Practical course: Basic biochemical methods and ischemic heart models

Gelatin zymography for detection of matrix-metalloproteinase-2 and -9 (MMP-2, MMP-9) from

myocardiam samples A practical manual

Krisztina Kupai, PharmD, PhD

2011

Supported by: HURO/0901/069/2.3.1

HU-RO-DOCS

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Table of content

INTRODUCTION OF MATRIX METALLOPROTEINASES (MMPS) .............. 3

INTRODUCTION OF ZYMOGRAPHY ............................................................... 4

SAMPLES FOR ZYMOGRAPHY ....................................................................... 6

Isolation procedure of rat heart to detect tissue MMP-2 ............................... 6

FLOW CHART OF GELATIN ZYMOGRAPHY ................................................. 7

SAMPLE HOMOGENIZATION........................................................................... 7

PROTEIN CONCENTRATION MEASUREMENT FROM SUPERNATANT WITH BCA KIT AND SAMPLE MIXING WITH LOADING BUFFER .............. 8

PREPARATION OF SEPARATING GEL ........................................................ 10

PREPARATION OF STACKING GEL ............................................................. 12

LOADING SAMPLES:....................................................................................... 12

RUNNING OF GELS ......................................................................................... 13

WASHING (renaturation) AND INCUBATION OF THE GELS .................... 14

GEL STAINING .................................................................................................. 14

REPRESENTATIVE GELATIN ZYMOGRAM GEL ........................................ 16

APPENDIX - PREPARING SOLUTIONS ........................................................ 17

REFERENCES ................................................................................................... 20

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INTRODUCTION OF MATRIX METALLOPROTEINASES (MMPS) MMPs are a family of at least 23 endopeptidases that act as

effectors of extracellular matrix remodelling in physiological and pathological conditions. MMPs can be subdivided into gelatinases (MMP-2 and -9), collagenases (MMP-1, -8, -13 and -18), stromelysins (MMP-3, -10 and -11) and other MMPs, according to their substrate affinity profile. Their activity is closely regulated by tissue inhibitors of metalloproteinases (TIMPs), a group of four endogenous antagonists that bind to the catalytic site of MMPs. MMPs have been shown to play significant roles in a number of physiological processes, including embryogenesis and angiogenesis, but also contribute to pathological processes such as tumour metastasis, inflammation and arthritis. Of this diverse family of enzymes, MMP-2 and -9 (also known as gelatinase A and gelatinase B, respectively) have emerged as important players in a number of cardiovascular diseases, including atherosclerosis, stroke, heart failure, ischaemic heart disease and aneurysm (Kupai et al.).

Zymogen, from the Greek zymo (ferment, leaven) and gen (new, beginning), is a term used to define an inactive enzyme precursor (or proenzyme).

Source: British Journal of Pharmacology (2007), 1–17

Mechanism of pro-matrix metalloproteinase 2 (pro-MMP-2) activation:

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The full-length MMP-2 can be activated in two ways. Proteolytic activation of MMP-2 by MT1-MMP/TIMP or by other proteases occurs by removal of the autoinhibitory propeptide domain (left arrow) resulting in an active truncated MMP-2. The presence of oxidative stress (ONOO) and cellular glutathione (GSH) causes the S-gluathiolation of the critical cysteine residue in the propeptide domain, disrupting its binding to the catalytic Zn2+ ion, resulting in an active full-length enzyme. MMP-2 also known as 72kDa collagenase IV or gelatinase A is synthesized as a 631 amino acid proenzyme which is activated by cleavage of the first 80 amino acids. MMP-9 or gelatinase B full length is 92 kDa (proenzyme,) and cleaved active enzyme is 84 kDa. ONOO: peroxynitrite; TIMP: tissue inhibitor of metalloproteinase INTRODUCTION OF ZYMOGRAPHY

