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Type: Journal article
Title: Solid-phase synthesis of europium-labeled human INSL3 as a novel probe for the study of ligand-receptor interactions
Author: Shabanpoor, F.
Hughes, R.
Bathgate, R.
Zhang, S.
Scanlon, D.
Lin, F.
Hossain, M.
Separovic, F.
Wade, J.
Citation: Bioconjugate Chemistry, 2008; 19(7):1456-1463
Publisher: Amer Chemical Soc
Issue Date: 2008
ISSN: 1043-1802
Statement of
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
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.
Keywords: Humans; Europium; Pentetic Acid; Insulin; Proteins; Receptors, G-Protein-Coupled; Chelating Agents; Ligands; Circular Dichroism; Staining and Labeling; Amino Acid Sequence; Protein Binding; Substrate Specificity; Stereoisomerism; Molecular Sequence Data
Rights: © 2008 American Chemical Society
RMID: 0020106249
DOI: 10.1021/bc800127p
Appears in Collections:Medical Sciences publications

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