiac myocytes (1-2-day-old rat hearts), in addition to the established Ca(2+) monitoring and/or videometric methods of tracking cell-shortening. This novel method was found to be non-intrusive to the contraction cycles. The decay of the transient IFFP signal (from 0.220+/-0.01 to 0.170+/-0.013) seems to be closely related to the extended phase of contractile activation. This fact was further supported when Ca(2+) exchanger inhibitor was introduced and significantly decreased (90%) the rate of beats of contraction and IFFP, but not the Ca(2+) beat rate changes. This result suggests that the IFFP indicator is probably associated with the physiological activation, rather than with Ca(2+) alterations. The IFFP measure monitors the average of effective changes in the micro-viscosity of the cytoplasm protein matrix, associated with cellular activation.     

Fluorescence polarization for mycotoxin determination

Over the last few years several laboratories have reported fluorescence polarization (FP) immunoassays for mycotoxins. These have included assays for fumonisins, deoxynivalenol and acetylated derivatives, aflatoxins, ochratoxin A, and zearalenone and related metabolites. Sensitivity in the FP assays may change dramatically over time, depending upon the antibody/tracer combination used. An important aspect of these homogeneous assays is the time required to reach an equilibrium endpoint. Although it is counterintuitive, the sensitivity of FP assays can actually be improved with shorter incubation times. However, the need for sensitivity must often be balanced against the need for the analyst to reproducibly time the incubation. The technical acumen of the analyst would be relatively more important in assays where measurements are taken before the system reaches equilibrium. In many cases the desired assays are those which reach equilibrium (and therefore give a stable endpoint) quickly, which may occur at the expense of sensitivity. It is for this reason the FP immunoassays are frequently not as sensitive as traditional ELISAs. Nevertheless, for many of the major mycotoxins rapid FP immunoassays can be developed, provided the appropriate combinations of antibody and tracer are used. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005 Financial support: United States Department of Agriculture     

Fluorescence polarization study of DNA--proflavine complexes

The binding of proflavine to DNA has been studied by measuring the polarization and intensity of emission of DNA–dye complexes. Such measurements also permit the determination of the fluorescence of the bound dye as a function of the degree of binding. Techniques of emission spectroscopy permit the study of complexing at high phosphate to dye ratios, and we have examined complexes formed at up to 12,300:1 phosphates to dye. At high phosphate to dye ratios, we find that equilibrium plots of the binding data show only one type of binding. Reports in the literature of multiple binding constants are shown to be due to the incorrect assumption that the fluorescence of the bound dye is independent of the amount bound. The emission properties can be qualitatively accounted for by assuming that nearest-neighbor interaction between bound dyes quenches the fluorescence. We report that, within experimental error, the binding constant is insensitive to the base content of the DNA. The DNA-dye complexes show a temperature dependent depolarization, the cause of which is, as yet, unknown. Heat denaturation of the DNA–dye complex may be followed on a Perrin plot.     

Fluorescence polarization as a tool to study lectin-sugar interaction

The binding ofRicinus communis agglutinin andAbrus agglutinin to 4-methylumbelliferyl β-D-galactopyranoside was studied by equilibrium dialysis, fluo-rescence quenching and fluorescence polarization. The number of binding sites and the association constant value obtained by fluorescence polarization for bothRicinus communis agglutinin andAbrus agglutinin are in close agreement with those obtained by the other methods. This indicates the potential of ligand-fluorescence polarization measurements in the investigation of lectin-sugar interactions.     

The fluorescein-mediated interaction of bovine serum albumin with fluorescent derivatives of prolactin and other polypeptides in polarization of fluorescence based assays

During the course of studies relating to the interaction of bovine prolactin with its receptor, it was observed that the fluorescence polarization of prolactin labeled with fluorescein isothiocyanate (fluorescein prolactin) increased from 0.10 to 0.15 upon the addition of bovine serum albumin. Dilution titration measurements show an apparent K_dissociation for the BSA-fluorescein-prolactin complex of 1.1 × 10^?7 M. The stoichiometry of the complex was shown to be approximately 2 mol of fluorescein-prolactin per mole of BSA. The fluorescence emission spectra of the fluorescein moiety in the fluorescein-prolactin is slightly red shifted and increased in intensity in the presence of BSA. The interaction between prolactin and BSA is dependent on the fluorescein attached to the prolactin since [¹²⁵I]prolactin does not form a complex with BSA under identical conditions. The fluorescence polarization of fluorescein-labeled growth hormone and α-lactalbumin also increased in the presence of BSA, suggesting that BSA may interact generally with fluorescein-labeled proteins to form complexes bridged through the fluorescein moiety.     

