Zymography of Metalloproteinases

Zymography is an electrophoretic technique enabling visualization of the number and approximate size of proteinases in a sample on the basis of their hydrolysis of a gel-embedded protein substrate such as proteoglycan (Barrett, 1966), gelatin (Itoh et al., 1998), and casein (Leber and Bakuil, 1997; Zeng et al., 2002). The technique is particularly useful for analyzing the proteinase composition of complex biological samples, because visualization depends directly on proteolytic activity (Feitosa et al., 1998; Jain et al., 2001). Zymography is also widely used to study various aspects of matrix metalloproteinase (MMP) function (Kjeldsen et al., 1993; Itoh et al., 1998). Gelatin zymography, a widely used technique in the study of MMPs, is described in this unit. In principle, it is also applicable for use with many proteinases if the enzyme retains activity after electrophoresis and renaturation. This unit presents a representative zymography assay using matrix metalloproteinase (MMP) and a gelatin or casein substrate (see Basic Protocol), as well as protocols for other substrates (see Alternate Protocol 1), real-time zymography (i.e., using a fluorescent protein substrate; see Alternate Protocol 2), and reverse zymography for the study of proteinase inhibitors (see Alternate Protocol 3). These procedures employ the 2-amino-2-methyl-1,3-propanediol (ammediol) buffer system introduced by Bury (1981), but many other SDS-PAGE systems (e.g., Laemmli; UNIT 10.1) have been successfully used by others (Hummel et al., 1996; Hawkes et al., 2001). NOTE: For an overview of matrix metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs), see UNIT 21.4. ZYMOGRAPHY OF MATRIX METALLOPROTEINASES USING A GELATIN OR CASEIN SUBSTRATE To provide a uniformly dispersed substrate, the peptide (e.g., gelatin) is included in the standard polyacrylamide gel solution and the gel is then poured and polymerized as usual. Since the substrate is entrapped in the pores of the polyacrylamide gel, it does not migrate out of the gel during electrophoresis. Proteinase samples are denatured with sodium dodecyl sulfate (SDS), but not boiled or reduced, and then electrophoresed. The separated proteinases are renatured within the gel by replacing the SDS with a nonionic detergent such as Triton X-100. The gel is then incubated in a buffer suitable for enzymatic activity, allowing the renatured proteinases to digest the gel-bound substrate protein in a zone around their electrophoresed position. These zones of digestion are visualized using Coomassie brilliant blue R250, with the areas of digestion appearing clear against a blue-stained background of undigested protein. Materials Acrylamide/bisacrylamide solution (see recipe) Separating gel buffer (see recipe) 10 casein or gelatin substrate solution (see recipes) Sucrose solution (see recipe) 10% (w/v) ammonium persulfate (see recipe) TEMED n-Butanol, H2O saturated Stacking gel buffer (see recipe) MMP sample 1 mM APMA (see recipe)

UNIT 21.15

BASIC PROTOCOL

Peptidases Contributed by Linda Troeberg and Hideaki Nagase

Current Protocols in Protein Science (2003) 21.15.1-21.15.12 Copyright 2003 by John Wiley & Sons, Inc.

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Supplement 33

2 sample loading buffer (see recipe) Proteinase standards (e.g., recombinant MMP-1, -2, or -3) 1 anode and cathode reservoir buffers (see recipes) Sample known to degrade gelatin or casein (e.g., recombinant culture supernatents) Molecular mass standards (e.g., BioRad low-range SDS-PAGE standards) Enzyme renaturing buffer (see recipe) Developing buffer, 37C (see recipe) Staining solution (see recipe) Destaining solution (see recipe) Electrophoresis apparatus (e.g., 9-cm 6-cm 0.75-mm minigel system) and comb Gel-loading pipet tips or Hamilton syringe Constant voltage power supply Sealed plastic container Additional equipment and reagents for preparing and running acrylamide gels (UNIT 10.1) 1. Choose an appropriate percentage separating gel and prepare according to Table 21.15.1. Mix the solution well and pour between electrophoresis plates to a height 1.5 to 2 cm from the top (UNIT 10.1). Overlay with water-saturated n-butanol and allow the gel to polymerize 30 min.
The choice of acrylamide percentage depends on the molecular weight of the proteinase to be visualized. Proteins with higher molecular masses resolve better on lower percentage gels, while those with lower molecular masses resolve better on higher percentage gels. For analysis of MMPs, 10% gels are usually appropriate, as they resolve proteins between 20 and 100 kDa. Alternatively, Novex casein and gelatin zymograms are commercially available from Invitrogen (Kleiner and Stetler-Stevenson, 1994). Also, different substrates can be used (see Alternate Protocol 1).

