期刊
ANALYTICAL BIOCHEMISTRY
卷 419, 期 1, 页码 53-60出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2011.07.031
关键词
Matrix metalloproteinase-9; Collagen IV; Surface plasmon resonance; Collagenolytic activity; Kinetic parameters
资金
- Ministry of Education, Culture, Sports, Science, and Technology of Japan [21350045]
- Grants-in-Aid for Scientific Research [21350045] Funding Source: KAKEN
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.
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