Chromatographic methods for characterization of angiotensin in brain tissue

Ang II antiserum with high sensitivity and specificity was produced. The native Ang II antiserum was purified by affinity chromatography on Affi-Gel 102 with covalently coupled [Ile5]Ang II, and purified Ang II antiserum was covalently coupled to Affi-Gel 10. The column with the covalently coupled Ang II antiserum was used for the specific enrichment of Ang II from brain extracts. The efficiency and usefulness of affinity chromatography for the purification of Ang II from biological sources were tested with 125I-labeled, 3H-labeled, and synthetic [Ile5]Ang II added to rat brains prior to extraction. In addition, the methodology was used for the purification of endogenous Ang II from pig brain. The described three-step procedure for the isolation and purification of Ang II including extraction, affinity chromatography, and HPLC is rapid and highly specific with high loading capacity. We have applied the method to the peptide Ang II in brain, but the methodology may also be used in general for the rapid purification of other neuropeptides. A combination of HPLC with specific radioimmunoassays for Ang I and Ang II was utilized to demonstrate that rat brain cells in culture devoid of the influence of the peripheral RAS were able to synthesize radioactively labeled Ang I and Ang II after incubation with [3H]isoleucine. And, finally, an HPLC system capable of separating Ang I, Ang II, and its metabolites was used to obtain insight into the degradation pattern of angiotensin peptides in the brain. Aminopeptidases appear to be the major angiotensin-degrading enzymes, and endopeptidases do not appear to be involved.