Solid-phase synthesis of europium-labeled human INSL3 as a novel probe for the study of ligand-receptor interactions.
Solid-phase synthesis of europium-labeled human INSL3 as a novel probe for the study of ligand-receptor interactions.
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DOI：
10.1021/bc800127p
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发表时间：
2008-06
影响因子：
4.7
通讯作者：
F. Shabanpoor;R. Hughes;R. Bathgate;Suode Zhang;D. Scanlon;F. Lin;M. Hossain;F. Separovic;J. Wade
中科院分区：
文献类型：
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作者：
F. Shabanpoor;R. Hughes;R. Bathgate;Suode Zhang;D. Scanlon;F. Lin;M. Hossain;F. Separovic;J. Wade
一个有效的固相合成协议已经开发，连同区域选择性顺序形成的三个二硫键，使专门的单镧系元素（铕）标记的人胰岛素样肽3（INSL 3）的制备与其G-蛋白偶联受体，RXFP 2，通过时间分辨荧光法的研究。将市售螯合剂二亚乙基三胺五乙酸（DTPA）偶联到固相上INSL 3 A链的N端，然后用EuCl 3保护螯合剂在随后的A链和B链结合过程中不产生未知的加合物，形成铕离子与DTPA的配位络合物.标记的肽用高效液相色谱纯化，在高产量与近中性pH缓冲液，以防止释放的Eu（3+）的螯合剂。使用时间分辨荧光测定法，进行饱和结合测定以确定稳定表达RXFP 2的HEK-293 T细胞中标记的INSL 3对RXFP 2的结合亲和力（pKd）。从饱和结合实验中获得的DTPA标记的INSL 3的解离常数（9.05 +/- 0.03，n = 3）与（125）I标记的INSL 3的解离常数（9.59 +/- 0.09，n = 3）相当。使用Eu-DTPA-INSL 3作为标记配体，测定人INSL 3的受体结合亲和力（p Ki）为9.27 +/- 0.06，n = 3，这同样与使用（125）I-INSL 3作为标记配体时获得的结果（9.34 +/- 0.02，n = 4）相似。这种新型镧系元素配位的DTPA标记的INSL 3具有优异的灵敏度、稳定性和高比活性，这些特性将特别有益于在结构-活性研究中高通量筛选INSL 3类似物。
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.