Anti-fluorescein antibody of high affinity and restricted heterogeneity as characterized by fluorescence polarization and quenching equilibrium techniques

Aspergillus fumigatus is an opportunistic human pathogenic fungus responsible for deadly lung infections in immunocompromised individuals. Galactofuranose (Galf) residues are essential components of the cell wall and play an important role in A. fumigatus virulence. The flavoenzyme UDP-galactopyranose mutase (UGM) catalyzes the isomerization of UDP-galactopyranose to UDP-galactofuranose, the biosynthetic precursor of Galf. Thus, inhibitors of UGM that block the biosynthesis of Galf can lead to novel chemotherapeutics for treating A. fumigatus-related diseases. Here, we describe the synthesis of fluorescently labeled UDP analogs and the development of a fluorescence polarization (FP) binding assay for A. fumigatus UGM (AfUGM). High-affinity binding to AfUGM was only obtained with the chromophore TAMRA, linked to UDP by either 2 or 6 carbons with Kd values of 2.6 ± 0.2 μM and 3.0 ± 0.7 μM, respectively. These values were ~6 times lower than when UDP was linked to fluorescein. The FP assay was validated against several known ligands and displayed an excellent Z′ factor (0.79 ± 0.02) and good tolerance to dimethyl sulfoxide.

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Synthesis of a high-affinity Fluorescent PPARγ ligand for high-throughput fluorescence polarization assays

Bioorganic & Medicinal Chemistry ◽

10.1016/s0968-0896(03)00494-2 ◽

2003 ◽

Vol 11 (20) ◽

pp. 4325-4332 ◽

Cited By ~ 13

Author(s):

Michael J DeGrazia ◽

Jerry Thompson ◽

John P.Vanden Heuvel ◽

Blake R Peterson

Keyword(s):

High Throughput ◽

Fluorescence Polarization ◽

High Affinity

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EVIDENCE THAT THE C-TERMINAL HEPARIN BINDING DOMAIN ("HEP II") DOMINATES HEPARIN-FIBRONECTIN INTERACTIONS

10.1055/s-0038-1643631 ◽

1987 ◽

Author(s):

M J Beneckv ◽

C G Kolvenbach ◽

D L Amrani ◽

M W Mosesson

Keyword(s):

Fluorescence Polarization ◽

Rotational Diffusion ◽

Fluid Phase ◽

Scatchard Analysis ◽

Heparin Binding ◽

Single Class ◽

High Affinity ◽

Heparin Interaction ◽

Heparin Affinity ◽

Binding Component

The interaction of glycosaminoglycans with plasma fibronectin (PFn) may play a role in the conversion of PFn from an “inert” dimeric circulating form to an “activated” multimeric form deposited on the cell surface or in the extracellular matrix. We carried out a quantitative comparison of heparin affinity for PFn and its proteolytic fragments in order to assess the relative importance of heparin interactions with PFn’s various reported heparin-binding domains. We employed affinity chromatography on PFn-sepharose to prepare a subset of fluorescein-labelled heparin molecules with high affinity for PFn, and confirmed that heparin binding to PFn is very sensitive to ionic strength. This suggests that the PFn-sepharose column selectively binds a fraction of highly sulfated heparin molecules. We quantified PFn-heparin affinity in the fluid-phase by monitoring a fluorescence polarization change that occurred as a consequence of the decrease in the rotational diffusion rate of fluorescently-labelled heparin molecules (13.8 kD) as they became “immobilized” by binding to PFn. Scatchard analysis of the heparin fluorescence polarization data obtained for PFn in Tris-buffered saline yielded a biphasic curve with Kd’s estimated at 5 and 130 nM, respectively A 190 kD thrombin fragment, containing the C-terminal "Hep II" domain but lacking the 29 kD N-terminal “Hep I” domain, yielded a linear plot displaying a single class of heparin-binding sites with a Kd of 130 nM Similar results were obtained for the C-terminal 150 kD Fn fragment which also contained the “Hep II” domain. In contrast, the 29 kD N-terminal “Hep I” Fn fragment bound heparin weakly (Kd =25 μM). The nature of the “high affinity (Kd= 5 nM) heparin binding component is uncertain; it may reflect heparin interaction with soluble multimers present in our PFn preparations Our observations suggest that the Kd=130 nM heparin binding component corresponds to heparin interaction with the C-terminal “Hep II” domain We conclude that the N-terminal “Hep I” domain does not participate significantly in heparin binding to soluble dimeric Fn under physiological conditions, whereas the C-terminal “Hep II” domain dominates such interactions

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Mapping Protein–Protein Interactions of the Resistance-Related Bacterial Zeta Toxin–Epsilon Antitoxin Complex (ε2ζ2) with High Affinity Peptide Ligands Using Fluorescence Polarization

Toxins ◽

10.3390/toxins8070222 ◽

2016 ◽

Vol 8 (7) ◽

pp. 222 ◽

Cited By ~ 5

Author(s):

María Fernández-Bachiller ◽

Iwona Brzozowska ◽

Norbert Odolczyk ◽

Urszula Zielenkiewicz ◽

Piotr Zielenkiewicz ◽

...

