Fyhrquist F, Tikkanen I, Grönhagen-Riska C, Hortling L, Hichens M
Clin Chem. 1984 May;30(5):696-700.
We describe a new principle for the determination of enzymes, here applied to angiotensin-converting enzyme (ACE, EC 3.4.15.1) in human serum. The enzyme inhibitor binding assay is based on specific binding of labeled inhibitor to the active center of the enzyme. Serum (10-15 microL) is incubated with 125I-labeled ACE inhibitor (" 351A ," a p- hydroxybenzamidine derivative of N-(1-carboxy-3-phenylpropyl)-L-lysyl-L-proline) at pH 7.0 at 37 degrees C for 2 h in a non-equilibrated system. Inhibitor bound to ACE is separated by adsorption to coated charcoal, the radioactivity remaining in the supernate is counted, and the ACE value is calculated from a standard curve. Sensitivity for ACE in serum is 200 U/L, corresponding to 5.0 ng of ACE purified from human lung. The coefficient of variation was 3.9% within assay, and 6.4% between assays for normal ACE activities. Correlation with a comparison spectrophotometric method (Am J Med 59: 363-372, 1975) for ACE assay was excellent (r = 0.98) in 59 samples from healthy subjects and from patients with various diseases including active sarcoidosis. The novel assay principle presented here is simple and specific, and can be extended to use with various biological fluids and tissues, and to other enzymes as well.
我们描述了一种测定酶的新原理，此原理在此应用于检测人血清中的血管紧张素转换酶（ACE，EC 3.4.15.1）。酶抑制剂结合测定法基于标记抑制剂与酶活性中心的特异性结合。将血清（10 - 15微升）与125I标记的ACE抑制剂（“351A”，N -（1 - 羧基 - 3 - 苯基丙基）-L - 赖氨酰 - L - 脯氨酸的对羟基苯甲脒衍生物）在pH 7.0、37℃下于非平衡体系中孵育2小时。结合到ACE上的抑制剂通过吸附到包被活性炭上进行分离，对上清液中剩余的放射性进行计数，并根据标准曲线计算ACE值。血清中ACE的检测灵敏度为200 U/L，相当于从人肺中纯化得到的5.0 ng ACE。对于正常ACE活性，批内变异系数为3.9%，批间变异系数为6.4%。在59份来自健康受试者以及包括活动性结节病在内的各种疾病患者的样本中，与用于ACE检测的比较分光光度法（《美国医学杂志》59: 363 - 372, 1975）的相关性极佳（r = 0.98）。本文提出的这种新型测定原理简单且特异，可扩展用于各种生物体液和组织，以及其他酶的检测。
