ALEXIS Biochemicals: MMP-2 Proenzyme (human) (recombinant)

ALX-201-095

Revised 06-Jul-07

MMP-2 Proenzyme (human) (recombinant)
SYNONYMS	Progelatinase A (human) (recombinant)
PRODUCT LINE	Cytoskeleton
PRODUCT CATEGORY	MMPs

Ordering Information

Product Numbers:	Format:	Size:	Unit Price:	Quantity:	Add To Cart
ALX-201-095-C010		10 µg	350.00 USD

Product Specification

MW:	~72kDa.
EC:	3.4.24.24
SOURCE/HOST:	Produced in Sf9 cells.
CONCENTRATION:	0.2mg/ml
PURITY:	≥95% (SDS-PAGE)
FORMULATION:	Liquid. In 50mM TRIS-HCl, pH 7.0, 200mM NaCl, 5mM CaCl₂, 0.05% Brij-35.
SPECIFIC ACTIVITY:	120-150mU/mg protein after AMPA 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.
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.

Product Description

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.

Product Specific Literature References

[1] A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases: C.G. Knight, et al.; FEBS Lett. 296, 263 (1992) Abstract

General Information

BACKGROUND/TECHNICAL INFORMATION

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-NH₂in 20% DMSO. Store at -20°C.

2 Activation:
An aliquot of 19.5ml 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. [1].
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) = (C_Mca-Pro-Leu/F_Mca-Pro-Leu) x (ΔF_{Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg}/V_enzyme) x V_total

C_Mca-Pro-Leu: Concentration of Mca-Pro-Leu used for calibration of the fluorimeter (µmoles/ml).
F_Mca-Pro-Leu: Fluorescence of Mca-Pro-Leu at the concentration C_Mca-Pro-Leuused for fluorimeter calibration.
ΔF_{Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg}: Change in fluorescence during peptide hydrolysis per min.
V_total: Volume of peptide hydrolysis reaction (2.5ml).
V_enzyme: Volume of added enzyme (0.001 to 0.002ml).

Due to autoproteolytic activity minor bands of activated enzyme may be visible in the preparation.

Swiss-Prot link P08253: MMP-2 (human)
AfCS Signalling Gateway link A001471: MMP-2 (mouse)

Further Categories Containing This Product:

Enzymes • Recombinant Proteins/Fusion Proteins

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