title: Real-time monitoring of matrix metalloproteinase-9 collagenolytic activity with a surface plasmon resonance biosensor.
abstract: 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.
authors.affiliation: Department of Chemistry, College of Humanities and Sciences, Nihon University, Sakurajousui, Setagaya, Tokyo 156-8550, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Shoji
authors.forename: Mitsutaka
authors.initials: M
authors.lastname: Kabeya
authors.forename: Masao
authors.initials: M
authors.lastname: Sugawara
mesh_terms: D015374:Biosensing Techniques; D020134:Catalytic Domain; D002942:Circular Dichroism; D024141:Collagen Type IV / Q000378:metabolism*; D003902:Detergents / Q000737:chemistry; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000737:chemistry; D040081:Protein Array Analysis; D011487:Protein Conformation; D012965:Sodium Chloride / Q000737:chemistry; D012996:Solutions / Q000737:chemistry; D013388:Succinimides / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods*; D013997:Time Factors
chemical_list: D024141:Collagen Type IV; D003902:Detergents; D012996:Solutions; D013388:Succinimides; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; D011092:Polyethylene Glycols; D012965:Sodium Chloride; D020780:Matrix Metalloproteinase 9; C515901:Brij 35