Chung A, Ryan J W, Pena G, Oza N B
Adv Exp Med Biol. 1979;120A:115-25. doi: 10.1007/978-1-4757-0926-1_12.
We have developed a sensitive, highly selective assay for human urinary kallikrein (HUK) that uses Pro-Phe-Arg-[3H]benzyl-amide as substrate. The substrate was prepared from Pro-Phe-Arg-3-iodo-benzylamide by dehalogenation in 3H2 gas. HUK is measured by its ability to release [3H]benzylamine. The pH optimum is 9.5. Urokinase, plasmin and thrombin do not interfere. The assay can measure as little as 5 ng of HUK in a 15 min incubation at 37 degrees C. Typically, we use 50 microliter of dialyzed urine for HUK assays. Reactions are terminated by adding 0.1 M NaOH, and reaction product is separated from substrate by partitioning with an equal volume of toluene. A sample of the toluene phase is submitted for liquid scintillation counting. As judged by separations obtained on molecular sieve chromatography (Sephacryl), only one urinary enzyme possesses the ability to hydrolyze our substrate. The enzyme MW 45,000, is inhibited by Trasylol but not by soya bean trypsin inhibitor (SBTI). It is reactive with and is inhibited by antibodies prepared against pure HUK.
我们已开发出一种灵敏、高选择性的人尿激肽释放酶（HUK）检测方法，该方法使用Pro-Phe-Arg-[3H]苄基酰胺作为底物。底物由Pro-Phe-Arg-3-碘苄基酰胺通过在3H2气体中脱卤制备而成。通过HUK释放[3H]苄胺的能力来测定其含量。最适pH为9.5。尿激酶、纤溶酶和凝血酶不产生干扰。在37℃孵育15分钟的情况下，该检测方法能够检测低至5纳克的HUK。通常，我们使用50微升透析尿液进行HUK检测。通过加入0.1 M氢氧化钠终止反应，并通过与等体积甲苯分配将反应产物与底物分离。取甲苯相样品进行液体闪烁计数。根据在分子筛色谱（Sephacryl）上获得的分离结果判断，只有一种尿酶具有水解我们底物的能力。该酶分子量为45,000，受抑肽酶抑制，但不受大豆胰蛋白酶抑制剂（SBTI）抑制。它与针对纯HUK制备的抗体发生反应并受其抑制。