2. Prepare a stacking gel by mixing the following: 142 l acrylamide/bisacrylamide 285 l stacking gel buffer 285 l sucrose solution 428 l H2O 14 l 10% (w/v) ammonium persulfate 3 l TEMED. Decant the n-butanol from the separating gel, gently rinse with water (e.g., from a squirt bottle) to remove any remaining alcohol, and immediately pour the stacking gel solution on top of the separating gel. Insert the well comb, taking care not to trap bubbles under the comb. Allow the stacking gel to polymerize 15 to 20 min at room temperature.
The stacking gel polymerizes much faster than the separating gel (5 to 10 min), so work as quickly as possible after adding TEMED. A stacking gel with the same percentage acrylamide is used irrespective of the acrylamide concentration in the separating gel. There should be at least 1-cm stacking gel below the comb to ensure proper stacking of proteins, which is a prerequisite for good resolution in the separating gel. Refer to UNIT 10.1 for more information.
Zymography of Metalloproteinases

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Table 21.15.1

Recipes for Zymogram Separating Gelsa,b

Stock solution Acrylamide/bisacrylamide solution Separating gel buffer 10 casein or gelatin substrate solution Sucrose solution Water 10% (w/v) ammonium persulfate TEMED

Final acrylamide concentration in gel 6% 0.8 ml 1.0 ml 0.4 ml 0.86 ml 0.94 ml 14 l 1.5 l 7.5% 1.0 ml 1.0 ml 0.4 ml 0.86 ml 0.74 ml 14 l 1.5 l 10% 1.32 ml 1.0 ml 0.4 ml 0.86 ml 0.42 ml 14 l 1.5 l 12% 1.61 ml 1.0 ml 0.4 ml 0.86 ml 0.13 ml 14 l 1.5 l

aAbbreviations: TEMED N,N,N,N-tetramethylethylenediamine. bRecipes given are sufficient for one 9-cm 6-cm 0.75mm minigel.

3. Optional: Activate proenzyme (zymogen) samples by incubating 1 to 4 hr at 37C in the presence of 1 mM APMA diluted from a 20 mM stock. Mix 5 to 20 l activated sample with an equal volume of 2 sample loading buffer.
Most MMP proenzymes can be activated using these conditions. See Nagase (1997) for a discussion of the current hypothesis of APMA activation. Note that this loading buffer contains no reducing agents or EDTA, which would inactivate matrix metalloproteinases (MMPs). Do not boil the sample, as this will irreversibly inactivate MMPs.

4. Optional: If the identity of the proteinase is known, prepare a standard curve of proteinase standards (i.e., at least five known concentrations) and coanalyze (see Figure 21.15.1). 5. Assemble the gel in the electrophoresis apparatus according to the manufacturers instructions and UNIT 10.1. Pour 1 anode and cathode reservoir buffers into the lower and upper chambers, respectively. 6. Load samples, proteinase standards (if proteinase is known), a sample known to degrade gelatin or casein (e.g., recombinant culture supernatants; positive control), unconditioned medium (negative control), and molecular mass standards into appropriate wells using gel-loading pipet tips or a Hamilton syringe.
Be especially careful to avoid spillage between lanes for samples that contain a high concentration of proteinase activity or leave empty lanes between such samples. Thoroughly wash the Hamilton syringe between samples.

7. Electrophorese using a constant voltage power supply at 150 V (nonlimiting current) until the bromophenol blue reaches the bottom of the gel (UNIT 10.1).
A 10% acrylamide gel should take 45 min to complete.

8. After electrophoresis, disassemble the gel apparatus and place the gels in a sealed plastic or glass container filled with 20 ml enzyme renaturing buffer. Wash 15 min at room temperature with gentle agitation on an orbital shaker. Repeat this procedure four times using fresh buffer each time, for a total washing time of 1 hr.
This process exchanges SDS within the gel with the nonionic detergent Triton X-100, facilitating renaturation of enzyme samples. Incubate one gel per container so that all areas of each gel are thoroughly washed. It is not necessary to remove the stacking gel.
Peptidases

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9. Transfer gel to 30 ml developing buffer and incubate 2 hr to overnight at 37C.

An appropriate incubation time should be empirically determined depending on the required level of sensitivity. As little as 5 pg MMP-2 can be detected using an overnight incubation. If incubating overnight, ensure that the container is airtight to prevent evaporation. Real-time zymography can also be performed (see Alternate Protocol 2).

10. Decant the developing buffer and add staining solution. Stain 20 to 30 min at room temperature and then destain thoroughly (>2 hr) using multiple changes of destaining solution until clear bands of MMP activity are visible against the dark blue background.
The gel can be stored in destaining solution up to a few months at room temperature. For long-term storage, gels can be dried using a commercial gel drier. Refer to UNIT 10.5 for more information on Coomassie brilliant blue R250 staining and drying gels.