Zymography is known as an electrophoretic technique, commonly based on sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE), which contains a substrate copolymerized within the polyacrylamide gel matrix (e.g gelatin), for the detection of an enzymatic activity. Samples are normally prepared by the standard SDS-PAGE treatment buffer, under non-reducing conditions, i.e. absence of heating and reducing agent [2-mercaptoetanol, dithiothreitol (DTT)]. After the electrophoretic run, the SDS is soaked out from the gel (zymogram) by incubation in a non-buffered Triton X-100 (or similar detergent), followed by incubation in an appropriate activation buffer, for an optimized length of time and temperature, depending on the type of enzyme being assayed and the type of substrate being degraded. The zymogram is subsequently stained, and areas of digestion are distinguished. Though many different types of zymography exist (according to the type of enzyme), not all are possible to mention in this protocol.

For the specific case of proteases (MMP-2, MMP-9) gelatin is one of the most frequently used substrate. In this case, visualization of the proteolytic activity appears as clear bands over a deep blue background, after Coomassie staining.

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Source: Recent Patents on Biotechnology 2009, 3, 175-184 Schematic overview of some zymographic techniques. (A) 1-D zymography consists in a SDS-PAGE, with a co-polymerized substrate. After run and enzyme activation, active bands corresponding to the enzymes are seen as white bands with a blue background. (B). For the 2-D zymogram, the sample must be first submitted to IEF and then to SDS-PAGE with the co-polymerized substrate. After run and incubation, white spots are evidence of enzymatic activity. (C) The spots obtained by 2-DZ, may be further analyzed by MALDI-TOF/MS, in order to identify the enzyme. (D) In-situ zymography (ISZ) is performed directly on tissue samples, allowing cellular localization of the enzymatic activity. (E) Real-time zymography (RTZ) allows continuous detection of the enzymatic activity over time. (F) Multiple layer substrate zymography (MLSZ) allows the simultaneous detection of different kind of enzymes from one gel. A gel (1) is run with the sample and then further electrotransferred to several zymograms (2, 3 and 4) containing different substrates. The net result is that with one run, it is possible to detect different enzymes. (G) Reverse zymography consists in incorporating not only a substrate to the gel, but also the enzyme. Thus, it is possible to detect enzyme inhibitors, visible as dark bands on a light background.

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SAMPLES FOR ZYMOGRAPHY Zymography is suitable for analysis of MMPs in complex

biological fluids (serum, synovial fluid) and tissue extracts (heart, liver, kidney, spleen, etc.).

Isolation procedure of rat heart to detect tissue MMP-2 Male Wistar were fed a standard rat chow diet. At age of 8

weeks hearts were excised and perfused on a Langendorff perfusion apparatus for 10 min to eliminate the blood from the heart. At the end of 10 min washing period hearts were freeze-clamped and crushed at liquid N2.

Isolated Langendorff rat heart preparation: As shown in

figures, this involves the cannulation of the aorta which is then attached to a reservoir containing oxygenated perfusion fluid. This fluid is then delivered in a retrograde direction down the aorta either at a constant flow rate (delivered by an infusion or roller pump) or a constant hydrostatic pressure (usually in the range of 60-100mmHg). In both instances, the aortic valves are forced shut and the perfusion fluid is directed into the coronary ostia thereby perfusing the entire ventricular mass of the heart, draining into the right atrium via the coronary sinus.

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FLOW CHART OF GELATIN ZYMOGRAPHY

SAMPLE HOMOGENIZATION Procedure:

1. Weigh out 30 mg pulverized heart tissue and add homogenization buffer 1:4 ratio (30 mg tissue+120 µl homogenization buffer)

2. Homogenize the sample 3x10 sec with Pestle homogenizator on ice

3. Centrifure the samples on 4 °C and 5000 g 4. Collect the supernatant which contains the MMPs

Pestle homogenizator

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PROTEIN CONCENTRATION MEASUREMENT FROM SUPERNATANT WITH BCA KIT AND SAMPLE MIXING WITH

