Use of RP-HPLC for Purification of a CCK-39 Radio-Ligand and of Its Photoactivable Derivative. Controls of Degradations

In order to characterize CCK receptors on guinea-pig pancreatic acini we developped a method of purification and control of degradation of CCK-39 radio-ligands and of their photoactivable derivatives. CCK-39 was radio-labeled with N-hydroxysuccinimide 3-(4-hydroxy 5 [125I] phenyl) propionate at pH=8.5. Mono-labeled CCK-39 was separated from di-and tri-labeled CCK-39 on a C-18 column and coupled with N-hydroxysuccinimidyl-4-azido-benzoate. The resulting photoactivable radio-ligand 125I-BH-[4-azidobenzoyl]-CCK-39 specifically bound to pancreatic membranes. After photolysis under UV irradiation and SDS-PAGE a major labeled protein of Mr 90 000 could be identified. Furthermore, RP-HPLC permitted the control of degradation of each ligand. Presence of endo-peptidasic and aminopeptidasic activities sensitive to EDTA and bacitracin were demonstrated.