Real-time monitoring of matrix metalloproteinase-9 collagenolytic activity with a surface plasmon resonance biosensor
Real-time monitoring of matrix metalloproteinase-9 collagenolytic activity with a surface plasmon resonance biosensor
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DOI：
10.1016/j.ab.2011.07.031
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发表时间：
2011-12-01
影响因子：
2.9
通讯作者：
Sugawara, Masao
中科院分区：
文献类型：
--
作者：
Shoji, Atsushi;Kabeya, Mitsutaka;Sugawara, Masao
我们描述了一种使用表面等离子共振 (SPR) 生物传感器实时监测天然三螺旋 IV 型胶原蛋白基质金属蛋白酶 9 (MMP-9) 溶胶活性的简单方法。 MMP-9 的蛋白水解活性通过固定在传感器表面的 IV 型胶原蛋白裂解导致的 SPR 信号减少来测量。通过SPR方法估算全长MMP-9及其催化结构域的动力学参数——催化常数(k(cat))、结合速率常数(k(a))和解离速率常数(k(d))。反应溶液中氯化钠和非离子去污剂 Brij-35 的存在导致 MMP-9 的胶原蛋白溶解活性降低，同时抑制了 MMP-9 和半胱胺修饰芯片之间的非特异性相互作用。 MMP-9与其催化结构域之间的动力学参数比较表明，MMP-9的缔合常数远大于催化结构域的缔合常数，这表明血红素样结构域、纤连蛋白II型重复基序和连接区（O-糖基化结构域）之间的相互作用在识别IV型胶原蛋白中发挥重要作用。 (C) 2011 Elsevier Inc. 保留所有权利。
We describe a simple method for real-time monitoring of matrix metalloproteinase-9 (MMP-9) collagenolytic activity for native triple helical collagen IV with a surface plasmon resonance (SPR) biosensor. The proteolytic activity of MMP-9 is measured as a decrease in the SPR signal resulting from the cleavage of collagen IV immobilized on the sensor surface. The kinetic parameters of full-length MMP-9 and its catalytic domain-catalytic constant (k(cat)), association rate constant (k(a)), and dissociation rate constant (k(d))-were estimated by the SPR method. The presence of sodium chloride and a nonionic detergent Brij-35 in a reaction solution led to the lower collagenolytic activity of MMP-9, whereas they suppressed the nonspecific interaction between MMP-9 and a cysteamine-modified chip. The comparison of kinetic parameters between MMP-9 and its catalytic domain revealed that the association constant of MMP-9 is much larger than that of the catalytic domain, suggesting that the interplay among hemopexin-like domain, fibronectin type II repeats motif, and linker region (O-glycosylated domain) plays an important role in recognizing collagen IV. (C) 2011 Elsevier Inc. All rights reserved.