A B
1 2 3 4 5 6 7 8 9 10
3 10 6

11

12

Band intensity (arbitrary units)

2 10 6

1 10 6

250

500 MMP-2 (pg)

750

1000

Zymography of Metalloproteinases

Figure 21.15.1 Recombinant proMMP-2 was activated by 1 hr incubation with 1 mM APMA at 37C and its active concentration confirmed by titration against TIMP-2 (Troeberg et al., 2002). Various concentrations of recombinant MMP-2 were analyzed on a 7.5% gelatin zymogram (upper panel) incubated at 37C for either 4 hr or 16 hr, A and B, respectively. Loads were as follows: lane 1, 2 pg; lane 2, 5 pg; lane 3, 10 pg; lane 4, 25 pg; lane 5, 50 pg; lane 6, 100 pg; lane 7, 200 pg; lane 8, 300 pg; lane 9, 400 pg; lane 10, 500 pg; lane 11, 600 pg; and lane 12, 1 ng. Empty lanes were left between the highest concentrations of MMP-2 to ensure accurate quantitation. Pixel volume was quantified by arbitrary units (given by the software) and plotted against the amount of MMP-2 (lower panel), with the 4 hr and 16 hr incubations represented with closed and open circles, respectively. The dashed lines show that band intensity is linearly related to MMP-2 concentration, but the linear range of enzyme concentration varies with the incubation time (i.e., a longer incubation results in more substrate being cleaved and subsequently the signal becomes saturated at lower concentrations of enzyme relative to a shorter incubation). In this example, the relationship is linear up to 1.3 106 intensity units. Empty lanes have left between the highest concentrations of MMP-2 to ensure accurate quantitation.

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11. If proteinase standards were included, construct a calibration curve using the known concentrations of the enzyme under study and the area of digestion produced. Compare this with the area of digestion of a test sample to quantify results.
The linear range for such analysis varies with the enzyme analyzed (and its ability to digest the protein substrate used) and with the incubation time. As little as 1 to 10 pg MMP-2 (Fig. 21.15.1; Kleiner and Stetler-Stevenson, 1994) or 32 pg MMP-9 (Leber and Balkwill, 1997) can be detected on gelatin zymograms. The area of digestion can be quantified using a densitometer and associated software. Specific analysis techniques will vary with the software available. The gel in Figure 21.15.1 was scanned using a Bio Rad GS-710 Calibrated Imaging Densitometer and analyzed using Phoretix 1D software (Non-Linear Dynamics). The image should be inverted to give black bands on a white background. Place equally sized rectangular areas around each band, choosing an area just large enough to encompass the largest band. Background can be subtracted either locally or globally. Suitable background subtraction techniques are best determined empirically depending on the gel (intensity of bands, darkness of background) and the software available. Pixel density within the areas is then quantified and plotted against the amount of MMP-2. Band intensity is linearly related to MMP-2 concentration over a range that varies with the incubation time. In Figure 21.15.1, the relationship is linear up to 1.3 106 units. Empty lanes have been left between the highest concentrations of MMP-2 to ensure accurate quantitation.

ZYMOGRAPHY OF MATRIX METALLOPROTEINASES USING SUBSTRATE PROTEINS OTHER THAN GELATIN Gelatin is widely used as a substrate for zymography because it is readily available, economical, and serves as a substrate for several MMPs (see Basic Protocol). However, any protein that is digested by the proteinase of interest and that remains soluble in the buffer system used for electrophoresis can be used as an alternative to gelatin. For example, proteins such as collagen (0.2 to 1 mg/ml; Gogly et al., 1998), fibronectin (2 mg/ml; Feitosa et al., 1998), and fibrinogen (2 mg/ml; Feitosa et al., 1998) have been used (all available from Sigma). When working with a novel enzyme, it may be beneficial to try several possible substrates to determine which yields the best results. Although able to detect low picogram quantities of enzymes (Gogly et al., 1998), collagen zymograms are technically difficult and not widely used. Collagen has a tendency to precipitate in buffers commonly used for electrophoresis and considerable care must be taken to prevent denaturation of the collagen to gelatin in order to prevent detection of gelatinase in addition to the collagenase activity. For example, the temperature of the gel must not exceed 25C at any time, including during electrophoresis. REAL-TIME ZYMOGRAPHY With Coomassie staining of zymograms (see Basic Protocol), it is necessary to estimate a suitable development time before irreversibly fixing and staining proteins. To eliminate this inherent uncertainty, the technique of real-time zymography has been developed, using a fluorescent protein as the substrate. Digestion is visualized directly in the developing buffer using a UV transilluminator with areas of digestion visible as dark bands against a fluorescent background. Fluorescent substrates include 0.5 mg/ml FITCgelatin (heat-denatured FITC-collagen) and 0.5 mg/ml FITC-casein (Hattori et al., 2002), which have been used for analyzing MMP-2, -3, and -9. FITC-gelatin zymograms allow for detection of as little as 16 pg MMP-2 and 3 pg MMP-9 after incubation for 24 hr. In fact, FITC-casein is five times more sensitive for MMP-3 than conventional casein zymography.

