Rapid analysis of matrix metalloproteinase-3 activity by gelatin arrays using a spectral surface plasmon resonance biosensor.
We developed a novel assay system using an array-based spectral surface plasmon resonance (SPR) biosensor for a high-throughput analysis of matrix metalloproteinase (MMP)-3 activity. Gelatin arrays were fabricated by immobilizing gelatin, a MMP-3 substrate, on amine-modified gold arrays. MMP-3 activity was determined by monitoring the shift of SPR wavelength caused by gelatin proteolysis. The gelatinolytic activity of MMP-3, which caused a decrease of the SPR wavelength, was verified by SPR spectroscopy, atomic force microscopy, and fluorescence-based protein arrays. MMP-3 activity increased by three non-ionic detergents in a dose-dependent manner, and Brij-35 was most effective. The array-based SPR biosensor was successfully applied to the rapid analysis of dose-dependent MMP-3 activity and its inhibition with tissue inhibitors of metalloproteinase 1 and GM6001, MMP inhibitors. Therefore, this new assay system using a spectral SPR biosensor is simple, label-free, and high-throughput, and is likely to have a strong potential for inhibitor screening.
Jung
Se-Hui
SH
Department of Molecular and Cellular Biochemistry and Vascular System Research Center, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 200-701, Korea.
Kong
Deok-Hoon
DH
Park
Jun Hyoung
JH
Lee
Seung-Taek
ST
Hyun
Jinho
J
Kim
Young-Myeong
YM
Ha
Kwon-Soo
KS
D015374:Biosensing Techniques / Q000379:methods*; D002460:Cell Line; D004151:Dipeptides / Q000494:pharmacology; D005780:Gelatin / Q000737:chemistry*; D006046:Gold / Q000737:chemistry; D057166:High-Throughput Screening Assays; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D011480:Protease Inhibitors / Q000494:pharmacology; D040081:Protein Array Analysis; D020349:Surface Plasmon Resonance / Q000379:methods*; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000494:pharmacology
D004151:Dipeptides; D055767:Immobilized Proteins; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D011480:Protease Inhibitors; D019715:Tissue Inhibitor of Metalloproteinase-1; D006046:Gold; D005780:Gelatin; D019278:Matrix Metalloproteinase 3