MMP-9 Colorimetric Drug Discovery Kit - AK-410 FORM: 25 mM (16.4 mg/ml) in DMSO (dimethylsulfoxide) STORAGE: -20 or -70°C QUANTITY: 0.82 mg PRESENTATION: 50 l in amber screw-cap microfuge tube.
BACKGROUND Matrix metalloproteinase-9 (MMP-9, gelatinase B, 92 kDa type IV collagenase) is a member of the MMP family of extracellular proteases. These enzymes play a role in many normal and disease states by virtue of their broad substrate specificities1. Targets of MMP-9 include native and denatured collagens, fibronectin, elastin, laminin, pro-TNF-, and 1-3 interleukins and their receptors . MMP-9 is secreted as a 92 kDa proenzyme (as measured by SDS-PAGE), and activated by cleavage to forms 82 kDa and smaller4. MMP-9 is an important target for inhibitor screening due to its involvement in diseases such as alopecia5, cancer, angiogenesis, and metastasis2,6-8.
PI-115-9090 INHIBITOR (NNGH; MW=316.4) FORM: 1.3 mM in DMSO STORAGE: -20 or -70°C QUANTITY: 50 µl PRESENTATION: Screw-cap microfuge tube.
KI-173 ASSAY BUFFER 50mM HEPES, 10mM CaCl 2 , 0.05% Brij-35, 1mM DTNB, pH7.5 FORM: Liquid in screw-cap plastic bottle The MMP-9 Colorimetric Drug Discovery Kit is a complete STORAGE: -20 or -70°C assay system designed to measure protease activity of MMPQUANTITY: 20 ml 9 using a thiopeptide as a chromogenic substrate (Ac-PLG-[29,10 mercapto-4-methyl-pentanoyl]-LG-OC 2 H 5 ) . The MMP KI-101 96-WELL MICROPLATE cleavage site peptide bond is replaced by a thioester bond in STORAGE: Room temperature. the thiopeptide. Hydrolysis of this bond by an MMP produces a sulfhydryl group, which reacts with DTNB [5,5’-dithiobis(2nitrobenzoic acid), Ellman’s reagent] to form 2-nitro-5- OTHER MATERIALS REQUIRED thiobenzoic acid, which can be detected by its absorbance at 412 nm (=13,600 M-1cm-1 at pH 6.0 and above11). The Microplate reader capable of measuring A 412 to 3-decimal assays are performed in a convenient 96-well microplate accuracy. format. The kit is useful to screen inhibitors of MMP-9, a Pipetman or multi-channel pipetman capable of pipetting 10potential therapeutic target. An inhibitor, NNGH12, is also 100 µl accurately (note: reagents can be diluted to increase included as a prototypic control inhibitor. Thiol inhibitors the minimal pipetting volume to >10 µl). should not be used with this kit, as they may interfere with the Ice bucket to keep reagents cold until use. colorimetric assay. Water bath or incubator for component temperature equilibration.
