Design of a photoaffinity label for the hormone binding site of neurophysin.

The photolabile peptide, L-methionyl-L-tyrosyl-p-azido-L-phenylalaninamide, was synthesized by solution methods. This peptide, as well as the analogous species containing tritiated methionine, were found to bind reversibly and specifically, in the dark, to bovine neurophysin II. The dissociation constant, stoichiometry, and pH-dependence of this noncovalent interaction are typical of those properties for hormone (oxytocin) and hormone-like ligand binding to neurophysin II. Under photolytic conditions, methionyl-tyrosyl-p-azidophenylalaninamide causes irreversible inhibition of the noncovalent ligand binding activity of neurophysin II. This inactivation was achieved to the extent of about 90%. Both the dark and light (photolytic) interactions of the photolabile peptide with neurophysin II indicate its reaction at the hormone binding site of the protein and thus its potential use to identify amino acid residues at this site by covalent photoaffinity labelling.