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-A(2)pr(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 nm, lambda(em) 393 nm) 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 mu M. Thus, it was concluded that the activity of r-MMP-7 can be reliably measured by;he 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 (Nephila 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. (C) 1997 Elsevier Science B.V.