Keyword(s):

Protein Interactions ◽

Fluorescence Polarization ◽

Peptide Ligands ◽

Protein Protein Interactions ◽

High Affinity ◽

Affinity Peptide

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Development of a Fluorescence Polarization Assay for the Molecular Chaperone Hsp90

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057104265995 ◽

2004 ◽

Vol 9 (5) ◽

pp. 375-381 ◽

Cited By ~ 70

Author(s):

Joungnam Kim ◽

Sara Felts ◽

Laura Llauger ◽

Huazhong He ◽

Henri Huezo ◽

...

Keyword(s):

Molecular Chaperone ◽

Fluorescence Polarization ◽

High Throughput Screening ◽

Cancer Therapeutics ◽

Heat Shock Protein 90 ◽

Hsp90 Inhibitors ◽

Amino Terminal ◽

High Affinity ◽

Measured Polarization ◽

Fluorescence Polarization Assay

Heat shock protein 90 (Hsp90) is a molecular chaperone with essential functions in maintaining transformation, and there is increasing interest in developing Hsp90 inhibitors as cancer therapeutics. In this study, the authors describe the development and optimization of a novel assay for the identification of Hsp90 inhibitors using fluorescence polarization. The assay is based on the competition of fluorescently (BODIPY) labeled geldanamycin (GM) for binding to purified recombinant Hsp90α (GM is a natural product that binds to the ATP/ADP pocket in the amino terminal of Hsp90). The authors show that GM-BODIPY binds Hsp90α with high affinity. Even at low Hsp90α concentrations (30 nM), the measured polarization value is close to the maximum assay range of 160 mP, making measurements very sensitive. Its performance, as judged by signal-to-noise ratios (> 10) and Z and Z′ values (> 0.5), suggests that this is a robust and reliable assay. GM, PU24FCl, ADP, and ATP, all known to bind to the Hsp90 pocket, compete with GM-BODIPY for binding to Hsp90α with EC50s in agreement with reported values. These data demonstrate that the Hsp90-FP-based assay can be used for high-throughput screening in aiding the identification of novel Hsp90 inhibitors.

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Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

PLoS ONE ◽

10.1371/journal.pone.0184843 ◽

2017 ◽

Vol 12 (9) ◽

pp. e0184843 ◽

Cited By ~ 45

Author(s):

Justin Hall ◽

Amy Brault ◽

Fabien Vincent ◽

Shawn Weng ◽

Hong Wang ◽

...

Keyword(s):

Fluorescence Polarization ◽

High Affinity ◽

Fluorescence Polarization Assay

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Fluorescence of actin-bound hydrophobic molecules

Canadian Journal of Biochemistry and Cell Biology ◽

10.1139/o83-124 ◽

1983 ◽

Vol 61 (9) ◽

pp. 981-988 ◽

Cited By ~ 3

Author(s):

L. D. Burtnick ◽

K. W. Chan

Keyword(s):