LOADING BUFFER

I. Measure the protein concentration of supernatant with BCA (bicinchoninic acid) method

In case of heart samples must be diluted (20x) before protein measurement. Follow the instuction of Pierce® BCA Protein Assay Kit (Cat No:23225)

Principle of protein concentration measurement with BCA

II. Calculete the loaded protein amount and mix the the samples with loading buffer:

1. We have homogenized a heart sample after centrifuge=supernatant and need to load 20 g protein per lane. Since we load 20 L per lane, this means that the final protein concentration of sample needs to be 20 g/20 L. 2. In case we want to load a sample only once, it is enough to prepare 1.5× volume of one load (30 L), which means that we should add 15 L non-reducing loading buffer. 3. The remaining 15 L should contain 1.5×20 g=30 g protein. Therefore, volume of the sample will be: V1=C1/30, where C1 is the protein concentration of your sample.

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4. Then we should add homogenization buffer to dilute the sample. Volume of which will be: V2=15-V1.

Taken together:

Loading volume: 20 L Loaded protein: 20 g Prepared volume: 30 L (1.5× load) Loading buffer: 15 L Sample: V1=C1/30 Homogenization buffer: V2=15-V1

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PREPARATION OF SEPARATING GEL The final acrylamide concentration in the separating gel (8%) for MMP-2 and MMP-9

STOCK SOLUTIONS VOLUME

30% acrylamide/ 0.8% bisacrylamide

4.0 ml

1.5 MTris HCl, pH 8.8 3.75 ml

ddH2O 5.75 ml

Gelatin Solution

(20 mg/mL, 1 % w/v SDS)

1.5 ml

10% w/v Ammonium Persulfate Solution (APS)

50 L

TEMED 10 L

MATERIALS: 25 mL glass baker pipettes

electrophoresis unit (casting mount, glasses, spacers, combs, seals) butanol

wipe, blotting paper PROCEDURE:

1. Prepare gelatin solution (See:Appendix). 2. Assemble electrophoresis unit. In order to avoid leakage, ensure

that spacers and glass plates are perfectly aligned. 3. Mark desired level of separating gel on unit (use comb). 4. Mix 30% acrylamide/0.8% bisacrylamide solution with Tris HCl, pH

8.8, gelatin solution and ddH2O. 5. Add 10% APS solution and TEMED to the mix quickly.

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6. Swirl to mix. Avoid bubbling. USE IMMEDIATELY as polymerization process has begun.

7. Using a pipette, pour a small amount into sandwich plates and watch for leakage. No leaks? - continue to fill to 1 mm above line.

8. Gently add butanol along top to remove bubbles (avoid “shooting” the butanol).

9. Allow gels to polymerize (approximately 20 minutes at 25 °C). Use this time to prepare stacking gel (without adding TEMED and 10% APS) TIP: leave pipette in the left over separating gel, when it is polymerized you will be able to lift it with the pipette.

10.A layer of H2O on top of the gel will be visible when polymerization is complete. Drain this layer from the unit with a small stripe of blotting paper.

11.Prepare the stacking gel

Combs and spacers for gel

Preparation of separating gel (Source:http://www.soonersci.com/catalog/page21.html)

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PREPARATION OF STACKING GEL MATERIALS: 25 mL glass baker Pipettes

STOCK SOLUTIONS VOLUME

30% acrylamide/ 0.8% bisacrylamide

0.65 ml

0.5 M Tris HCl pH 6.8 1.25 ml

ddH2O 3.05

10% SDS 50 L

10% w/v Ammonium Persulfate Solution (APS)

25 L

TEMED 8 L

PROCEDURE:

1. Mix 30% acrylamide/0.8% bisacrylamide solution with Tris HCl, pH 6.8 and ddH2O.

2. Add 10% SDS, 10% APS and TEMED. 3. Swirl to mix. Avoid bubbling. USE IMMEDIATELY as polymerization

process has begun. 4. Place comb in units and then use pipettes to pour stacking gel. 5. Place stacking gel solution on ice and continue to “top up” the gel

until polymerization is complete.

