Affinity purification of synenkephalin-containing peptides, including a novel 23.3-kilodalton species

Affinity chromatography has been used for rapid and high-yield purification of synenkephalin (proenkephalin 1-70) containing peptides present in bovine adrenal medulla (BAM) chromaffin granular lysate. A column of CN-Br-activated Sepharose 4B coupled to synenkephalin antiserum bound synenkephalin immunoreactivity which was eluted by a stepwise gradient of 50 mM ammonium acetate containing 20% (vol/vol) acetonitrile over the pH range 7-3. Synenkephalin immunoreactivity emerged as two peaks, eluting at pH 5.5 and 4.5. Characterization of the two peaks by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting indicated that the pH 5.5 peak contained principally low-molecular-weight proenkephalin species (8.6 and 12.6 kilodaltons), whereas the pH 4.5 peak contained, in addition, high-molecular-weight proenkephalin species (18.2 and 23.3 kilodaltons). The 8.6- and 12.6-kilodalton species were isolated from the pH 5.5 peak by TSK gel filtration HPLC, whereas the pH 4.5 peak was further purified by passage over successive affinity columns coupled to antiserum against BAM 22P (proenkephalin 182-203) and [Met5]-enkephalin-Arg6-Gly7-Leu8. The former column retains the 23.3-kilodalton species, whereas the latter column retains the 18.2-kilodalton species. The 23.3-kilodalton peptide represents a novel putative proenkephalin intermediate (proenkephalin-1-206), containing [Leu5]-enkephalin at the C-terminus.