Integrity of tritiated orphanin FQ/nociceptin: implications for establishing a reliable binding assay

In the course of establishing a reliable and reproducible binding assay for the orphanin FQ/nociceptin (OFQ/N) ligand-receptor system we used reversed phase-high-performance liquid chromatography (HPLC) (RP-HPLC) to monitor t he integrity of [H-3]OFQ/N obtained from three different manufacturers. Thi s means of analysis revealed that the stability of [H-3]OFQ/N during storag e varied considerably depending on the manufacturer. Furthermore, the integ rity of [H-3]OFQ/N was significantly compromised in the presence of COS-7 c ell membranes. Interestingly, if the peptide was added to COS-7 membranes a fter they had been exposed to low pH it remained intact, suggesting that th e peptide's breakdown during binding is, in part, enzymatically mediated. A lthough a variety of protease inhibitors were tested, none proved completel y effective at protecting the tritiated peptide. The intention of the studi es presented here was to evaluate OFQ/N binding components, namely the avai lable [H-3]OFQ/N ligands, in an effort to standardize the binding condition s for this receptor ligand system. Consequently, this study underscores the importance of monitoring the integrity of the trace ligand being used in a given binding assay. (C) 2000 Elsevier Science Inc. All rights reserved.