ALEXIS Biochemicals: MMP-13 (human) (recombinant) (His)

ALX-201-097

Revised 04-Sep-08

MMP-13 (human) (recombinant) (His)
SYNONYMS	Procollagenase-3 (human) (recombinant) (His)
PRODUCT LINE	Cytoskeleton
PRODUCT CATEGORY	MMPs

Ordering Information

Product Numbers:	Format:	Size:	Unit Price:	Quantity:	Add To Cart
ALX-201-097-C010		10 µg	560.00 USD

Product Specification

MW:	~52kDa (unglycosylated)
EC:	3.4.24.-
SOURCE/HOST:	Produced in Sf9 cells.
CONCENTRATION:	0.2mg/ml
PURITY:	≥95% (SDS-PAGE)
FORMULATION:	Liquid. In 50mM TRIS-HCl, pH 7.5, 150mM NaCl, 5mM CaCl₂, 0.05% Brij-35.
SPECIFIC ACTIVITY:	≥200mU/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 38°C, pH 7.5.
APPLICATION:	Antigen standard in immunochemical analyses. The active enzyme may be used to study the degradation of extracellular matrix proteins, to screen inhibitors of matrix metalloproteinases and to characterize inhibitor actions.
SHIPPING:	SHIPPED ON DRY ICE
SHORT TERM STORAGE:	-20°C
LONG TERM STORAGE:	-80°C
HANDLING:	Avoid freeze/thaw cycles.

General Information

MMP-13 is inhibited by tissue inhibitors of matrix metalloproteinases (TIMP) and by chelators of divalent cations as EDTA or o-phenanthroline.

BACKGROUND/TECHNICAL INFORMATION

Activation of MMP-13 proenzyme and measurement of catalytic activity

1 Preparation and stability of solutions:
∙ APMA-solution: 40mM p-aminophenyl mercuric acetate (APMA) in dimethylsulfoxide. 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 in 20% dimethylsulfoxide. Store at -20°C.
∙ Stock solution of unquenched peptide: 10μM solution of (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-NH₂ (Mca-Pro-Leu) in 20% dimethylsulfoxide. Store at -20°C.

2 Activation:
An aliquot of 19.5μl MMP-13 proenzyme is mixed with 0.5µl APMA solution and the mixture is incubated for 90 min. at 37°C.

3 Assay protocol:
The activity of MMP-13 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 min. 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 (mmoles/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.

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

Swiss-Prot link P45452: MMP-13 (human) (precursor)
AfCS Signalling Gateway link A001465: MMP-13 (mouse)

General 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

Further Categories Containing This Product:

Enzymes • Recombinant Proteins / Fusion Proteins

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