Zymography of Metalloproteinases

Linda Troeberg1, Hideaki Nagase1

1 Imperial College London, London, United Kingdom
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 21.15
DOI:  10.1002/0471140864.ps2115s33
Online Posting Date:  November, 2004
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Zymography is an electrophoretic technique enabling visualization of the number and approximate size of peptidases in a sample on the basis of their hydrolysis of a protein substrate within the gel. The technique is particularly useful for analyzing the peptidase composition of complex biological samples because visualization depends directly on proteolytic activity. This unit presents a representative zymography protocol for the study of matrix metallopeptidases (MMPs).

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Table of Contents

  • Basic Protocol 1: Zymography of Matrix Metalloproteinases Using a Gelatin or Casein Substrate
  • Alternate Protocol 1: Zymography of Matrix Metalloproteinases Using Substrate Proteins other than Gelatin
  • Alternate Protocol 2: Real‐Time Zymography
  • Alternate Protocol 3: Reverse Zymography for Study of Proteinase Inhibitors
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Zymography of Matrix Metalloproteinases Using a Gelatin or Casein Substrate

  • Acrylamide/bisacrylamide solution (see recipe)
  • Separating gel buffer (see recipe)
  • 10× casein or gelatin substrate solution (see recipe)
  • Sucrose solution (see recipe)
  • 10% (w/v) ammonium persulfate (see recipe)
  • n‐Butanol, H 2O saturated
  • Stacking gel buffer (see recipe)
  • MMP sample
  • 1 mM APMA (see recipe)
  • 2× sample loading buffer (see recipe)
  • Proteinase standards (e.g., recombinant MMP‐1, ‐2, or ‐3)
  • 1× anode and cathode reservoir buffers (see recipe)
  • 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, 37°C (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)
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  •   FigureFigure 21.15.1 Recombinant proMMP‐2 was activated by 1 hr incubation with 1 mM APMA at 37°C and its active concentration confirmed by titration against TIMP‐2 (Troeberg et al., ). Various concentrations of recombinant MMP‐2 were analyzed on a 7.5% gelatin zymogram (upper panel) incubated at 37°C 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.
  •   FigureFigure 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 37°C. 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 µl 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‐µl 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‐µl 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 µl 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.
  •   FigureFigure 21.15.3 A sample containing 100 pg recombinant proMMP‐2/MMP‐2 (rE) and 2 µl conditioned medium from TPA‐treated HT1080 (H) were run on a 7.5% gelatin zymogram as in Figure . The effects of various proteinase inhibitors on activity were investigated. Compared with zymograms incubated in the absence of inhibitors (none), addition of 20 µM E‐64, 10 µM 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.
  •   FigureFigure 21.15.4 MMP samples were analyzed on a 7.5% acrylamide gelatin zymogram, incubated in developing buffer overnight at 37°C. 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 37°C 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 α2M to MMP‐2 for 30 min at 37°C reduces the activity of both the 68‐ and 45‐kDa proteolytically active species (lane 3). α2M differs from TIMP‐2 in that the α2M/MMP‐2 complex is stable in sample loading buffer. Preincubation of MMP‐2 with TIMP‐2 protects it from association with α2M (lane 4), since the MMP‐2/TIMP‐2 complex is not proteolytically active and hence unable to bind to α2M. 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 α2M 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 α2M (lane 6). The truncated catalytic domain, however, interacts poorly at TIMP‐2 and becomes associated with α2M (Itoh et al., ), shifting its zone of digestion from 45 kDa. A zone of lysis can often be observed associated with α2M. Lane M shows protein molecular mass markers.


Literature Cited

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  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.
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