Grasso Giuseppe, Fragai Marco, Rizzarelli Enrico, Spoto Giuseppe, Yeo Kwon Joo
Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici, Bari, Italy.
J Am Soc Mass Spectrom. 2007 May;18(5):961-9. doi: 10.1016/j.jasms.2007.02.003. Epub 2007 Mar 23.
Matrix metalloproteinases (MMPs) are cell-secreted soluble and membrane-tethered enzymes that are capable of degrading extracellular matrix proteins, but also can process a number of bioactive molecules. They are involved in the cleavage of cell surface receptors, but are also thought to play a major role on cell behaviors as well as in diverse physiological and pathological processes, including embryonic development, wound repair, inflammatory diseases, and cancer. For these reasons, it is obvious that a control over MMPs activity is highly desirable. Consequently, the frantic search for new inhibitors has been coupled to the development of high-throughput methods able to rapidly screen the effect of possible MMP inhibitors on the activity of these enzymes. In this scenario, solid-state-based methods play a major role because of their compatibility with array formats that are able to extract more information from smaller sample volumes and offer some important advantages that are not available in the standard solution assays. In this work, the catalytic domain of MMP-12 was immobilized on a gold substrate and the surface coverage was measured by FT-SPR experiments. A new experimental procedure was developed to freeze-dry the anchored molecules and their activity was measured by ESI-MS. The kinetics parameters obtained for the immobilized enzyme are in good accordance with those reported for similar systems in solution. Inhibition of the immobilized molecules was also carried out, demonstrating the applicability of the method for rapid screening of MMP inhibitors.
基质金属蛋白酶（MMPs）是细胞分泌的可溶性和膜结合酶，它们不仅能够降解细胞外基质蛋白，还能加工多种生物活性分子。它们参与细胞表面受体的裂解，但也被认为在细胞行为以及多种生理和病理过程中发挥重要作用，包括胚胎发育、伤口修复、炎症性疾病和癌症。基于这些原因，显然非常需要控制MMPs的活性。因此，在开发能够快速筛选可能的MMP抑制剂对这些酶活性影响的高通量方法的同时，人们也在疯狂寻找新的抑制剂。在这种情况下，基于固态的方法发挥了重要作用，因为它们与阵列形式兼容，能够从更小的样本体积中提取更多信息，并提供一些标准溶液测定中没有的重要优势。在这项工作中，MMP-12的催化结构域被固定在金基底上，并通过傅里叶变换表面等离子体共振（FT-SPR）实验测量表面覆盖率。开发了一种新的实验程序来冻干固定的分子，并通过电喷雾质谱（ESI-MS）测量其活性。固定化酶获得的动力学参数与溶液中类似系统报道的参数非常吻合。还对固定化分子进行了抑制实验，证明了该方法在快速筛选MMP抑制剂方面的适用性。