ALTERNATE PROTOCOL 1

ALTERNATE PROTOCOL 2

Peptidases

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In addition to the substrates mentioned above, a short peptide sequence known to be hydrolyzable by the proteinase of interest can be tagged with the fluorescent 4-methylcoumaryl-7-amide (MCA) moiety. For example, trypsin and chymotrypsin activity have been detected using Boc-Gln-Ala-Arg-MCA and Suc-Ala-Ala-Pro-Phe-MCA respectively (Yasothornsrikul and Hook, 2000). In this case, activity is visible as zones of fluorescence against a dark background.
ALTERNATE PROTOCOL 3

REVERSE ZYMOGRAPHY FOR STUDY OF PROTEINASE INHIBITORS A variation of zymography (see Basic Protocol) can be used to analyze inhibitors of matrix metalloproteinases such as tissue inhibitors of metalloproteinases (TIMPs). In this version, reverse zymography, a proteinase (commonly MMP-2) is embedded in the gel in addition to the protein substrate (e.g., gelatin). The TIMPs are then electrophoresed, and during the development step, the renatured proteinase digests the background substrate throughout the gel, except at positions in the gel where inhibitors are present. Upon staining, the digested background appears pale blue, while areas of inhibition are visible as reddish-purple zones. It is always necessary to run a companion SDS-PAGE gel (without gelatin or proteinase) to check that these zones represent true areas of inhibition, and not just Coomassie-stained proteins; inhibitors will be visible on the reverse zymogram, but absent on the companion SDS-PAGE gel. As little as 1 pg TIMP-2 and 40 pg TIMP-1 can be detected using MMP-2 and gelatin (Oliver et al., 1997). This technique has been used to investigate the presence of TIMPs in biological samples (Noha et al., 2000), the role of TIMP-2 in MMP-2 activation (Itoh et al., 1998), and various aspects of TIMP function (Yeow et al., 2002). FITC-labeled proteins (see Alternate Protocol 2) can also be used for reverse zymography. In this case, visualization is directly dependent on inhibition of proteolytic activity and thus permits easier discrimination between inhibitors and contaminating protein bands than gelatin-based techniques (Hattori et al., 2002). REAGENTS AND SOLUTIONS
Use Milli-Q-purified water or equivalent for the preparation of all buffers. For common stock solutions, see APPENDIX 2E; for suppliers, see SUPPLIERS APPENDIX.

Acrylamide/bisacrylamide solution 30% acrylamide (Severn Biotech) 0.8% bisacrylamide (Severn Biotech) Store up to 6 months at 4C.
This reagent is commercially available as 37.5:1 acrylamide/bisacrylamide.

Ammonium persulfate, 10% (w/v) Dissolve 0.5 g ammonium persulfate in a final volume of 5 ml water. Store up to 1 month at 20C. Anode reservoir buffer, 4, 1 Dissolve 26.28 g of 2-amino-2-methyl-1,3-propanediolHCl (ammediol; 84 mM) in 900 ml water and adjust to pH 8.23 with HCl. Adjust volume to 1000 ml with water. Store up to 1 month at 4C. Dilute to 1 with water as required.
The anode reservoir is the lower chamber and is indicated by red markings.

Zymography of Metalloproteinases

APMA, 20 mM Dissolve 7 mg (20 mM) 4-aminophenylmercuric acetate (APMA; ICN Biochemicals) in 1 ml of 80 mM NaOH with vortexing. Store up to 1 month protected from light at 20C.

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Casein substrate solution, 10 Dissolve 800 mg casein (BDH) in 100 ml of 0.1 N NaOH. Heat the solution to 37C with occasional vortexing until no undissolved material is visible. Store up to several months at 20C. Cathode reservoir buffer, 4, 1 Dissolve 17 g ammediol (41 mM), 12 g glycine (40 mM ), and 4 g SDS (0.4%) in 900 ml water. Adjust volume to 1000 ml with water. Check that the pH is 9.39 and adjust with NaOH or HCl as necessary. Store up to 1 month at 4C. Dilute to 1 with water as required.
The cathode reservoir is the upper chamber and is usually indicated by black markings.

Destaining solution Combine 1.5 liters methanol, 3.5 liters water, and 50 ml formic acid. Store up to several months at room temperature. Developing buffer Dissolve 6.055 g Tris base (50 mM), 11.69 g NaCl (200 mM), 0.7 mg ZnCl2, 0.74 g CaCl22 H2O (5 mM), and 0.2 g NaN3 (0.02%) in 900 ml water. Adjust to pH 7.5 with HCl and adjust volume to 1000 ml with water. Store up to 1 month at 4C. Enzyme renaturing buffer Add 25 ml Triton X-100 to developing buffer (see recipe). Store up to 1 month at 4C.
Final concentrations are 200 mM NaCl, 5 mM CaCl2, 5 M ZnCl2, 2.5% (v/v) Triton X-100, 0.02% (w/v) NaN3, and 50 mM TrisCl, pH 7.5.

