Discriminative disulfide-bonding affinity labeling of opioid receptor subtypes

The affinity-labeling technique is an extremely important method in receptor biochemistry. The 3-nitro-2-pyridinesulfenyl (Npys) group, attached to a mercapto group, can react only with a free thiol group (the beta -mercapto group of cysteine residue) of the target receptor molecules, forming a disulfide bond. This disulfide bonding is mediated through the thiol-disulfide exchange reaction. Unlike other labeling methods, the approach utilizing such chemically activated thiol-containing ligands is able to reproduce an unlabeled protein by treatment with dithiothreitol, a reducing reagent This provides several unique aspects for the studies elucidating the structure-function relationships between the peptide and the receptor. Based on the SNpys affinity technique, we have achieved the discriminative disulfide-bonding affinity labeling of the three different subtypes of opioid receptors: mu, delta and kappa. This article reviews our novel affinity techniques in the in vitro receptor biochemistry. (C) 2001 Elsevier Science BN. All rights reserved.