Biochemistry

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Authors: Kazushi Imai & Yasunori Okada 

Introduction

To detect activities of MMP-1, MMP-2, MMP-3, MMP-7 and MMP-9, use radio-labeled fibrillar collagen (for MMP-1), gelatin (for MMP-2 and MMP-9) and carboxymethylated transferrin (for MMP-3 and MMP-7) substrates for the assays (1), although these can be substituted with commercially available FITC-labeled substrates and fluorogenic synthetic peptides for the MMPs. Zymography using gelatin or casein substrate gels is also useful to monitor gelatinolytic activity of MMP-2 and MMP-9 or caseinolytic activity of MMP-3 and MMP-7 in each column fraction, although zymography is not suitable for calculation of purification fold and recovery of proteinases.

Procedure

Collagenase assay for MMP-1

  1. Mix a small amount of [14C]-labeled collagen (2~3 ml) with unlabeled collagen in 3% (vol/vol) acetic acid (45 ml; 3 mg/ml) to make the count ~3000 c.p.m. in 50 μl.
  2. Dialyze the mixture against 0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.02% (wt/vol) NaN3 and then 0.01 M Tris-HCl, pH 7.5, 0.2 M NaCl, 0.02% (wt/vol) NaN3 at 4˚C, and store the collagen at 4˚C.
  3. For collagen fibril assay, take 50 μl of the labeled collagen solution into 400 μl long centrifuge tubes (150 μg/tube), and incubate with 10 μl of each or combined fraction in TNC or TNC+B buffer, pH 7.5 (225 μl) in the presence or absence of 1 mM APMA (15 μl of 20 mM APMA) at 37°C (total volume of 300 μl).
  4. After incubation, spin the tubes at 10,000 g for 15 min using Beckman microcentrifuge and take 200 μl of the supernatant for counting using a scintillation counter.
    • ▲CRITICAL For the controls, add 250 μl of TNC or TNC+B buffer, pH 7.5 (blank), 10 μl of 100 μg/ml trypsin and 240 μl of the buffer (negative control) or 10 μl of 4 mg/ml bacterial collagenase and 240 μl of the buffer (positive control) in the assay. Activity is calculated as follows: (count of enzyme solution digestion – count of trypsin digestion) / (count of bacterial collagenase digestion – count of trypsin digestion) x 150 μg/min. Although we have used [14C]-labeled collagen as a substrate, collagenase assays using FITC-labeled type I collagen (collagenase assay kit for collagenase, Chondrex, http://www.chondrex.com), fluorogenic peptide substrate (MMP-1 Substrate I, Fluorogenic, MERC, http://www.merckbioscience.com/) or FRET substrate (SensoLyte Plus™ 520 MMP-1 assay kit, AnaSpec, http://www.anaspec.com) are commercially available and can be used to detect MMP-1 activity.

Gelatinase assay for MMP-2 and MMP-9

  1. Prepare heat-denatured gelatin by incubation of the [14C]-labeled collagen at 60˚C for ~30 min.
  2. Take 50 μg of the gelatin solution into 400 μl long centrifuge tubes, and incubate with enzyme solution in TNC or TNC+B buffer, pH 7.5 (135 μl) in the presence or absence of 1 mM APMA (15 μl of 20 mM APMA) at 37˚C (total volume of 200 μl).
  3. After incubation, add 100 μl of cold 45% (vol/vol) trichloroacetic acid. Keep samples in ice for at least 10 min and spin at 10,000 g for 15 min.
  4. Take 100 μl of the supernatant for counting using a scintillation counter.
    • ▲CRITICAL As for controls, add 150 μl of TNC or TNC+B buffer, pH 7.5 (blank) or 10 μl of 100 μg/ml trypsin and 140 μl of the buffer (positive control) in the assay. Activity is calculated as follows: (count of enzyme solution digestion – count of blank) / (count of trypsin digestion – count of blank) x 150 μg/min. Although we have used [14C]-labeled gelatin as a substrate, quenched fluorescent peptide substrate (EnzChek for gelatinases, Invitrogen, http://www.invitrogen.com/) is commercially available and can be used to detect MMP-2 and MMP-9 activities.

