Measurement of Total Opioid Peptides in Rat Brain and Pituitary by Radioimmunoassay Directed at the α-N-Acetyl Derivative

A sensitive assay, which cross-reacts with and is specific for diverse opioid peptides, is described. This is based on the prior acetylation of samples and subsequent radioimmunoassay with an antiserum highly specific for the acetylated NH₂ terminus of opioid peptides. The result is a procedure that can be used to investigate multiple forms of opioid peptides in extracts of biological material. The sensitivity of the assay is ?15 fmol of β-endorphin per incubation tube, i.e., ? 100-fold greater sensitivity than the radioreceptor assay used in our laboratory. The peptide concentration required for 50% displacement of trace ranged from 0.65 nM (β-endorphin) to 1.6 nM (Met-enkephalin). The assay apparently shows an absolute requirement for a free (or acetylated) NH₂ terminus corresponding to either a Leu- or Met-enkephalin sequence. Use of the assay with and without prior acetylation of sample provides a method for estimation of the ratio of acetylated:nonacetylated opioid peptides in crude or fractionated extracts. The procedure is used to investigate the forms of opioid peptide found in rat brain and pituitary.