Gelatin substrate solution, 10 Add 800 mg porcine-skin gelatin (Sigma) to 100 ml water in a glass container. To ensure a uniformly stained background, dissolve the protein substrate fully before incorporation into the gel. Heat in a microwave oven until the solution just boils. Do not allow to boil over. Swirl thoroughly to ensure that all of the protein is in solution. Store up to one week at 4C or several months at 20C. Sample loading buffer, 2 Dissolve 0.2 g SDS (2%) and 0.01 g bromophenol blue (0.1%) in 5 ml stacking gel buffer (see recipe) and add 5 ml glycerol (40%). Store up to 1 month at 20C. Separating gel buffer Dissolve 4.63 g ammediolHCl (110 mM) and 0.02 g sodium azide (NaN3; 0.02%) in 90 ml water. Adjust pH to 8.96 with HCl and adjust volume to 100 ml with water. Store up to 1 month at 4C. Stacking gel buffer Dissolve 3.51 g ammediolHCl (84 mM ) and 0.02 g NaN3 (0.02%) in 90 ml water. Adjust pH to 8.37 with HCl and adjust volume to 100 ml with water. Store up to 1 month at 4C. Staining solution Dissolve 2.5 g Coomassie brilliant blue R-250 (0.125%) in a mixture of 1.25 liters methanol, 0.5 liters acetic acid, and 0.75 liters water. Store up to several months at room temperature.

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Sucrose solution Dissolve 50 g sucrose and 0.02 g NaN3 in 50 ml water. Add 30 l toluene and adjust volume to 100 ml with water. Store up to 1 month at 4C. COMMENTARY Critical Parameters and Troubleshooting
See UNIT 10.1 for a thorough discussion of important technical considerations as well as problems that can occur with preparing and running SDS polyacrylamide gels. The following is a brief list of problems that are specific to zymograms. 1. Background too pale. Inadequate incorporation of the substrate protein into the gel can cause problems. Remake the gelatin solution, taking care that the correct amount of protein has been added. Gelatin solutions should not be stored at 4C for more than 1 week, to prevent contamination with bacteria, which may secrete proteolytic enzymes. 2. Background streaky or blotchy. To achieve a uniformly stained background, it is crucial that the substrate protein be completely dissolved prior to casting the gel. Hold the protein solution up to the light and swirl to check for any undissolved material. Gelatin solutions should be heated briefly in the microwave or incubated at 37C with occasional vortexing to ensure that all protein is dissolved. 3. No zones of digestion are evident. As a positive control, incorporate appropriate quantities of enzymes that are known to digest the substratee.g., MMP-2 (100 pg for an overnight incubation) or MMP-9 (100 pg for an overnight incubation) for gelatin zymograms, and MMP-1 (500 pg for an overnight incubation) for casein zymograms. If these positive controls are visible, the test sample may be devoid of the enzymes that digest the particular protein substrate or the proteinase concentration may be lower than expected; either concentrate the solution by nondenaturing means or increase the incubation time. If these positive controls are not visible, check that the sample loading buffer does not contain any reagents that would inactivate the proteinasese.g., for MMPs, this buffer should not contain EDTA. Also check that the samples have not been boiled, as this will irreversibly inactive the proteinases, and that the renaturing solution contains the correct amount of Triton X-100, which is required to renature the proteinases after electrophoresis. Gels must be washed at
Zymography of Metalloproteinases

least four times for 15 min each to achieve adequate renaturation. 4. Smeared zones of digestion. Smearing is a sign that too much substrate protein has been digested. Decrease the incubation time or load less enzyme.

Anticipated Results
Figure 21.15.2 shows zymograms of various recombinant MMPs using gelatin (panel A) or casein (panel B) substrate stained with Coomassie brilliant blue. The substrate background stains dark blue, while the MMPs show up as lightly stained zones of digestion, having degraded the protein substrate around the position to which they have electrophoresed. On gelatin zymograms, as little as 10 pg MMP-2 generates a visible zone of digestion (Fig. 21.15.2A, lane 3), while 500 pg MMP-1 and -3 are required to generate a clear zone of digestion. This indicates that gelatin is a better substrate for MMP-2 and -9 than MMP-1 and -3. Similar amounts of MMP-1 and -3 are required if casein is used as the substrate protein (Fig. 21.15.2B), but since MMP-2 does not hydrolyze casein effectively, casein zymograms are more useful for analyzing MMP-1 and -3, while excluding MMP-2 activity. The molecular mass of proteinases can be estimated from their position relative to molecular mass markers; however, samples are not boiled or reduced prior to electrophoresis, so their migration may not accurately reflect their molecular mass. Additionally, inclusion of a protein substrate in the gel retards the migration of proteins through the gel in a nonlinear manner (Hummel et al., 1996). Accurate assessment of molecular mass is thus not possible on a zymogram. Samples of known MMPs can be run alongside test samples for more accurate estimation of molecular mass, but alignment of a band of interest with a known MMP cannot be taken as proof of identity. Further characterization, such as immunoblotting (UNIT 10.10) or removal of the proteinase from the test sample by immunoprecipitation (UNIT 9.8), is required for unequivocal identification. MMP proenzymes are inactive in solution, but visible on zymograms. As shown in Figure 21.15.2A, zymography permits visualization of

