Isolation and purification of angiotensin II using affinity and high-pressure liquid chromatography

Abstract

Peptides have been found in a variety of tissues including brain. To purify the peptide angiotensin II, a three-step method for the isolation and purification has been developed using extraction, affinity chromatography, and high-pressure liquid chromatography. Angiotensin II antiserum purified by affinity chromatography was covalently coupled to Affi-gel 10 (Affi-gel 10-AB). The efficiency and usefulness of this column for the purification of angiotensin II from biological sources were tested with 125I- and 3H-labeled (Ile 5)-angiotensin II added to rat brains prior to extraction. After extraction, the recoveries for both peptides were 74 and 75%, respectively. Recovery after the purification on Affi-gel 10-AB was 84 and 82%. Thirty-two percent of the radioactivity was not retained and 50% of the radioactivity could be eluted with 0.1 m Na citrate buffer containing 1 m NaCl using a stepwise pH gradient. Characterization by HPLC of the unretained radioactivity from the Affi-gel 10-AB column showed one peak for [ 125I]angiotensin II, coeluting with the [ 125I]angiotensin II standard and two minor peaks. Only 30% of unretained [ 3H]angiotensin II could be identified as intact [ 3H]angiotensin II on HPLC. Both [ 125I]angiotensin II and [ 3H]angiotensin II elutable at pH 5.0 and 4.0 on Affi-gel 10-AB could be demonstrated as highly purified [ 125I]angiotensin II and [ 3H]angiotensin II on HPLC with a purity of more than 90%. On HPLC, the recovery was 81% for [ 125I]angiotensin II and 99% for [ 3H]angiotensin II. The recovery for the entire three-step procedure was about 60%. The loading capacity of the Affi-gel 10-AB column for (Ile 5)-angiotensin II was 550 ng. With the method described, angiotensin II could be purified very specifically and in a high purity. The method may also be used for the rapid purification of other neuropeptides.