REFERENCES: 1. L.J. McCawley and L.M. Matrisian Curr. Opin. Cell Biol. 2001 13 534 2. M. Egeblad and Z. Werb Nat. Rev. Cancer 2002 2 163 3. B.-C. Sheu et al. Cancer Res. 2001 61 237 4. J. P. O’Connell et al. J. Biol. Chem. 1994 269 14967 5. F. Jarrousse et al. Int. J. Dermatol. 2001 40 385 6. G. Bergers et al. Nat. Cell Biol. 2000 2 737 7. J.M. Sivak and M.E. Fini Prog. Retin. Eye Res. 2002 21 1 8. C. Chang and Z. Werb Trends Cell Biol. 2001 11 S37 9. H. Weingarten and J. Feder Anal. Biochem. 1985 147 437 10. H. Weingarten et al. Biochemistry 1985 24 6730 11. L. Yu and E.A. Dennis Methods Enzymol. 1991 197 65 12. L.J. MacPherson et al. J. Med. Chem. 1997 40 2525
Note on storage: Store all components except the microplate (room temperature) at -70°C for the highest stability. The MMP-9 enzyme should be handled carefully in order to retain maximal enzymatic activity. It is stable, in diluted or concentrated form, for several hours on ice. As supplied, MMP-9 enzyme is stable for at least 5 freeze/thaw cycles. To minimize the number of freeze/thaw cycles, aliquot the MMP-9 into separate tubes and store at -70°C. When setting up the assay, do not maintain diluted components at reaction temperature (e.g. 37°C) for an extended period of time prior to PLEASE READ ENTIRE BOOKLET BEFORE running the assay. PROCEEDING WITH THE ASSAY. CAREFULLY NOTE THE HANDLING AND STORAGE CONDITIONS OF EACH To start assay: KIT COMPONENT. PLEASE CONTACT ENZO LIFE 1. Briefly warm kit components P-125-9090 and PI-115-9090 SCIENCES TECHNICAL SERVICES FOR ASSISTANCE IF to RT to thaw DMSO. NECESSARY. 2. Dilute inhibitor (NNGH, PI-115-9090) 1/200 in assay buffer KI-173 as follows. Add 1 µl inhibitor into 200 µl assay buffer, in a separate tube. Warm to reaction temperature COMPONENTS OF AK-410 KIT (e.g. 37°C). 3. Dilute substrate P-125-9090 1/25 in assay buffer to SE-244-9090 MMP-9 ENZYME (HUMAN, RECOMBINANT) required total volume (10 l are needed per well). For FORM: E. coli recombinant human MMP-9 catalytic domain example, for 15 wells dilute 6.4 l P-125-9090 into 153.6 l (amino acids 88-438; calculated MW 39 kDa), 2.68 U/µl. assay buffer, in a separate tube. Warm to reaction One U=100 pmol/[email protected]
37°C, 100 µM thiopeptide. Purity temperature (e.g. 37°C). >95% by SDS-PAGE. 4. Dilute MMP-9 enzyme 1/60 (Note: new dilution factor) in STORAGE: -70°C; Avoid freeze/thaw cycles assay buffer to required total volume (20 µl are needed per QUANTITY: 130 U well). Warm to reaction temperature (e.g. 37°C). PRESENTATION: 48.5 µl in screw-cap microfuge tube. 5. Pipet assay buffer into each desired well of the 1/2 volume microplate as follows: P-125-9090 SUBSTRATE (thiopeptide; MW=655.9)
Blank (no MMP-9)=90 µl Assay Buffer Control (no inhibitor)=70 µl Assay Buffer Inhibitor NNGH=50 µl Assay Buffer Test inhibitor=varies (see Table 1, below)
Figure 1. Plot of OD vs. time
6. Allow microplate to equilibrate to assay temperature (e.g. 37°C). 7. Add 20 µl MMP-9 (diluted in step 4) to the control, inhibitor NNGH, and test inhibitor wells. Final amount of MMP-9 will be 0.9 U per well (9 mU/l). Remember to not add MMP-9 to the blanks! 8. Add 20 µl NNGH inhibitor (diluted in step 2) to the inhibitor NNGH wells only! Final inhibitor concentration=1.3 µM.
Note: 1.3 µM NNGH will inhibit MMP-9 by approximately 96% under these conditions (see Figure 2).
9. Add desired volume of test inhibitor to appropriate wells. See Table 1, below. 10. Incubate plate for 30-60 minutes at reaction temperature (e.g. 37°C) to allow inhibitor/enzyme interaction. 11. Start reaction by the addition of 10 µl P-125-9090 substrate (diluted and equilibrated to reaction temperature in step 3). Final substrate concentration=100 µM. 12. Continuously read plates at A 412nm in a microplate reader. Record data at 1 min. time intervals for 10 to 20 min. 13. Perform data analysis (see below).
