Grasso Giuseppe, D'Agata Roberta, Rizzarelli Enrico, Spoto Giuseppe, D'Andrea Luca, Pedone Carlo, Picardi Andrea, Romanelli Alessandra, Fragai Marco, Yeo Kwon Joo
Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici, Via C. Ulpiani 27, 70126, Bari, Italy.
J Mass Spectrom. 2005 Dec;40(12):1565-71. doi: 10.1002/jms.929.
Matrix metalloproteinases (MMPs) are a family of Zn-dependent endo-peptidases known for their ability to cleave several components of the extracellular matrix, but which can also cleave many non-matrix proteins. There are many evidences that MMPs are involved in physiological and pathological processes, and a huge effort has been put in the development of possible inhibitors that could reduce the activity of MMPs, as it is clear that the ability to monitor and control such activity plays a pivotal role in the search for potential drugs aimed at finding a cure for several diseases such as pulmonary emphysema, rheumatoid arthritis, fibrotic disorders and cancer.A powerful method currently available to study enzyme-inhibitor interactions is based on the use of the surface plasmon resonance (SPR) technique. When MMP interactions are studied, a procedure by which inhibitors are normally anchored on sensor chips and SPR technique is used in order to study their interaction with MMPs molecules is usually followed. This is because it is currently believed that MMPs cannot be anchored on the sensor-chip surface without losing their activity. However, this approach gives rise to problems, as the anchoring of low-molecular-weight inhibitors on gold surfaces easily affects their ability to interact with MMPs. For this reason, the anchoring of MMPs is highly desirable.A new experimental protocol that couples the Fourier transform-SPR (FT-SPR) technique with electrospray ionization-mass spectroscopy (ESI-MS) is described here for the evaluation of the activity of MMP-1 catalytic domain (cdMMP-1) anchored on gold surfaces. The cdMMP-1 surface coverage is calculated by using FT-SPR and the enzyme activity is estimated by ESI-MS. The proposed method is label-free.
基质金属蛋白酶（MMPs）是一类锌依赖性内肽酶，以其能够切割细胞外基质的多种成分而闻名，但也能切割许多非基质蛋白。有许多证据表明MMPs参与生理和病理过程，并且人们已经付出巨大努力来开发可能降低MMPs活性的抑制剂，因为很明显，监测和控制这种活性的能力在寻找旨在治愈多种疾病（如肺气肿、类风湿性关节炎、纤维化疾病和癌症）的潜在药物中起着关键作用。目前可用于研究酶-抑制剂相互作用的一种强大方法是基于表面等离子体共振（SPR）技术的应用。在研究MMP相互作用时，通常遵循一种程序，即先将抑制剂固定在传感器芯片上，然后使用SPR技术来研究它们与MMP分子的相互作用。这是因为目前认为MMPs无法固定在传感器芯片表面而不丧失其活性。然而，这种方法会产生问题，因为低分子量抑制剂固定在金表面很容易影响它们与MMPs相互作用的能力。因此，非常需要固定MMPs。本文描述了一种将傅里叶变换-SPR（FT-SPR）技术与电喷雾电离质谱（ESI-MS）相结合的新实验方案，用于评估固定在金表面的MMP-1催化结构域（cdMMP-1）的活性。通过FT-SPR计算cdMMP-1的表面覆盖率，并通过ESI-MS估计酶活性。所提出的方法无需标记。