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proenzyme forms of MMPse.g., proMMP-2 in lane 2 (band a). MMPs are maintained in their proenzyme form by coordination of the catalytic zinc atom in the MMP active site by a cysteine residue in the propeptide region. They are activated by proteolytic processing of the propeptide, which generates an active lowermolecular-mass form (Nagase, 1997). Zymography, however, permits visualization of these normally inactive proenzymes. Treatment of MMPs with SDS perturbs their tertiary structure, disrupting the interaction of the propeptide with the catalytic zinc atom. Upon renaturation of the enzymes after electrophoresis, the proenzymes are autocatalytically activated and the protein substrate digested in a region corresponding to the molecular mass of the zymogen. This characteristic of zymography can be useful and has permitted study of proMMP-2 activation by zymography (Itoh et al., 1998). Care must be taken not to infer in vivo activity from a zymogram band that actually corresponds with a proenzyme inactive in solution. Also, this zymogram-dependent

activation should not be confused with in vitro activation by proteinases or APMA, which is carried out prior to electrophoresis. For example, 72-kDa proMMP-2 (Fig. 21.15.2A, band a) is converted to 68-kDa active MMP-2 (lane 3) by treatment with APMA prior to electrophoresis. Furthermore, MMPs are often present in biological samples in inactive complexes with their endogenous inhibitors, TIMPs. These complexes dissociate upon treatment with SDS and, as MMP and TIMP electrophorese to different positions and remain separated during development of the zymogram, TIMPcomplexed MMPs appear as active enzymes on zymograms (see also Fig. 21.15.4; Itoh et al., 1998). High-molecular-mass MMP complexes Gels of biological samples often contain bands of proteolytic activity at masses in excess of any known MMPs, which are often actually complexes of MMP-9. MMP-9 (92 kDa) forms a disulfide-bonded homodimer of 200 kDa

B
250 kDa 150 100 75 50 37 25

M 6

9 10

9 10

Figure 21.15.2 Various recombinant MMPs and conditioned culture media from different cell lines were analyzed on 7.5% acrylamide gelatin (A) or casein (B) zymograms, and developed overnight at 37C. Lane 1 shows 500 ng of 43-kDa recombinant MMP-1. Lane 2 shows 100 pg of 72-kDa proMMP-2 (band a) and the 68-kDa active form of MMP-2 (band b) purified from the culture medium of human cervical fibroblasts. Lane 3 contains 10 pg of 68-kDa APMA-activated MMP-2. Lane 4 shows 250 ng of 18.5-kDa recombinant MMP-3 catalytic domain. Lanes 5 to 10 show culture media collected from MMP-expressing cells. In lane 5, 2 l of HT1080 (human fibrosarcoma) culture supernatant treated with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) produce zones of digestion likely to correspond to the 92-kDa proMMP-9 (band c), 83-kDa active form of MMP-9 (band d), 72-kDa proMMP-2, and 68-kDa MMP-2. Lanes 6 to 8 show 2-l loads of three fibroblast cell lines that produce mainly proMMP-2 and MMP-2 (lane 6, human embryonic lung fibroblasts; lane 7, human fibrosarcoma; lane 8, human uterine cervical fibroblasts). Lane 9 shows a 2-l load of cultured human rheumatoid synovial fibroblasts, which produce 92-kDa proMMP-9 and 83-kDa MMP-9, in addition to proMMP-2/MMP-2, whereas only proMMP-2 is visible in the 2 l culture medium from cultured human chondrocytes shown in lane 10. Protein molecular mass markers are shown in lane M. Fewer bands of digestion are evident on the casein zymogram (B), since MMP-2 and -9 digest this substrate poorly; however, MMP-1 and -3 are clearly visible on both casein and gelatin zymograms.

Peptidases

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none

EDTA

E-64

pepA

AEBSF 250 kDa 150 100 75 50 37 25

M rE M

rE

rE

M rE

M rE H

Figure 21.15.3 A sample containing 100 pg recombinant proMMP-2/MMP-2 (rE) and 2 l conditioned medium from TPA-treated HT1080 (H) were run on a 7.5% gelatin zymogram as in Figure 21.15.2. The effects of various proteinase inhibitors on activity were investigated. Compared with zymograms incubated in the absence of inhibitors (none), addition of 20 M E-64, 10 M pepstatin A (pepA), or 1 mM AEBSF to the renaturing and developing buffers had no effect on enzyme activity. Addition of 20 mM EDTA, however, abolished all zones of gelatin digestion, confirming that the enzymes are metalloproteinases. Lane M contains protein molecular-mass markers.

