Isolation and purification of angiotensin II using affinity and high-pressure liquid chromatography.
Isolation and purification of angiotensin II using affinity and high-pressure liquid chromatography.
复制标题
使用亲和和高压液相色谱分离和纯化血管紧张素 II。
DOI：
10.1016/0003-2697(86)90346-5
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发表时间：
1986
影响因子：
2.9
通讯作者：
M. Phillips
中科院分区：
文献类型：
--
作者：
K. Hermann;B. Kimura;M. Phillips
在包括脑在内的多种组织中发现了肽。为了纯化肽血管紧张素II，已经开发了使用萃取、亲和层析和高压液相层析的用于分离和纯化的三步方法。将通过亲和层析纯化的血管紧张素II抗血清共价偶联至Affi-gel 10（Affi-gel 10-AB）。用125 I-和3 H-标记的（Ile 5）-血管紧张素II在提取前加入大鼠脑中，测试了该柱从生物源中纯化血管紧张素II的效率和有用性。提取后，两种肽的回收率分别为74%和75%。在Affi-gel 10-AB上纯化后的回收率为84%和82%。32%的放射性未保留，50%的放射性可使用含1 m NaCl的0.1 m柠檬酸钠缓冲液（采用逐步pH梯度）洗脱。通过HPLC对Affi-gel 10-AB色谱柱的未保留放射性进行表征，发现[125 I]血管紧张素II的一个峰（与[125 I]血管紧张素II标准品共洗脱）和两个次要峰。在HPLC上，只有30%的未保留的[3 H]血管紧张素II可以被鉴定为完整的[3 H]血管紧张素II。[125 I]血管紧张素II和[3 H]血管紧张素II均可在Affi-gel 10-AB上在pH 5.0和4.0下洗脱，在HPLC上可证明为高纯度的[125 I]血管紧张素II和[3 H]血管紧张素II，纯度超过90%。在HPLC上，[125 I]血管紧张素II的回收率为81%，[3 H]血管紧张素II的回收率为99%。整个三步程序的回收率约为60%。Affi-gel 10-AB柱对（Ile 5）-血管紧张素II的上样量为550 ng。采用所述方法，可以非常特异性地以高纯度纯化血管紧张素II。该方法也可用于其他神经肽的快速纯化。
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 with125I- and3H-labeled (Ile5)-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 (Ile5)-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.