A semi-synthetic ion channel platform for detection of phosphatase and protease activity.
A semi-synthetic ion channel platform for detection of phosphatase and protease activity.
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DOI：
10.1021/nn901231h
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发表时间：
2009-11-24
期刊：
影响因子：
17.1
通讯作者：
Yang, Jerry
中科院分区：
文献类型：
--
作者：
Macrae, Michael X.;Blake, Steven;Jiang, Xiayun;Capone, Ricardo;Estes, Daniel J.;Mayer, Michael;Yang, Jerry
关键词：
灵敏的方法来探测酶的活性是重要的临床分析和阐明这些蛋白质在复杂的生化网络中的作用。本文介绍了一种半合成离子通道平台，用于高灵敏度检测两种不同类型酶的活性。在第一种情况下，该方法使用单离子通道电导测量来跟踪在碱性磷酸酶（AP）存在下附着在gramicidin a （gA，一种离子通道形成肽）c端上的磷酸基团的酶催化水解。该磷酸基的酶解去除gA孔入口的负电荷，导致产物与原始的gA-磷酸底物相比，单离子通道电导率明显降低。该技术采用标准的商业双分子层设置，利用酶的催化周转和通过单个gA孔的离子通量的放大特性来检测溶液中活性AP的皮摩尔浓度。此外，该技术使研究酶的动力学成为可能，并通过跟踪gA-磷酸底物在不同浓度的AP存在下随时间转化为产物，提供了观察到的速率常数（kcat）和米切里斯常数（KM）的估计。在第二种情况下，用炭疽致死因子（LF）修饰gA的底物进行蛋白水解裂解提供了一种检测LF活性的灵敏方法。举例说明基于离子通道的传感用于检测潜在的生物战剂的实用性。这种基于离子通道的平台代表了一种强大的、新颖的方法来监测溶液中两种不同类型酶的飞摩尔到皮摩尔的活性。此外，该平台有潜力实现小型化，具有成本效益的生物分析分析，补充目前建立的分析。
Sensitive methods to probe the activity of enzymes are important for clinical assays and for elucidating the role of these proteins in complex biochemical networks. This paper describes a semi-synthetic ion channel platform for detecting the activity of two different classes of enzymes with high sensitivity. In the first case, this method uses single ion channel conductance measurements to follow the enzyme-catalyzed hydrolysis of a phosphate group attached to the C-terminus of gramicidin A (gA, an ion channel-forming peptide) in the presence of alkaline phosphatase (AP). Enzymatic hydrolysis of this phosphate group removes negative charges from the entrance of the gA pore, resulting in a product with measurably reduced single ion channel conductance compared to the original gA-phosphate substrate. This technique employs a standard, commercial bilayer setup and takes advantage of the catalytic turnover of enzymes and the amplification characteristics of ion flux through individual gA pores to detect picomolar concentrations of active AP in solution. Furthermore, this technique makes it possible to study the kinetics of an enzyme and provides an estimate for the observed rate constant (kcat) and the Michaelis constant (KM) by following the conversion of the gA-phosphate substrate to product over time in the presence of different concentrations of AP. In the second case, modification of gA with a substrate for proteolytic cleavage by anthrax lethal factor (LF) afforded a sensitive method for detection of LF activity, illustrating the utility of ion channel-based sensing for detection of a potential biowarfare agent. This ion channel-based platform represents a powerful, novel approach to monitor the activity of femtomoles to picomoles of two different classes of enzymes in solution. Furthermore, this platform has the potential for realizing miniaturized, cost-effective bioanalytical assays that complement currently established assays.
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影响因子：
15
作者：
Cockroft, Scott L.;Chu, John;Ghadiri, M. Reza
通讯作者：
Ghadiri, M. Reza
影响因子：
64.8
作者：
Cornell, BA;BraachMaksvytis, VLB;Pace, RJ
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影响因子：
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Kramer, Richard H.
影响因子：
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通讯作者：
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影响因子：
158.5
作者：
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通讯作者：
ZAMCHECK, N