Flow injection analysis for measurement of activity of matrix metalloproteinase-7 (MMP-7).
A simple and convenient method for measuring the activity of a recombinant human matrix metalloproteinase 7 (MMP-7, matrilysin) was developed by flow injection analysis (FIA). For this method, purified recombinant MMP-7 zymogen expressed in E. coli and the substrate peptide (MOCAc-Pro-Leu-Gly-Leu-A2pr(DNP)-Ala-Arg-NH2) were used. Following the incubation of substrate peptide with activated r-proMMP-7, the resulting fluorescent product peptide (MOCAc-Pro-Leu-GLY) was monitored with a fluorescence detector (lambda ex 328 mm, lambda em 393 mm) without chromatographic separation. In this FIA system, the analysis time is 2 min and the standard curve is linear from 5 to 100 pmol of the product peptide injected. In order to use this FIA system as a method for screening inhibitors against MMP-7, the effects of CaCl2, EDTA and of the tissue inhibitor of metalloproteinase-1, and -2, were tested. A synthetic PRCGXPD-containing peptide (BS-10) was also observed to inhibit MMP-7 activity, with an IC50 value of 104 microM. Thus, it was concluded that the activity of r-MMP-7 can be reliably measured by the proposed system. Furthermore, to confirm the utility of this FIA system as a screening method, the inhibitory activity of the MMP-related substance in Joro spider (Nephilia clavata) venom was measured by this method. This inhibitory activity was observed in an extract of a venom diluted 1000-fold. Thus, the FIA method is not only simple and quick, but also sensitive enough to screen and analyze the inhibitory properties of a large number of test compounds.
Itoh
M
M
Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Setsunan University, Osaka, Japan.
Osaki
M
M
Chiba
T
T
Masuda
K
K
Akizawa
T
T
Yoshioka
M
M
Seiki
M
M
D004791:Enzyme Inhibitors / Q000032:analysis*; D004926:Escherichia coli; D017022:Flow Injection Analysis; D006801:Humans; D020783:Matrix Metalloproteinase 7; D008666:Metalloendopeptidases / Q000032:analysis* / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins / Q000032:analysis*; D013111:Spider Venoms / Q000737:chemistry; D013997:Time Factors
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D013111:Spider Venoms; D008666:Metalloendopeptidases; D020783:Matrix Metalloproteinase 7