FIGURE 4.
Purification, characterization, and MMP-14 inhibitory activity of N-TIMP2 variants. A, size-exclusion chromatography for clone N-TIMP2B. B, mass spectrometry analysis for clone N-TIMP2B after size-exclusion chromatography. C, SDS-PAGE analysis on 15% polyacrylamide gel under reducing conditions for the purified clones N-TIMP2WT, N-TIMP2A, N-TIMP2B, N-TIMP2C, and N-TIMP2D. D, MMP-14 inhibition by N-TIMP2WT. E, MMP-14 inhibition by N-TIMP2 variants. D and E, MMP-14CAT was incubated with N-TIMP2WT and N-TIMP2 variants at various concentrations. The substrate Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 was added, and the fluorescent signal upon substrate cleavage by MMP-14CAT was measured. The slopes (cleavage velocities) at each inhibitor concentration were measured, and the curves were fitted by the Morrison equation (Equation 1) to obtain the Ki values.