Fluorescence polarization studies on myosin-substrate interaction

The degree of polarization of the intrinsic fluorescence of purified myosin was estimated. On addition of ATP, polarization of the fluorescence of myosin increased when excited at wavelengths longer than 300 nm. In kinetic studies, coupled with the decay of the increased intensity of fluorescence of myosin, the increased polarization of the fluorescence decreased when the ATP was depleted. The decay of the increased polarization of fluorescence of myosin was specific to MgATP. According to the theory of polarization of the fluorescence of proteins, it is likely that some tryptophan residues of myosin, which are responsible for the increase in the fluorescence intensity and polarization when myosin interacts with substrates, reduce their local freedom of rotation.     

Fluorescence polarization applied to cellular microrheology

The fluorescence polarization of probe molecules gives information on the "fluidity" of probe environment. Although the data cannot be related with absolute values of microviscosity, the method is largely used for probing the "fluidity" of lipid regions of biological membranes. Therefore, fluorescence polarization is of interest in clinical research, for membrane alterations are associated with either pathological processes of red cells, platelets, leukocytes or important cell functions.     

Fluorescence polarization immunoassay employing immobilized antibody

The use of an antibody immobilized on latex or silver colloid in fluorescence polarization immunoassay (FPI) is assessed. In FPI it is possible to detect antigens of high molecular weight because the molecular weight of the antibody is effectively increased. In the assay for rabbit immunoglobulin G a limit of detection lower by two orders of magnitude and an assay range wider by one order of magnitude can be obtained in comparison with conventional FPI. The detection limit is 10(-10) mol l-1 and the total assay time for one sample is 8 min. This assay combines a low detection limit with a short assay time.     

Fluorescence imaging using a fluorescent protein with a large Stokes shift

Keima is a far-red fluorescent protein endowed with a large Stokes shift. It absorbs light maximally at around 440 nm and emits maximally at around 620 nm. While the original Keima is obligately tetrameric (tKeima), the dimeric and monomeric versions (mKeima and dKeima, respectively) have been generated. More recently, a tandem dimer of Keima (tdKeima) has been developed as the brightest version. Here we describe examples, which show the usefulness of Keima for dual-color fluorescence imaging technologies, such as fluorescence cross-correlation spectroscopy (FCCS) and two-photon laser scanning microscopy (TPLSM). Keima can be used in conjunction with existing fluorescent proteins in which the Stokes shift is much smaller, with the idea that while two fluorescent proteins are excited by a single laser each will fluoresce a different color.     

Fluorescence polarization competition assay: the range of resolvable inhibitor potency is limited by the affinity of the fluorescent ligand

For the development of fluorescence polarization (FP) competition assays, there is a widespread belief that tight-binding fluorescent ligands should be avoided to identify inhibitors of low or intermediate potency in the screening of small-molecule compound libraries. It is demonstrated herein that this statement is a misconception; in fact, the higher the affinity of the fluorescent ligand, the wider the range of inhibitor potency that can be resolved. An approximate estimate for the low end of inhibitor K(i) values that can be resolved is the K(d) value of the fluorescent ligand. Because FP competition assays are typically conducted under nonstoichiometric titration conditions, it is suggested that a fluorescent ligand of highest affinity that also has an adequate quantum yield to satisfy such conditions be selected.     

A tubulin-based fluorescent polarization assay for paclitaxel

This paper reports the development, characterization, and application of a separation-free, homogeneous, and simple to perform fluorescence polarization bioassay for paclitaxel. The bioassay is based on the binding interaction of paclitaxel to tubulin, the receptor protein on which this drug acts. The bioassay was carried out in a competitive format where the paclitaxel and a synthetic fluorescent-labeled paclitaxel (Rh-Tx) competed for tubulin binding, causing a change in fluorescence polarization, which was an inverse function of the paclitaxel concentration. The bioassay had a dynamic range from 0.03 to 0.35 microM, which falls in the therapeutic range (0.01-10 microM), and a limit of detection of 23 nM. The bioassay was selective against other naturally occurring taxanes such as baccatin III and 10-deacetylbaccatin III. However, there was interference from cephalomannine. The excellent sensitivity, accuracy, reproducibility, and simplicity make this analytical technique suitable for routine and high-throughput analyses and might be helpful in providing better care for patients. The bioassay was successfully applied to the determination of paclitaxel in human plasma.     