Binding Sites ◽

Fluorescence Polarization ◽

Sulfonic Acid ◽

Fold Increase ◽

Net Charge ◽

Emission Anisotropy ◽

High Affinity ◽

Deoxyribonuclease I ◽

Hydrophobic Site ◽

Polarization Of Luminescence

2-(N-methylanilino)naphthalene-6-sulfonic acid (MANS) binds to G-actin at a single high affinity hydrophobic site (Kd = 41 μM). Salt-induced polymerization of MANS–G-actin results in a general enhancement of sample emission intensities at all wavelengths. At 430 nm, KCl-induced polymerization yields a 2.3-fold enhancement, while MgCl2-induced polymerization gives a 2.0-fold increase. Polymerization of MANS–G-actin in the absence of agitation produces MANS–F-actin samples that have fluorescence polarization values at 430 nm of 0.33. Subsequent mixing or sonication of such MANS–F-actin samples results in a dramatic drop in fluorescence polarization values to 0.14. After cessation of mixing, the polarization values do not recover to their initial levels. Circular polarization of luminescence studies on MANS–actin demonstrate that agitation of MANS–F-actin samples drastically alters emission anisotropy values. 9-Anthroyl choline (9AC) binds to G-actin at a single hydrophobic site (Kd = 68 μM). The fluorescence of 9AC–actin is sensitive to salt-induced polymerization and depends upon the identity of the salt employed. KCl causes a drop in the fluorescence intensity at 490 nm to 70% of the value for 9AC–G-actin, while MgCl2 produces a 30% increase in intensity. Polarization experiments with 9AC–actin produced qualitatively the same results as did those with MANS–actin. Differences in the behaviours of MANS–actin and 9AC–actin in response to polymerization by KCl and MgCl2 and in response to the binding of deoxyribonuclease I suggest that the binding sites on actin for MANS and 9 AC do not overlap completely. The binding of MANS and 9 AC to actin may be modulated by the opposite signs of the net charge borne by each of these probes.

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Identification of a High-Affinity Anti-Phosphoserine Antibody for the Development of a Homogeneous Fluorescence Polarization Assay of Protein Kinase C

CrossRef Listing of Deleted DOIs ◽

10.1177/108705710000500106 ◽

2000 ◽

Vol 5 (1) ◽

pp. 23-30 ◽

Cited By ~ 22

Author(s):

Jinzi J. Wu ◽

Donna R. Yarwood ◽

Quynhchi Pham ◽

Matthew A. Sills

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Protein Interactions ◽

Tyrosine Kinases ◽

Fluorescence Polarization ◽

Rapid Screening ◽

Dependent Manner ◽

High Affinity ◽

Kinase C ◽

Fluorescence Polarization Assay

In the last few years, fluorescence polarization (FP) has been applied to the development of robust, homogeneous, high throughput assays in molecular recognition research, such as ligand-protein interactions. Recently, this technology has been applied to the development of homogeneous tyrosine kinase assays, since there are high-affinity anti-phosphotyrosine antibodies available. Unlike tyrosine kinases, application of FP to assay development for serine/threonine kinases has been impeded because of lack of high-affinity anti-phosphoserine/threonine antibodies. In the present study, we report the discovery of a high-affinity, monoclonal anti-phosphoserine antibody, 2B9, with a Kd of 250 ± 34 pM for a phosphoserine-containing peptide tracer, fluorescein-RFARKGS(PO4)LRQKNV. Our data suggest that 2B9 is selective for fluorescein-RFARKGS(PO4)LRQKNV. The antibody and tracer have been used for the development of a competitive FP assay for protein kinase C (PKC) in 384-well plates. Phosphatidylserine, which enhances the kinase activity of PKC in a Ca2+-dependent manner and has a structure similar to that of phosphoserine, did not interfere with binding of the peptide tracer to the antibody in the FP assay. The data indicate that the FP assay is more sensitive and robust than the scintillation proximity assay for PKC. The FP assay developed here can be used for rapid screening of hundreds of thousands of compounds for discovery of therapeutic leads for PKC-related diseases.

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Neuronal and Extraneuronal Uptake of 3H-Norepinephrine in the Heart of the Active Bat: An Electron Microscopic Radioautographic Study

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100073052 ◽

1973 ◽

Vol 31 ◽

pp. 522-523

Author(s):

Martin Hagopian ◽

Michael D. Gershon ◽

Eladio A. Nunez

Keyword(s):

Smooth Muscle ◽

Monoamine Oxidase ◽

Vascular Smooth Muscle ◽

Cardiac Muscle ◽

Maximum Rate ◽

External Medium ◽

Extraneuronal Uptake ◽

Electron Microscopic ◽

Cardiac Tissues ◽

High Affinity

The ability of cardiac tissues to take up norepinephrine from an external medium is well known. Two mechanisms, called Uptake and Uptake respectively by Iversen have been differentiated. Uptake is a high affinity system associated with adrenergic neuronal elements. Uptake is a low affinity system, with a higher maximum rate than that of Uptake. Uptake has been associated with extraneuronal tissues such as cardiac muscle, fibroblasts or vascular smooth muscle. At low perfusion concentrations of norepinephrine most of the amine taken up by Uptake is metabolized. In order to study the localization of sites of norepinephrine storage following its uptake in the active bat heart, tritiated norepinephrine (2.5 mCi; 0.064 mg) was given intravenously to 2 bats. Monoamine oxidase had been inhibited with pheniprazine (10 mg/kg) one hour previously to decrease metabolism of norepinephrine.

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