Tri-peptide chloromethylketones have been utilized extensively to irreversibly inhibit various serine proteases (1-5). Among the most common chloromethylketones are FPRCK (Phe-Pro-Arg-chloromethylketone; commonly referred to as PPACK), which is a rapid thrombin inhibitor and EGRCK (Glu-Gly-Arg-chloromethylketone; commonly referred to as GGACK), which is a rapid factor Xa inhibitor (1). Both FPRCK and EGRCK are used extensively during protein isolation procedures to inhibit serine protease activity and prevent further conversion of zymogens to active enzymes. Recently, the modification of these tri-peptide chloromethylketones with reporting groups, such as fluorescent probes (6-8,14), radioactive labels (9) or thioreactive-labels (10), has provided a unique approach to the study of various serine proteases. These probes are useful because they allow a means of reporting molecular changes in an enzyme, and not its zymogen, while also inhibiting the enzymatic activity.
The use of biotin as a reporting group has been used extensively with antibodies in ELISA based assays and in western blotting. The biotin, in conjunction with avidin, creates a highly sensitive method for detecting antibodies, and therefore, antigens. By modifying the tripeptide-chloromethylketones with a biotin group, the sensitivity of the avidin/biotin system can be extended to study serine proteases without the need for specific antibodies to the active enzymes.
Biotinylated tripeptide chloromethyl ketones can be used in a variety of ways (11-13). First, the compounds can be reacted with unwanted serine proteases in a sample or preparation, and can then be removed along with the protease using avidin-Sepharose (11). Second, the biotinylated-serine protease can be visualized on a blot without the use of specific antibodies (11). Third, the biotinylated serine protease can be quantitated in an active-site specific immunoassay (12,13,15), such as the tPA-CASSIA (see Assay Kits). The spacer utilized on these compounds has been optimized to allow good reactivity of the biotinylated FPRCK and the biotinylated EGRCK in the above mentioned procedures.
In addition to biotinylated chloromethyl ketones, fluorescein labelled compounds are also available. The fluorescein labelled compounds are useful in both Western blot and fluorescent imaging applications.
Biotinylated and fluorescein labelled FPRCK and EGRCK are prepared by the method of Williams et al. (11).
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2、可逆抑制剂：包括
a、竞争性抑制剂，抑制剂与底物竞争性结合酶反应中心，使Km增大，而Vmax不变 b、非竞争性抑制剂，酶与抑制剂结合后还能与底物结合，但活性降低，使Vmax减小，而Km不变
c、反竞争性抑制剂，酶只能与底物结合后才能与抑制剂结合，Vmax与Km都减小
可逆抑制剂可用透析等方法除去，使酶恢复作用
1、测定酶比活力：底物需要过量么？测定时间多长？是否可以加入过量的底物，然后测定3min吸光度的增加值，从吸光度的变化值计算比活力。
2、在酶抑制剂筛选的过程中，是否需要保证底物过量？还是要水浴一定时间让反应完全？我看到文献说用终浓度为0.1μmol/mL的底物，终浓度为45U/ml的酶，我想问，假设总体积为1ml，那么终浓度为45U/ml的酶岂不是每分钟能转化4.8μmol的底物？那么0.1μmol/mL的底物不就几秒钟就反应完了？那么怎么测定初速度？
1.对于抑制剂筛选工作（求ic50）是不是体系内酶与底物的量（底物应该是过量的）对实验结果影响不大。
2.如果要求算Km值，是不是需要知道反应产物的绝对量。反应时间文献上都是5分钟，反应速度就用反应产物量除以反应时间即可。
3.酶是进口分装的，规格5U，一次用不完，用PBS稀释后如何保存
谢谢
《血管紧张素转换酶抑制剂在心血管病中应用的中国专家共识》.PDF(242.69k)
