MMP-1 proenzyme (human fibroblasts)

≥100mU/mg protein (Y. Masui, et al.; Biochem. Med. 17 , 215 (1977) ) . One unit is defined as the amount of enzyme that hydrolyzes 1μmol Dnp-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH per min. at 37°C, pH 7.0. APMA activation 1 hour at 37°C; enzyme dilution for assay 1:10/5-10μl for enzyme activity assay – incubation time 30min.

Activity: We recommend to dilute this enzyme preparation at least 1:10 and to take 5μl or less for the determination of activity. Specific activity can be assayed with the synthetic substrate N- (2,4) -dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg (Dnp-peptide) (Masui et al.) . Substrate concentration should be 0.5mg/ml in buffer 50mM TRIS-HCl, pH 7.0, 200mM NaCl, 5mM CaCl 2 , 1μM ZnCl 2 , 0.05% BRIJ35, 0.05% sodium azide, containing 0.05mg/ml albumin. One unit MMP catalyzes the hydrolysis of 1μmol Dnp-peptide/min at 37°C and pH 7.0. We recommend to employ the fluorogenic substrate (7-Methoxycoumarin-4-yl) acetyl-Pro-Leu-Gly-Leu-N-β-Dnp-L-α, β-diaminopropionyl-Ala-Arg-NH 2) (Knight et al. 1992) . The hydrolysis of the Gly-Leu bond separates the highly fluorescent (7-Methoxycoumarin-4-yl) acetyl group from the 2,4-dinitrophenyl resulting in an increase of fluorogenic intensity. The substrate should be kept as a 9.15mM stock solution in DMSO (10mg/ml) . In the assay the substrate concentration should be ~25mM. The assay can be performed in a 96-well microtiter plate (200μl per well) suitable for fluorogenic measurements (excitation wavelength of 328nm; emission wavelength of 393nm) . Activation: Do not dilute enzyme for activation! Activation is required by trypsin (2μl trypsin; 1mg/ml) for 10-20 min at 37°C and stopped by the addition of 10μl trypsin-inhibitor (2mg/ml) or aprotinin or TLCK. Activation can also be done by 2mM (final concentration) APMA for 60 min. at 37°C. Inhibitors: Only the activated and not the latent forms of wild-type MMP-1 protein is able to form a complex with TIMP-1. Quite in contrast to MMP-2 (gelatinase A) and MMP-9 (gelatinase B), in MMP-1 the C-terminal hemopexin domain does not interact with TIMP-1. The integrity of the catalytic domain of MMP-1 and its ability to bind Zn 2+ is absolutely required for complex formation with TIMP-1, which further underlines the importance of this region for proper regulation of enzymatic activity of MMP-1 (Vallon et al. 1997) . Therefore, the enzyme is also inhibited by chelators of divalent cations like EDTA or o-phenantroline.