LOADING SAMPLES: MATERIALS: 1000 mL running buffer 20 L pipette

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PROCEDURE:

1. Prepare and cool down running buffer. 2. Map out gel lanes for sample loading. 3. When gel is polymerized, remove combs by pulling straight up. 4. Remove gel plates and snap onto electrode assembly. 5. Fill up the lower and the upper buffer container with running buffer. 6. Load samples. 7. Load protien molecular weight and purified MMP-2 as an internal

standard

RUNNING OF GELS 1. Connect electrodes properly 2. Set voltage at 90 V. 3. For 8% gel run for 0.5 hour after dye front disappears. (The exact

duration of electrophoresis will vary from person to person.) 4. Use this time to prepare Triton X-100 solution and incubation

buffer.

Electrophoretic tank&steps electrophoresis (Source:http://www.topac.com/vertical_casting.html)

1. With plates in position place central running module in the casting base. 2. Turn both cams to pull module down onto silicone sealing units. 3. Pour gel between plates. 4. Place appropriate comb between plates and allow gels to set. 5. After carefully removing combs release module from casting base. 6 Place the module in the outer buffer tank and fill the two buffer chambers.

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7. Load sample and replace lid. 8. Unit is now ready for electrophoresis.

Sample separation on SDS-gelatin gel after electrophoresis

WASHING (renaturation) AND INCUBATION OF THE GELS MATERIALS: 1000 mL 2.5% v/v Triton X-100 aqueous

solution 1000 mL incubation buffer 1000 mL dd H2O water bath (set at 37oC) PROCEDURE:

1. Set incubator at 37oC. 2. Disassemble gel apparatus. 3. Cut the bottom left corner of gel #1, and both the top and bottom

left corners for gel #2. (Ensure that the gel is oriented correctly so that you don’t accidentally cut the right side corner!)

4. Wash gels for 40 min in 300-500 mL 2.5% Triton X-100 solution at room temperature.

5. Place gels in incubation buffer (500 mL per gel). 6. Incubate gels for 20 h at 37oC.

GEL STAINING MATERIALS: 0.05% Coomassie Brilliant Blue G-250 solution destaining solution

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PROCEDURE:

1. Even before staining, gelatinolytic activity should be visible (hold the gel up against a dark background to visualize).

2. Put gels in Coomassie Brilliant Blue solution. Place on shaker for 1 - 2 hours.

3. Put gels in destaining solution. Place on shaker for 2 hours 4. Scan the scan 5. Gelatinolytic activities should be detected as transparent bands

against the blue background of Coomassie Brilliant Blue stained gelatin.

Pour the in Coomassie Brilliant Blue to gels Rocking the freezer box on the tilt table will ensure that the gel gets evenly stained. Destaining of the gel will remove any Coomassie blue dye that is not bound to protein.

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REPRESENTATIVE GELATIN ZYMOGRAM GEL

Zymogram gel at the end of procedure: gelatinolytic activities should be detected as transparent bands against the blue background of Coomassie Brilliant Blue stained gelatin

MMPs are initially secreted as an inactive proenzyme or zymogen, which supposely have no gelatinolytic activity. So how the zymography works to show the pro-MMP2, pro-MMP9 binds, since the proenzymes cannot digest the gelatin?

The enzymes are separated in denaturing conditions (SDS), refolded in Triton (to remove the SDS), then incubated. The zymogens forms are activated by this process of denaturation and renaturation, and so may be visualized in the zymogram. In this way, they migrate according to the molecular weight (pro-form migrate less than the active form) and both are seen at the gel. This is the main advantage of zymography, you can visualize both forms.

