使用 FA-PGG 和 HHL 作为底物的两种常用血管紧张素转换酶抑制测定的性能,Journal of Dairy Research

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使用 FA-PGG 和 HHL 作为底物的两种常用血管紧张素转换酶抑制测定的性能
Journal of Dairy Research ( IF 1.6 ) Pub Date : 2006-02-14 , DOI: 10.1017/s0022029905001639
Samah M Shalaby ₁ , Mila Zakora , Jeanette Otte

Affiliation

在开发对血管紧张素转换酶 (ACE) 具有高抑制活性的发酵乳制品和蛋白质水解物时，至关重要的是要有可靠的测定方法来测量 ACE 活性。在本研究中，测试了基于三肽合成 N-衍生物作为底物的两种常用测定法在测定 ACE 活性方面的可靠性本身以及胰蛋白酶乳清蛋白消化物和卡托普利的抑制作用。在一项测试中，ACE 活性是根据在与 ACE 孵育 30 分钟期间从马尿酸-His-Leu (HHL) 释放的马尿酸量计算得出的，在 HPLC 分离反应产物后进行量化。在另一个测定中，ACE 活性直接测量为 ACE 催化呋喃丙烯酰-Phe-Glu-Glu (FA-PGG) 水解的前 30 分钟内 340 nm 处吸光度的降低速率。在没有抑制剂的情况下，两种测定均显示出良好的性能，重复样品之间的相对标准偏差约为 7%。在抑制剂溶液存在的情况下，两种测定的相对标准偏差对于不同稀释的抑制剂在 1% 和 18% 之间变化。两种测定都给出了抑制 ACE 50% 所需的抑制剂浓度值，这与之前报道的乳清蛋白消化物和卡托普利的值相似。然而，两种测定的不同结果强调了控制实际 ACE 活性以进行测定之间比较的重要性。讨论了每种测定的局限性。考虑到使用 FA-PGG 作为底物的检测步骤更少，因此每个样品消耗的时间和化学品更少，并且所需的设备更简单，因此建议使用此检测方法筛选透明肽样品。

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Performance of two commonly used angiotensin-converting enzyme inhibition assays using FA-PGG and HHL as substrates

In development of fermented dairy products and protein hydrolysates with high inhibitory activity towards angiotensin-converting enzyme (ACE), it is crucial to have a reliable assay for measuring the ACE activity. In the present study, the performance of two commonly used assays based on synthetic N-derivates of tripeptides as substrates were tested with respect to reliability in determination of ACE activity per se and to the inhibitory effect of a tryptic whey protein digest and captopril. In one test, the ACE activity was calculated from the amount of hippuric acid liberated from hippuryl-His-Leu (HHL) during 30 min of incubation with ACE, as quantified after HPLC separation of reaction products. In the other assay, the ACE activity was measured directly as the rate of decrease in the absorbance at 340 nm during the first 30 min of ACE catalysed hydrolysis of furanacroloyl-Phe-Glu-Glu (FA-PGG). Both assays, in the absence of inhibitor, showed a good performance with relative standard deviation between replicate samples around 7%. In the presence of inhibitor solutions, relative standard deviations for both assays varied between 1 and 18% for the variously diluted inhibitors. Both assays gave values for the concentration of inhibitor needed to inhibit ACE by 50% similar to those previously reported for whey protein digests and captopril. Different results from the two assays, however, emphasize the importance of controlling the actual ACE-activity for comparison between assays. The limitations of each assay are discussed. Considering the fewer steps in the assay using FA-PGG as substrate, and thus less time and chemicals consumed per sample, and the simpler equipment needed, this assay is recommended for the screening of clear peptide samples.

更新日期：2006-02-14

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