2M/MMP-2 complexes

250 kDa 150 100 75

proMMP-2 active MMP-2 MMP-2 catalytic domain

50 37 25 1 2 3 4 M 5 6

Zymography of Metalloproteinases

Figure 21.15.4 MMP samples were analyzed on a 7.5% acrylamide gelatin zymogram, incubated in developing buffer overnight at 37C. Lane 1 shows 100 pg MMP-2 activated with 1 mM APMA, which results in a mixture of the 69-kDa full-length active form and the truncated 45-kDa catalytic domain. Preincubation of this sample with an equimolar amount of TIMP-2 for 1 hr at 37C has no effect on the observed pattern of digestion because the enzyme/inhibitor complex dissociates in sample loading buffer (lane 2). Addition of a 50-fold molar excess of 2M to MMP-2 for 30 min at 37C reduces the activity of both the 68- and 45-kDa proteolytically active species (lane 3). 2M differs from TIMP-2 in that the 2M/MMP-2 complex is stable in sample loading buffer. Preincubation of MMP-2 with TIMP-2 protects it from association with 2M (lane 4), since the MMP-2/TIMP-2 complex is not proteolytically active and hence unable to bind to 2M. Lane 5 shows a mixture of 72-kDa proMMP-2, 68-kDa MMP-2, and 45-kDA MMP-2 catalytic domain, preincubated with TIMP-2. Addition of 2M to this sample has no effect on the zones of hydrolysis observed for proMMP-2 as the zymogen is proteolytically inactive (lane 6). The MMP-2/TIMP-2 complex is similarly inactive and does not interact with 2M (lane 6). The truncated catalytic domain, however, interacts poorly at TIMP-2 and becomes associated with 2M (Itoh et al., 1998), shifting its zone of digestion from 45 kDa. A zone of lysis can often be observed associated with 2M. Lane M shows protein molecular mass markers.

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(Kjeldsen et al., 1993). Additionally, MMP-9 forms SDS-stable disulfide-bonded complexes with a 25-kDa protein called neutrophil gelatinase-associated protein (NGAL), producing a complex of 125 kDa (Kjeldsen et al., 1993). Specific inhibitors to confirm class of proteinase To confirm that the observed proteinase is a metalloproteinase, specific proteinase inhibitors can be added to the renaturing and developing buffers. As shown in Figure 21.15.3, metalloproteinases (MMP-2 and -9 in this example) are inhibited by addition of 20 mM EDTA to the renaturing and developing buffers, but unaffected by addition of L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane (E64), which inhibits cysteine proteinases at 20 M; phenylmethylsulfonyl fluoride (PMSF) or 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), which inhibit serine proteinases at 1 mM; or pepstatin A, which inhibits aspartic proteinases at 10 M. 2-Macroglobulin (2M) is a large plasma proteinase inhibitor that only binds to active proteinases and is therefore useful for identifying whether matrix metalloproteinases (MMPs) extracted from tissues or found in conditioned media are proteolytically active (Itoh et al., 1995). Most active endoproteinases from all four classes of proteinases (UNIT 21.1) readily cleave the bait region of 2M, which triggers a conformational change in the inhibitor and traps the proteinase within the molecule, hence rendering the proteinase inactive against large protein substrates (Barrett, 1981). Most 2M/proteinase complexes are SDS-stable so addition of 2M reduces activity at the normal zymogram position of the enzyme. While 2M complexes are inactive against protein substrates in solution, addition of SDS to the enzyme solution reduces the band at the enzymes molecular weight as the enzyme runs together with the 2M on the gel near the top of the zymogram where 2M runs. Figure 21.15.4 shows the effects of 2M on migration of active MMP-2 and inactive MMP-2/TIMP-2 complexes. Treatment of MMP-2 with 2M reduces the enzymes zone of digestion at 68 kDa (compare lane 1 with lane 3). Preincubation of MMP-2 with TIMP-2 generates a proteolytically inactive complex and hence protects the enzyme from interaction with 2M (compare lanes 2 and 4). Addition of 2M has no effect on the zone of digestion for either proMMP-2 or proMMP-2/TIMP-2, as both are proteolytically inactive (comparing lanes 5 and 6).

It is crucial to add the inhibitors at the stated concentrations during all renaturing steps as well as in the development step to ensure reliable results.

Time Considerations
Preparation and polymerization of separating and stacking gels requires 1 to 2 hr. Sample preparation takes 10 to 15 min. Gels take 1 hr to load and run. Washing of gels in Triton X-100 (renaturation) takes 1 hr, and the development step varies between 2 and 20 hr, depending on the sensitivity required. Staining takes 30 min and destaining takes 2 hr.

