Department of Radiology, University of Pennsylvania, Philadelphia, PA 19104, USA.
Anal Biochem. 2012 Mar 15;422(2):96-102. doi: 10.1016/j.ab.2011.12.009. Epub 2011 Dec 9.
There has been recent growth in the development of activatable near-infrared (NIR) fluorescent probes for molecular imaging, generally designed by placing fluorochromes on a cleavable substrate in close proximity to one another, such that they self-quench, but fluoresce on separation via enzymatic cleavage of the substrate. Although these probes offer excellent contrast, the detection of enzyme activity has largely only been described qualitatively. In order to assess the effectiveness of a probe, it is useful to have a quantitative measure, such as the enzyme-substrate kinetic parameters. We have developed an assay to determine kinetic parameters and applied it to an intramolecularly quenched molecule, Pyro-PtdEtn-BHQ, a NIR fluorescent probe specific to phosphatidylcholine-specific phospholipase C. The development of this assay includes corrections for intermolecular quenching, calibration, optimization of reaction mixtures, and determination of kinetic and inhibition parameters. This assay can easily be extended to analyze and compare the efficiency of other fluorescent activatable phospholipase probes as suitable molecular imaging agents.
近年来，用于分子成像的可激活近红外（NIR）荧光探针的发展取得了进展，通常通过将荧光团放置在彼此靠近的可裂解底物上来设计，使得它们自猝灭，但通过酶裂解底物分离时会发出荧光。尽管这些探针提供了出色的对比度，但酶活性的检测在很大程度上仅被定性描述。为了评估探针的有效性，最好有一个定量指标，例如酶-底物的动力学参数。我们开发了一种测定动力学参数的方法，并将其应用于一种分子内猝灭的分子 Pyro-PtdEtn-BHQ，这是一种针对磷脂酰胆碱特异性磷脂酶 C 的 NIR 荧光探针。该测定方法的开发包括对分子间猝灭的校正、校准、反应混合物的优化以及动力学和抑制参数的确定。该测定方法可以很容易地扩展到分析和比较其他荧光可激活磷脂酶探针作为合适的分子成像剂的效率。
