Activation generates two molecular species. One species begins at the N-terminus with Tyr156 of full-length MMP-24, while the other species is two aa residues shorter and starts with Leu158. Both species extend to Ser351 of MMP-24 and contain an additional His-tag sequence at the C-terminus.
Product Details
| Alternative Name: | Matrix metalloproteinase 24 |
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| MW: | ~23kDa |
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| Source: | Produced in E. coli. |
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| EC: | 3.4.24.- |
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| UniProt ID: | Q9Y5R2 |
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| Concentration: | 0.2mg/ml |
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| Formulation: | Liquid. In 50mM TRIS-HCl, pH 7.5, containing 150mM NaCl and 5mM CaCl2. |
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| Purity: | ≥90% (SDS-PAGE) |
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| Specific Activity: | ≥120mU/mg protein. One unit is defined as the amount of enzyme that hydrolyzes 1µmol Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 per min. at 37°C, pH 7.5. |
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| Shipping: | Dry Ice |
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| Long Term Storage: | -80°C |
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| Protocol: |
Measurement of catalytic activity
1 Preparation and stability of solutions:
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Peptide hydrolysis buffer: 50mM TRIS-HCl, pH 7.5, 150mM NaCl, 5mM CaCl2, 0.025% Brij 35. Solution is stable for several weeks at 4°C.
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Stock solution of peptide substrate: 100µM solution of Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg in 20% DMSO. Store at -20°C.
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Stock solution of unquenched peptide: 10µM solution of (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-NH2 (Mca-Pro-Leu) in 20% DMSO. Store at -20°C.
2 Assay protocol:
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The activity of MMP-24 is measured fluorimetrically with a synthetic internally quenched fluorescent substrate according to Knight et al.
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An excitation wavelength of 328nm and an emission wavelength of 393nm are set in an appropriate fluorimeter. The instrument is calibrated with the unquenched peptide Mca-Pro-Leu at a concentration corresponding to between 2 and 10% hydrolysis of the protease substrate. Kinetic reactions are conveniently carried out in a constant volume of 2.5ml.
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The substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg is diluted in peptide hydrolysis buffer to a concentration of 0.8µM and equilibrated at a temperature of 37°C.
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Aliquots of 2 to 4µl of the activation mixture are than added and the increase in fluorescence is recorded over a time interval between 2 and 12 minutes.
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Activity units per ml enzyme solution are calculated according to the following equation:
Activity (U/ml) = (CMca-Pro-Leu/FMca-Pro-Leu) x (δFMca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg/Venzyme) x Vtotal
CMca-Pro-Leu: Concentration of Mca-Pro-Leu used for calibration of the fluorimeter (µmoles/ml).
FMca-Pro-Leu: Fluorescence of Mca-Pro-Leu at the concentration CMca-Pro-Leu used for fluorimeter calibration.
δFMca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg: Change in fluorescence during peptide hydrolysis per min.
Vtotal: Volume of peptide hydrolysis reaction (2.5ml).
Venzyme: Volume of added enzyme (0.002 to 0.004ml).
3 Inhibitors:
The catalytic domain of MMP-24 is inhibited by tissue inhibitors of MMP-2 (TIMP-2) and by chelators of divalent cations like EDTA or o-phenanthroline. |
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| Regulatory Status: | RUO - Research Use Only |
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Product Literature References
A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases: C.G. Knight, et al.; FEBS Lett.
296, 263 (1992),
Abstract;