Figure 2. Example of inhibitor data:
NOTE: Retain microplate for future use of unused wells.
Inhibition of MMP-9 by NNGH TABLE 1. Example of Samples. Blank Control Inhibitor NNGH Test inhibitor*
Assay buffer 90 µl 70 µl 50 µl X µl
MMP-9 (45 mU/l) 0 20 µl 20 µl 20 µl
Inhibitor (6.5 µM) 0 0 20 µl Y µl
Substrate (1 mM) 10 µl 10 µl 10 µl 10 µl
Total Volume 100 µl 100 µl 100 µl 100 µl
0.221 no inh. 0.206 mOD
*Test inhibitor is the experimental inhibitor. Dissolve/dilute inhibitor into assay buffer and add to appropriate wells at desired volume “Y”. Adjust volume “X” to bring the total volume to 100 µl.
100nM inh. 1.3µM inh.
Example of plate:
well# A1 B1 C1 D1 E1 F1 G1 H1...
sample Blank Blank Control Control Inhibitor NNGH Inhibitor NNGH Test inhibitor Test inhibitor...
control slope = 4.69E-03 OD/min inhibitor slope = 2.04E-04 OD/min inhibitor % activity remaining = (2.04E-04/4.69E-03) x 100 = 4.34%
DATA ANALYSIS Plotting
Inhibition of MMP-9 by NNGH
1. Plot data as OD versus time for each sample (see Fig. 1). 2. Determine the range of time points during which the reaction is linear. Typically, points from 1 to 10 min are sufficient. 3. Obtain the reaction velocity (V) in OD/min: determine the slope of a line fit to the linear portion of the data plot using an appropriate routine. 4. Average the slopes of duplicate samples.
60 40 20
5. If the blank has a significant slope, subtract this number from all samples. 6. To determine inhibitor % remaining activity: Inhibitor % activity remaining = (V inhibitor / V control) x 100 See Figure 2 for example.
100nM NNGH 1.3µM NNGH
USE OF PRODUCT This product contains research chemicals. As such, they should be used and handled only by or under the supervision of technically qualified X mol substrate/min=(V x vol.)/( x l ) individuals. This product is not intended for diagnostic or human use. Where V is reaction velocity in OD/min, vol. is the reaction
7. To find the activity of the samples expressed as mol substrate/min, employ the following equation:
volume in liters, is the extinction coefficient of the reaction product (2-nitro-5-thiobenzoic acid)(13,600 M-1cm-1), and l is the path length of light through the sample in cm (for 100l in the supplied microplate, l is 0.5 cm). Note: The above equation determines enzyme activity in terms of moles of thiopeptolide substrate converted per minute. Under these conditions, the secondary substrate DTNB is saturating, and the velocity of DTNB conversion to 2-nitro-5-thiobenzoic acid is not rate-limiting.
WARRANTY Enzo Life Sciences International, Inc. makes no warranty of any kind, expressed or implied, which extends beyond the description of the product in this brochure, except that the material will meet our specifications at the time of delivery. Enzo Life Sciences International, Inc. makes no guarantee of results and assumes no liability for injuries, damages or penalties resulting from product use, since the conditions of handling and use are beyond our control.
See Figure 3 for activity and kinetic calculations. Figure 3. Example calculation for activity: Activity of a control sample = (4.81E-03OD/min x 1E-04L)/(13,600M-1cm-1 x 0.5cm)= 7.07E-11 mol/min at 37°C, 100M thiopeptolide
Enzo Life Sciences International, Inc. 5120 Butler Pike Plymouth Meeting, PA 19462-1202
Example graph for K m and V max determination:
Phone: 800-942-0430 (610) 941-0430 Fax: (610) 941-9252 email: [email protected]
1.4E-12 1.2E-12 1.0E-12
4.0E-13 2.0E-13 0.0E+0 0
400 600 800 [thiopeptolide] (uM)
K m =56.1 µM V max =1.8 pmol/sec