Reverse zymography using fluorogenic substrates for protease inhibitor detection
Reverse zymography using fluorogenic substrates for protease inhibitor detection
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DOI：
10.1002/elps.200306142
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发表时间：
2005-03
期刊：
影响因子：
2.9
通讯作者：
Q. T. Le;A. Ohashi;S. Hirose;N. Katunuma
中科院分区：
文献类型：
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作者：
Q. T. Le;A. Ohashi;S. Hirose;N. Katunuma
描述了一种利用凝胶中的荧光蛋白酶底物检测蛋白酶抑制剂的新颖、灵敏的方法。蛋白酶抑制剂在含有共聚肽底物，即 4-甲基-香豆基-7-酰胺 (MCA) 的十二烷基硫酸钠 (SDS)-聚丙烯酰胺凝胶上进行分离。作为凝胶中掺入的底物，胰蛋白酶使用 Boc-Phe Ser-Arg-MCA，α-胰凝乳蛋白酶使用 Suc-Ala-Ala-Pro-Phe-MCA，木瓜蛋白酶使用 Z-Phe-Arg-MCA。电泳后，用目标蛋白酶溶液清洗并孵育凝胶，使底物被蛋白酶裂解，并释放荧光 7 氨基-4 甲基香豆素 (AMC)，并在紫外透照仪下检测到。未切割的肽-MCA 底物保留在抑制剂所在的位置，并在浅绿色荧光背景凝胶上显示为深蓝色条带。与之前的其他方法相比，这种新方法具有几个优点，包括：（i）可以在更短的时间内大大提高灵敏度，这可能有助于在少量粗材料中发现新的蛋白酶抑制剂； (ii) 由于孵育时间很短并且不需要时间进行染色和脱色步骤，所以程序非常简单和快速； (iii)由于这些探针使用底物特异性/目标蛋白酶，因此它们是检测和区分各种目标蛋白酶的未知蛋白酶抑制剂的极好工具。
A novel, sensitive method for detecting protease inhibitors by using fluorescent protease substrates in gels is described. The protease inhibitors were separated on sodium dodecyl sulfate (SDS)‐polyacrylamide gels containing a copolymerized peptide substrate, namely 4‐methyl‐coumaryl‐7‐amide (MCA). As the incorporated substrates in the gel, Boc‐Phe Ser‐Arg‐MCA was used for trypsin, Suc‐Ala‐Ala‐Pro‐Phe‐MCA for α‐chymotrypsin, and Z‐Phe‐Arg‐MCA for papain. After electrophoresis, washing and incubating the gel with the target protease solutions allowed the substrate to be cleaved by the protease, and the release of the fluorescent 7 amino‐4 methyl‐coumarin (AMC), which was detected under a UV transilluminator. The uncleaved peptide‐MCA substrate remained where the inhibitors were present, and was visualized as dark blue bands on the light‐green fluorescent background gel. This new method offers several advantages over other previous methods including: (i) greatly increased sensitivity can be achieved in a shorter period of time, which may be useful for discovering new protease inhibitors in small amounts of crude material; (ii) the procedure is quite simple and quick since the incubation period is very short and no time is needed for staining and destaining steps; (iii) since these probes using substrate specificity/target proteases, they are excellent tools for detection and discrimination of unknown protease inhibitors for various target proteases.