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https://hdl.handle.net/2440/75332
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dc.contributor.author | Shabanpoor, F. | - |
dc.contributor.author | Hughes, R. | - |
dc.contributor.author | Bathgate, R. | - |
dc.contributor.author | Zhang, S. | - |
dc.contributor.author | Scanlon, D. | - |
dc.contributor.author | Lin, F. | - |
dc.contributor.author | Hossain, M. | - |
dc.contributor.author | Separovic, F. | - |
dc.contributor.author | Wade, J. | - |
dc.date.issued | 2008 | - |
dc.identifier.citation | Bioconjugate Chemistry, 2008; 19(7):1456-1463 | - |
dc.identifier.issn | 1043-1802 | - |
dc.identifier.issn | 1520-4812 | - |
dc.identifier.uri | http://hdl.handle.net/2440/75332 | - |
dc.description.abstract | An efficient solid-phase synthesis protocol has been developed which, together with regioselective sequential formation of the three disulfide bonds, enabled the preparation of specifically monolanthanide (europium)-labeled human insulin-like peptide 3 (INSL3) for the study of its interaction with its G-protein-coupled receptor, RXFP2, via time-resolved fluorometry. A commercially available chelator, diethylene triamine pentaacetic acid (DTPA), was coupled to the N-terminus of the INSL3 A-chain on the solid phase, and then a coordination complex between europium ion and DTPA was formed using EuCl 3 to protect the chelator from production of an unidentified adduct during subsequent combination of the A- and B-chains. The labeled peptide was purified in high yield using high-performance liquid chromatography with nearly neutral pH buffers to prevent the liberation of Eu (3+) from the chelator. Using time-resolved fluorometry, saturation binding assays were undertaken to determine the binding affinity (p K d) of labeled INSL3 for RXFP2 in HEK-293T cells stably expressing RXFP2. The dissociation constant of DTPA-labeled INSL3 (9.05 +/- 0.03, n = 3) that was obtained from saturation binding experiments was comparable to that of (125)I-labeled INSL3 (9.59 +/- 0.09, n = 3). The receptor binding affinity (p K i) of human INSL3 was determined to be 9.27 +/- 0.06, n = 3, using Eu-DTPA-INSL3 as a labeled ligand, which again is similar to that obtained when (125)I-INSL3 was used as labeled ligand (9.34 +/- 0.02, n = 4). This novel lanthanide-coordinated, DTPA-labeled INSL3 has excellent sensitivity, stability, and high specific activity, properties that will be particularly beneficial in high-throughput screening of INSL3 analogues in structure-activity studies. | - |
dc.description.statementofresponsibility | Fazel Shabanpoor, Richard A. Hughes, Ross A. D. Bathgate, Suode Zhang, Denis B. Scanlon, Feng Lin, Mohammed Akhter Hossain, Frances Separovic, and John D. Wade | - |
dc.language.iso | en | - |
dc.publisher | Amer Chemical Soc | - |
dc.rights | © 2008 American Chemical Society | - |
dc.source.uri | http://dx.doi.org/10.1021/bc800127p | - |
dc.subject | Humans | - |
dc.subject | Europium | - |
dc.subject | Pentetic Acid | - |
dc.subject | Insulin | - |
dc.subject | Proteins | - |
dc.subject | Receptors, G-Protein-Coupled | - |
dc.subject | Chelating Agents | - |
dc.subject | Ligands | - |
dc.subject | Circular Dichroism | - |
dc.subject | Staining and Labeling | - |
dc.subject | Amino Acid Sequence | - |
dc.subject | Protein Binding | - |
dc.subject | Substrate Specificity | - |
dc.subject | Stereoisomerism | - |
dc.subject | Molecular Sequence Data | - |
dc.title | Solid-phase synthesis of europium-labeled human INSL3 as a novel probe for the study of ligand-receptor interactions | - |
dc.type | Journal article | - |
dc.identifier.doi | 10.1021/bc800127p | - |
pubs.publication-status | Published | - |
Appears in Collections: | Aurora harvest Medical Sciences publications |
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