Carboxymethylated transferrin (Cm-Tf) assay for MMP-3 and MMP-7

  1. Reduce human transferrin (Tf) (20 mg) by incubation with 20 mM dithiothreitol in 20 ml of 0.2 M Tris-HCl, pH 8.6 containing 8 M urea for 4 h at 37˚C.
  2. Apply the reduced Tf to a column of Sephadex G-50 (Φ 2.5 cm x 20 cm) equilibrated in 0.2 M Tris-HCl, pH 8.6 containing 8 M urea at room temperature to remove dithiothreitol, and combine the void volume.
  3. Incubate the pooled sample with 1 mCi of [3H]iodoacetic acid for 30 min at 23˚C, and then with 30 mM iodoacetic acid for 30 min at 23˚C to complete carboxymethylation.
  4. After the reactions, add the same volume of cold 10% (vol/vol) trichloroacetic acid to the radiolabeled Tf and obtain the precipitate by centrifugation at 21,000 g for 5 min at 4˚C using a benchtop centrifuge. Repeat this precipitation step three times.
  5. Dissolve the precipitate in 50 mM Tris-HCl, pH 7.5, 0.15 M NaCl at a concentration of 3 mg/ml. Store the substrate at -20˚C in 1 ml portions.
  6. For the assay, take 10 μl of [3H]Cm-Tf into 1.5 ml Eppendorf tubes, and incubate with 10 μl of each or combined fraction in TNC or TNC+B buffer, pH 7.5 in the presence or absence of 1 mM APMA (10 μl of 3 mM APMA) at 37˚C (total volume of 30 μl). After incubation, add 200 μl of cold 3.3% (vol/vol) trichloroacetic acid. Keep samples at room temperature for 15 min and spin at 10,000 g for 5 min. Take 100 μl of the supernatant for counting by a scintillation counter.
    • ▲CRITICAL As for controls, add 10 μl of TNC or TNC+B buffer, pH 7.5 (negative control) or 10 μl of 100 μg/ml trypsin in TNC buffer, pH 7.5 (positive control) in the assay. Activity is calculated as follows: (count of enzyme solution digestion – count of blank) / (count of trypsin digestion – count of blank) x 30 μg/min. Although we have used [3H]-labeled Cm-Tf as a substrate, fluorogenic peptide substrates (Oncogene, http://www.apoptosis.com or MERCK, http://www.merckbioscience.com) are commercially available and can be used to detect MMP-3 and MMP-7 activities.

Zymography

  1. Make SDS-PAGE gels (1 or 2 mm in thickness) containing 0.2% (wt/vol) gelatin or 0.1% (wt/vol) casein.
  2. Take 20~40 µl of samples from column fractions, and incubate them with equal volume of SDS-PAGE sample buffer without 2-mercaptoethanol for ~30 min at 37˚C. Subject the samples to SDS-PAGE at 4˚C.
  3. After electrophoresis, wash the gels in 2.5% (vol/vol) Triton X-100 in TNC buffer, pH 7.5 for 15 min twice and then incubate in the incubation buffer at 37˚C for 12 – 24 h under shaking.
  4. After incubation, stain with Coomassie Brilliant Blue R and destain the gels.
    • ▲CRITICAL Rinsing the gels with 2.5% (vol/vol) Triton X in TNC buffer, pH 7.5 is necessary to remove SDS from the gels.
    • ▲CRITICAL Activation of proMMPs with APMA is not necessary for zymography, since proMMPs are spontaneously activated during SDS-PAGE probably because of their conformational changes. Proteolytic bands corresponding to proMMPs and active MMPs are seen as transparent bands within dark blue background.
    • ▲CRITICAL Inhibitor studies can be done by adding appropriate inhibitors (e.g., 15 mM EDTA for MMPs, 2 mM phenylmethane sulfonyl fluoride for serine proteinases, 5 mM N-ethylamaleimide for cysteine proteinases and 0.5 mM pepstatin A for aspartic acid proteinases) into the incubation buffer during incubation of the gels.

References

  1. Okada, Y., Nagase, H. & Harris, E.D., Jr. A metalloproteinase from human rheumatoid synovial fibroblasts that digests connective tissue matrix components. Purification and characterization. J Biol Chem 261, 14245-55 (1986).

Author information

Kazushi Imai, Department of Biochemistry, Nippon Dental University, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan

Yasunori Okada, Department of Pathology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-0016, Japan

Source: Protocol Exchange (2008) doi:10.1038/nprot.2008.85. Originally published online 3 April 2008.

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