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APPENDIX - PREPARING SOLUTIONS

I.Separating Gel Solution – 1.5 M Tris HCl, pH 8.8

BIO-RAD 161-0798

II,Stacking Gel Solution – 0.5 M Tris HCl/SDS, pH 6.8

BIO-RAD 161-0799

III.30% Acrylamide / 0.8% Bisacrylamide

**NOTE: Acrylamide monomer should be weighed out in fume hood as it is neurotoxic.

BIO-RAD 161-0156

IV.10% (w/v) Ammonium Persulfate Solution

MATERIALS: 100 mg ammonium persulfate (Sigma A-6761)

1 mL ddH2O

1.Dissolve 100 mg of APS in 1 mL ddH2O.

NOTE - should be prepared freshly

V.Gelatin Solution

MATERIALS: 100 mg gelatin (type A, from porcine skin, SIGMA cat # G-8150)

4.5 mL ddH2O 0.5 mL 10 % w/v SDS aqueous solution

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glass beaker + stir bar 1. Add gelatin to the H2O. 2. Gently heat solution until gelatin dissolves (beaker will be warm to

touch). 3. Add 10% w/v SDS aqueous solution to reach final desired volume.

VI. _Running Buffer

(25mM Tris, 192 mM glycine, 0.1% SDS, pH=8.3)

BIO-RAD 161-0732 or Pierce 28378

Or prepare it:

MATERIALS: 2000 mL graduated cylinder

28.83 g glycine

6.0 g Tris base

2.0 g SDS

2000 mL ddH2O

1. Dissolve Tris base and glycine in 1000 mL of ddH2O. 2. Bring solution to 1950 mL with ddH2O. 3. Add SDS. 4. Bring solution to 2000 mL total volume with ddH2O.

VII.Non reducing Loading (Sample) Buffer

Bio-Rad 161-0764

VIII.Triton X-100 solution (2.5% v/v)

MATERIALS: 1000 mL glass beaker + stir bar

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25 mL Triton X-100

1000 mL ddH2O

1. Add 25 mL of Triton-X solution SLOWLY to 900 mL of ddH2O while continuosly mixing. (NOTE: Triton X-100 is a viscous fluid pour into solution slowly)

2. Bring total volume up to 1000 mL with ddH2O.

IX.Incubation Buffer (50 mM Tris HCl, 0.15 M NaCl, 10 mM CaCl2)

MATERIALS: 8.766 g NaCl

1.47g CaCl2*2H2O

6.057 g Tris base

0.5 g NaN3 (weigh in fumehood – toxic)

1000 mL ddH2O

1. Add NaCl, CaCl2, Tris base, and 0.5 g NaN3 to 1000 mL of ddH2O. 2. Adjust to pH 7.8-8.0 with cc. HCl.

X.Coomassie Brilliant Blue (0.05%)

MATERIALS: 500 mL glass beaker

250 mg Coomassie Brilliant Blue G-250 (Sigma B-1131)

125 mL methanol

50 mL glacial acetic acid

325 mL ddH2O

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XI.Destaining Solution

MATERIALS: 1000 mL glass beaker

40 mL methanol

80 mL acetic acid

880 mL ddH2O

XII.Homogenization Buffer

MATERIALS: 500 ml ddH2O

0.335 g (50 mM) Tris base (Merck 648310-500 GM)

1 ml (0.5%) Triton

1. In 500 ml beaker dissolve compounds in 500 ml dd H2O. 2. Adjust to pH 7.4 with 1 M HCl. 3. Aliquot into 15 ml Falcon tubes.

REFERENCES

1. Kupai K.; J Pharmacol Toxicol Methods. 2010 Mar-Apr;61(2):205-9; Matrix metalloproteinase activity assays: Importance of zymography.

2. Laemmeli,E.K.; Nature. 227:680-685, 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4.

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3. Jeff Wilkesman, Liliana Kurz, Recent Patents on Biotechnology 2009, 3, 175-184; Protease Analysis by Zymography: A Review on Techniques and Patents

4. AK Chow, J Cena, R Schulz, British Journal of Pharmacology (2007) 1–17; Acute actions and novel targets of matrix metalloproteinases in the heart and vasculature