Literature Cited
Barrett, A.J. 1966. Chondromucoprotein-degrading enzymes. Nature 211:1188-1190. Barrett, A.J. 1981. Alpha 2-macroglobulin. Methods Enzymol. 80C:737-754. Bury, A.F. 1981. Analysis of protein and peptide mixtures. Evaluation of three sodium dodecyl sulfate-polyacrylamide gel electrophoresis buffer systems. J. Chromatogr. 213:491-500. Feitosa, L., Gremski, W., Veiga, S.S., Elias, M.C., Graner E., Mangili, O.C., and Brentani, R.R. 1998. Detection and characterization of metalloproteinases with gelatinolytic, fibronectinolytic and fibrinogenolytic activities in brown spider (Loxosceles intermedia) ven om. Toxicon 36:1039-1051. Gogly, B., Groult, N., Hornebeck, W., Godeau, G., and Pellat, B. 1998. Collagen zymography as a sensitive and specific technique for the determination of subpicogram levels of interstitial collagenase. Anal. Biochem. 255:211-216. Hattori, S., Fujisaki, H., Kiriyama, T., Yokoyama, T., and Irie, S. 2002. Real-time zymography and reverse zymography: A method for detecting activities of matrix metallopeptidases and their inhibitors using FITC-labeled collagen and casein as substrates. Anal. Biochem. 301:27-34. Hawkes, S.P., Hongxia, L., and Taniguchi, G.T. 2001. Zymography and reverse zymography for detecting MMPs and TIMPs. In Matrix Metallopeptidase Protocols (I.M. Clark, ed.) pp. 399410. Humana, Totowa, N.J. Hummel, K.M., Penheiter, A.R., Gathman, A.C., and Lilly, W.W. 1996. Anomalous estimation of protease molecular weights using gelatin-containing SDS-PAGE. Anal. Biochem. 233:140-142. Itoh, Y., Binner, S., and Nagase, H. 1995. Steps involved in the activation of pro-matrix metallopeptidase 2 (progelatinase A) and tissue inhibitor of metallopeptidases (TIMP)-2 by 4-aminophenylmercuric acetate. Biochem. J. 308:645-651.

Peptidases

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Itoh, Y., Ito, A., Iwata, K., Tanzawa, K., Mori, Y., and Nagase, H. 1998. Plasma membrane-bound tissue inhibitor of metallopeptidases (TIMP)-2 specifically inhibits matrix metallopeptidase 2 (gelatinase A) activated on the cell surface. J. Biol. Chem. 273:24360-24367. Jain, A., Nanchahal, J., Troeberg, L., Green, P., and Brennan, F. 2001. Production of cytokines, vascular endothelial growth factor, matrix metallopeptidases, and tissue inhibitor of metallopeptidases 1 by tenosynovium demonstrates its potential for tendon destruction in rheumatoid arthritis. Arthritis Rheum. 44:1754-1760. Kjeldsen, L., Johnsen, A.H., Sengelv, H., and Borregaard, N. 1993. Isolation and primary structure of NGAL, a novel protein associated with human neutrophil gelatinase. J. Biol. Chem. 268:1042510432. Kleiner, D.E. and Stetler-Stevenson, W.G. 1994. Quantitative zymography: Detection of picogram quantities of gelatinases. Anal. Biochem. 218:329-329. Leber, T.M. and Balkwill, F.R. 1997. Zymography: A single step staining method for quantitation of proteolytic activity on substrate gels. Anal. Biochem. 249:24-28. Nagase, H. 1997. Activation mechanisms of matrix metallopeptidases. Biol. Chem. 378:151-160. Noha, M., Yoshida, D., Watanabe, K., and Teramoto, A. 2000. Suppression of cell invasion on human malignant glioma cell lines by a novel matrixmetallopeptidase inhibitor SI-27: In Vitro study. J. Neurooncol. 48:217-223.

Oliver, G.W., Leferson, J.D., Stetler-Stevenson, W.G., and Kleiner, D.E. 1997. Quantitative reverse zymography: Analysis of picogram amounts of metallopeptidase inhibitors using gelatinase A and B reverse zymograms. Anal. Biochem. 244:161-166. Troeberg, L., Tanaka, M., Wait, R., Shi, Y.E., Brew, K., and Nagase, H. 2002. E. coli expression of TIMP-4 and comparative kinetic studies with TIMP-1 and TIMP-2: Insights into the interactions of TIMPs and matrix metallopeptidase 2 (gelatinase A). Biochem. 41:15025-15035. Yasothornsrikul, S. and Hook, V.Y. 2000. Detection of proteolytic activity by fluorescent zymogram in-gel assays. Biotechniques 28:1166-1168. Yeow, K.M., Kishnani, N.S., Hutton, M., Hawkes, S.P., Murphy, G., and Edwards, D.R. 2002. Sorbys fundus dystrophy tissue inhibitor of metallopeptidases-3 (TIMP-3) mutants have unimpaired matrix inhibitory activities, but affect cell adhesion to the extracellular matrix. Matrix Biol. 21:75-88. Zeng, Z.S., Shu, W.P., Cohen, A.M., and Guillem, J.G. 2002. Matrix metallopeptidase-7 expression in colorectal cancer liver metastases: Evidence for involvement of MMP-7 activation in human cancer metastases. Clin. Cancer Res. 8:144-148.

Contributed by Linda Troeberg and Hideaki Nagase Imperial College London London, United Kingdom

This work is supported by a Wellcome Trust grant no. 057508. The authors thank Abhilash Jain, Nidhi Sofat, and Yoshifumi Itoh for providing conditioned medium from cultured human wrist joint synovium, human chondrocytes, and TPA-treated HT1080, respectively.

Zymography of Metalloproteinases

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