Enzymatic and Radioimmunoassay Procedures Combined with Electrophoresis and HPLC for the Recovery and Characterization of Substance P in Human Brain

Immunoreactive substance P was recovered from human brain (hypothalamus and substantia nigra) by acetic acid extraction, ion exchange chromatography (SP-Sephadex), molecular sieving (Sephadex C-50) and column electrophoresis in agarose suspension. The chemical nature of the active material was further studied with various biochemical techniques including agarose suspension electrophoresis, HPLC and different kinds of enzyme radioimmunoassays. By combining these techniques it was possible to confirm structure identity between the recovered active component and substance P previously isolated from bovine brain. Thus, the major activity reacting with the substance P antibodies was indistinguishable from the synthetic bovine analogue in all chromatographic systems including analytical electrophoresis at different pH:s and HPLC. Furthermore, digestion of the active material with post-proline cleaving enzyme and trypsin yielded fragments identical with those expected from the bovine peptide as confirmed by specific radioimmunoassays in conjuction with electrophoresis or HPLC. The result also indicates the usefulness of the present procedures for identifying peptides structures available only in minute amounts.