32P Labeled Opioid Peptides with High Affinity for the δ-Opioid Receptor

We describe a mild and convenient labeling method for obtaining opioid radioligands which exhibit high specific activity together with a high affinity for the δ-opioid receptor. We chemically synthesized and tested the affinity of enkephalin- and deltorphin-like peptides that contain a phosphorylation site at their C-terminus. The peptide YdAGFLTPRRASLGC (peptide B), labeled to 700 Ci/mmol in the presence of cAMP-dependent protein kinase and [γ-32P]ATP, hound to the receptor with high affinity (Kd = 3.62 ± 0.29 nM). This peptide was also chemically coupled to bovine serum albumin and provided a multivalent opioid protein (B-BSA) with interesting properties: compared with peptide B, B-BSA was a better substrate for the kinase (100% 32P incorporation, sp act ≥7000 Ci/mmol when labeled) and a better ligand for the receptor (Kd = 0.20 ± 0.02 nM). The concept of peptide extension by a short phosphorylatable sequence should be more generally applicable to other small peptidic hormones or neurotransmitters and provide useful probes for biochemical studies and expression cloning of membrane receptors.