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MMP-2 proenzyme (human), (recombinant)

 
ALX-201-095-C010 10 µg 489.00 USD
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Product Specification

Alternative Name:Matrix metalloproteinase 2, Gelatinase A, 72 kDa Type IV collagenase
 
MW:~72kDa.
 
Source:Produced in Sf9 cells.
 
EC:3.4.24.24
 
UniProt ID:P08253
 
Concentration:0.2mg/ml
 
Formulation:Liquid. In 50mM TRIS-HCl, pH 7.0, 200mM NaCl, 5mM CaCl2, 0.05% Brij-35.
 
Purity:≥95% (SDS-PAGE)
 
Specific Activity:≥120mU/mg protein after APMA activation. 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.
 
Application Notes:Active MMP-2 is used to study the degradations of proteins of the extracellular matrix, including fibrillar collagens. MMP-2 is inhibited by tissue inhibitors of matrix metalloproteinases (TIMP), α2-macroglobulin and by chelators of divalent cations as EDTA or o-phenanthroline.
 
Shipping:Shipped on Dry Ice
 
Short Term Storage:-20°C
 
Long Term Storage:-80°C
 
Use/Stability:Stable for several weeks when stored at -20°C.
 
Handling:Avoid freeze/thaw cycles.
 
Protocol: Activation of MMP-2 proenzyme and measurement of catalytic activity
1) Preparation and stability of solutions:
APMA-solution: 40mM p-aminophenyl mercuric acetate (APMA) in DMSO. Store at -20°C.
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.
Stock solution of peptide substrate: 100µM solution of Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 in 20% DMSO. Store at -20°C.
Stock solution of unquenched peptide: 10µM solution of Mca-Pro-Leu-NH2 in 20% DMSO. Store at -20°C.

2) Activation:
An aliquot of 19.5µl MMP-2 proenzyme is mixed with 0.5µl APMA solution and is incubated for 60 min. at 37°C.

3) Assay protocol: The activity of MMP-2 is measured fluorimetrically with a synthetic internally quenched fluorescent substrate according to Knight et al. 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. 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. Aliquots of 1 to 2µl of the activation mixture are than added and the increase in fluorescence is recorded over a time interval between 2 and 12 minutes.

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 (0.001 to 0.002ml).
Due to autoproteolytic activity minor bands of activated enzyme may be visible in the preparation.

 

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;

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