Medintz Igor L, Clapp Aaron R, Brunel Florence M, Tiefenbrunn Theresa, Uyeda H Tetsuo, Chang Eddie L, Deschamps Jeffrey R, Dawson Philip E, Mattoussi Hedi
Center for Bio/Molecular Science and Engineering, Code 6900, US Naval Research Laboratory, Washington, DC 20375, USA.
Nat Mater. 2006 Jul;5(7):581-9. doi: 10.1038/nmat1676. Epub 2006 Jun 25.
Proteases are enzymes that catalyse the breaking of specific peptide bonds in proteins and polypeptides. They are heavily involved in many normal biological processes as well as in diseases, including cancer, stroke and infection. In fact, proteolytic activity is sometimes used as a marker for some cancer types. Here we present luminescent quantum dot (QD) bioconjugates designed to detect proteolytic activity by fluorescence resonance energy transfer. To achieve this, we developed a modular peptide structure which allowed us to attach dye-labelled substrates for the proteases caspase-1, thrombin, collagenase and chymotrypsin to the QD surface. The fluorescence resonance energy transfer efficiency within these nanoassemblies is easily controlled, and proteolytic assays were carried out under both excess enzyme and excess substrate conditions. These assays provide quantitative data including enzymatic velocity, Michaelis-Menten kinetic parameters, and mechanisms of enzymatic inhibition. We also screened a number of inhibitory compounds against the QD-thrombin conjugate. This technology is not limited to sensing proteases, but may be amenable to monitoring other enzymatic modifications.
蛋白酶是催化蛋白质和多肽中特定肽键断裂的酶。它们在许多正常生物过程以及包括癌症、中风和感染在内的疾病中都起着重要作用。事实上，蛋白水解活性有时被用作某些癌症类型的标志物。在此，我们展示了一种发光量子点（QD）生物共轭物，其设计目的是通过荧光共振能量转移来检测蛋白水解活性。为实现这一目标，我们开发了一种模块化肽结构，使我们能够将用于蛋白酶半胱天冬酶-1、凝血酶、胶原酶和胰凝乳蛋白酶的染料标记底物连接到量子点表面。这些纳米组装体中的荧光共振能量转移效率易于控制，并且在过量酶和过量底物条件下都进行了蛋白水解测定。这些测定提供了包括酶促速度、米氏动力学参数和酶抑制机制在内的定量数据。我们还针对量子点-凝血酶共轭物筛选了多种抑制性化合物。这项技术不仅限于检测蛋白酶，还可能适用于监测其他酶促修饰。
