Huang Wen-Yao, Wan Xiaojuan, Zhang Shuilan
Department of Laboratory Medicine, the 5th Municipal Hospital of Shenzhen, Shenzhen, China.
Clin Lab. 2009;55(3-4):106-13.
To determine the Km, Vmax, and Ki of BOC-Leu-Ser-Thr-Arg-pNA (B3644) in the determination of kallikrein, and then to establish the procedure for plasma prekallikrein/kallikrein determination. Method Chromogenic substrate assays on a semi-auto spectrophotometer were employed.
The apparent activities of seven substrates: B3644, S2238, S2251, ChromozymPCa, T6140, ChromozymPK, and T1637, for porcine kallikrein were 0.788, 0.691, 0.659, 0.636, 0.438, 0.396, and 0.145, respectively, that of B3644 being the highest. Amidolysis caused by kaolin activated human normal plasma was independent of coagulation factors II, X, XI, and protein C, but dependent on prekallikrein and to some extent on factor XII. The kallikrein's, thrombin's, and plasmin's Km and Vmax for the B3644 were determined and calculated to be 235 micromol/L and 337 nmol x s(-1) x U(-1), 469 micromol/L and 63 nmol x s(-1) x U(-1), and 70 micromol/L and 358 nmol x s(-1) x U(-1), respectively. At the same time, the kallikrein, thrombin, and plasmin Ki values for antithrombin III (AT-III) or limabean trypsin inhibitor (LBTI) were detected to be 840 or 2.50. 32 or 0.32, and, 108 or 1.55 U/L, [d1] respectively. With B3644, the recovery percentages of kallikrein and kaolin activated plasma were 100%. Intra-assay CV values ranged from 2.3% to 4.6%. By using B3644 and introducing AT-III or LBLI into the determination system, an optimized procedure for prekallikrein/kallikrein determination was obtained. With this procedure, the mean percentage values of plasma prekallikrein in normal subjects, pregnant women (gestation > or =24 weeks), and in patients with hypertension, hepatic failure, or with advanced cancers were determined to be 113.5, 151.6 (P<0.01), 173.2 (P<0.01), 43.5 (P<0.005), and 88.7 (P<0.05), respectively.
B3644 was identified as a suitable substrate for the determination of plasma prekallkrein/kallikrein activity. A sensitive and reliable chromogenic substrate assay for human plasma prekallikrein was developed by using this novel substrate.
测定BOC-亮氨酸-丝氨酸-苏氨酸-精氨酸-对硝基苯胺（B3644）在激肽释放酶测定中的米氏常数（Km）、最大反应速度（Vmax）和抑制常数（Ki），进而建立血浆前激肽释放酶/激肽释放酶的测定方法。方法采用半自动分光光度计进行发色底物分析。
七种底物：B3644、S2238、S2251、ChromozymPCa、T6140、ChromozymPK和T1637对猪激肽释放酶的表观活性分别为0.788、0.691、0.659、0.636、0.438、0.396和0.145，其中B3644的活性最高。高岭土激活的人正常血浆引起的酰胺水解不依赖于凝血因子II、X、XI和蛋白C，但依赖于前激肽释放酶，且在一定程度上依赖于因子XII。测定并计算了激肽释放酶、凝血酶和纤溶酶对B3644的Km和Vmax，分别为235微摩尔/升和337纳摩尔·秒⁻¹·单位⁻¹、469微摩尔/升和63纳摩尔·秒⁻¹·单位⁻¹、70微摩尔/升和358纳摩尔·秒⁻¹·单位⁻¹。同时，检测到抗凝血酶III（AT-III）或利马豆胰蛋白酶抑制剂（LBTI）对激肽释放酶、凝血酶和纤溶酶的Ki值分别为840或2.50、32或0.32、108或1.55单位/升。使用B3644时，激肽释放酶和高岭土激活血浆的回收率均为100%。批内变异系数（CV）值在2.3%至4.6%之间。通过使用B3644并在测定系统中引入AT-III或LBLI，获得了一种优化的前激肽释放酶/激肽释放酶测定方法。采用该方法，测定正常受试者、孕妇（孕周≥24周）、高血压患者、肝功能衰竭患者或晚期癌症患者血浆前激肽释放酶的平均百分比值分别为113.5、151.6（P<0.01）、173.2（P<0.01）、43.5（P<0.005）和88.7（P<0.05）。
B3644被确定为测定血浆前激肽释放酶/激肽释放酶活性的合适底物。利用这种新型底物开发了一种灵敏可靠的人血浆前激肽释放酶发色底物测定方法。
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