Purification and identification of multiple forms of dynorphin in human placenta

Immunoreactive dynorphin (ir-Dyn) and opiate-like peptides (OLP) were measured in acid (HC1) extracts of human placenta by the use of an antibody to synthetic Dyn-(1-13) and of the displacement of [3H]-naloxone binding to rat brain homogenates, respectively. The placenta contained 57.6 pmoles per g of ir-Dyn and 134.4 pmoles per g of naloxone binding equivalents. After passage of the extract through cartridges of Sep Pak C18, half of the OLP was eluted with ir-Dyn at 35% acetonitrile (ACN), the rest being eluted at 60% ACN. Both fractions obtained from Sep Pak were chromatographed separately on Sephadex G-50, the OLP of the 35% ACN fraction coeluting with the ir-Dyn speak and that of the 60% ACN fraction being eluted at the same volume as synthetic beta-endorphin. Conversely, the fraction of OLP coeluting with synthetic leucine-enkephalin (Leu-Enk) in these two chromatographies was minimal. The Dyn-immunoreactive material was further purified by high pressure liquid chromatography on reverse phase micro-Bondapak C18 columns to give three distinct peaks corresponding to synthetic Dyn-(1-11), Dyn-(1-13) and Dyn-(1-12), respectively. Our results indicate that the human placenta contains several forms of ir-Dyn which account for about half of its endogenous OLP.