Fluorescence polarization competition immunoassay for tyrosine kinases

To increase the sensitivity and throughput of protein tyrosine kinase (PTK), simple, homogeneous, nonradioactive, direct and indirect fluorescence polarization (FP) protein tyrosine kinase immunoassays have been developed that are compatible with high-throughput and ultrahigh-throughput screening for developing drugs. In the direct method, a fluorescinylated peptide substrate is incubated with the kinase, ATP, and antiphosphotyrosine antibody. The phosphorylated peptide product is immunocomplexed with the antiphosphotyrosine antibody, resulting in an increase in the polarization signal. Since the direct method can be used only with a peptide substrate and requires large amounts of antiphosphotyrosine antibody, a modified indirect method, wherein a phosphorylated peptide or protein produced by kinase reaction will compete with a fluorescent phosphopeptide used as a tracer for immunocomplex formation with phosphotyrosine antibody, was developed. In this format kinase activity will result in loss of the polarization signal. Both the direct and indirect FP-PTK immunoassays have been compared with a more commonly used (32)PO(4) transfer assay and validated using lymphoid T-cell protein tyrosine kinase (Lck). In both assays, Lck activity showed a similar dependence on ATP, Lck enzyme, and peptide substrate concentration, comparable to the (32)PO(4) transfer assay. Inhibition by staurosporine and the Lck inhibitor 4-amino-5-(methylphenyl)-7-(tert-butyl)pyrazolo[3, 4-d]pyrimidine in these two FP assays was similar to that obtained in the (32)PO(4) transfer assay. The advantages of these FP-PTK assays over the other kinase assays, besides high sensitivity, are use of inexpensive nonisotropic substrate; environmental safety; homogeneous nature of FP kinase assays that are done in the same tube (or in a well of 96- or 384-well microtiter plates), without separation, precipitation, or washing; and increase of throughput.     

Fluorescence polarization: analysis of carbohydrate-protein interaction.

Fluorescence polarization has been widely used for the studies on the molecular motion in solution and has been applied to immunoassays for proteins, therapeutic drug monitoring in clinical pharmacy, and assays for environmentally toxic compounds. Because fluorescence polarization is most readily applicable to the kinetic analysis of the binding reaction between a substance having small molecular mass and a receptor molecule, this method is easily applied to the analysis of carbohydrate-lectin binding. In this tutorial Thematic Review, we briefly introduce the principles of fluorescence polarization and some applications of fluorescence polarization technique to glycobiology.     

Fluorescence polarization studies of lysozyme and lysozyme-saccharide complexes

The fluorescence polarization properties of hen egg white lysozyme and of an iodine oxidized derivative of lysozyme in which tryptophan-108 was selectively modified, were investigated. Using the addition law of anisotropy of mixed systems, the contribution of tryptophan-108 to the anisotropy spectrum of lysozyme and lysozyme-chitotetraose complex was separated. The rate of fluorescence polarization was studied as a function of pH. The major contribution to this rate is shown to arise from internal rotations of the indole side-chain of tryptophan-108 as well as from structural changes around tryptophan-62 and 63. From the dependence of the fluorescence polarization of lysozyme and IL with saccharide concentration, the existence of the simultaneous binding of two saccharide molecules to the enzyme cleft was inferred. At low chitotetraose concentration, the subsites A, B and C are occupied with an association constant of 8 × 10⁴m^?1 whereas at high saccharide concentration, both subsites A–B–C and E–F are occupied. The association constants of a series of saccharides to subsites E–F were measured and all found to be around 2 × 10²m^?1. The dependence of the rate of depolarization with saccharide concentration was determined and showed that, upon binding of the first saccharide molecule to subsites A, B and C, the rate of internal rotation of tryptophan-108 and tryptophan-62 and 63 was much reduced whereas upon further binding of a saccharide molecule in subsites E–F the rates are enhanced. This behaviour was interpreted as an indication that the binding of saccharide in subsites E–F induces changes in conformation of the enzyme which affect the entire active site architecture.     

Fluorescence polarization of trypsin digested photosystem II membranes

Fluorescence polarization of photosystem II particles treated with trypsin and incubated with high salt-medium (2M NaCl) was investigated. The presence of atrazine and TMPD in normal and salt-washed particles induced a decrease in the polarization ratios. Similar results were obtained at low concentrations of trypsin. On the basis of our observations we suggest that the presence of these perturbing agents causes a reorganisation of the membrane components and alters pigment-pigment and pigment-protein interactions. The results of fluorescence polarization demonstrate trypsin entry into the membrane after the digestion of the peripheral proteins.Abbreviations Chl chlorophyll - Mes 2-(N-morpholino)-ethanesulfonic acid - OEC oxygen evolving complex - PS II photosystem II - TMPD N, N, N - N tetramethyl-p-phenyl-enediamine - DPH 1, 6-diphenyl-1, 3, 5-hexatriene