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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

2


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



3

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), 117

Mechanism of pro-matrix metalloproteinase 2 (pro-MMP-2)
activation:
4

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 Zn
2+
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.

5



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.


6

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 N
2
.





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.
7

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 l
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
8

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 1.520 g=30 g protein.
Therefore, volume of the sample will be: V
1
=C
1
/30, where C
1
is the
protein concentration of your sample.
9

4. Then we should add homogenization buffer to dilute the
sample. Volume of which will be: V
2
=15-V
1
.
Taken together:
Loading volume: 20 L
Loaded protein: 20 g
Prepared volume: 30 L (1.5 load)
Loading buffer: 15 L
Sample: V
1
=C
1
/30
Homogenization buffer: V
2
=15-V
1

10

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
ddH
2
O 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 ddH
2
O.
5. Add 10% APS solution and TEMED to the mix quickly.
11

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 H
2
O 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)
12

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
ddH
2
O 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 ddH
2
O.
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


13

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.
14

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 H
2
O
water bath (set at 37
o
C)
PROCEDURE:
1. Set incubator at 37
o
C.
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 dont 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 37
o
C.

GEL STAINING

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


15

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.



16

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.





17

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 ddH
2
O
1.Dissolve 100 mg of APS in 1 mL ddH
2
O.
NOTE - should be prepared freshly

V.Gelatin Solution
MATERIALS: 100 mg gelatin (type A, from porcine skin, SIGMA
cat # G-8150)
4.5 mL ddH
2
O
0.5 mL 10 % w/v SDS aqueous solution
18

glass beaker + stir bar

1. Add gelatin to the H
2
O.
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 ddH
2
O

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

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
19

25 mL Triton X-100
1000 mL ddH
2
O

1. Add 25 mL of Triton-X solution SLOWLY to 900 mL of ddH
2
O while
continuosly mixing. (NOTE: Triton X-100 is a viscous fluid pour
into solution slowly)
2. Bring total volume up to 1000 mL with ddH
2
O.

IX.Incubation Buffer (50 mM Tris HCl, 0.15 M NaCl, 10 mM CaCl
2
)
MATERIALS: 8.766 g NaCl
1.47g CaCl
2
*2H
2
O
6.057 g Tris base
0.5 g NaN
3
(weigh in fumehood toxic)
1000 mL ddH
2
O

1. Add NaCl, CaCl
2
, Tris base, and 0.5 g NaN
3
to 1000 mL of ddH
2
O.
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 ddH
2
O
20


XI.Destaining Solution
MATERIALS: 1000 mL glass beaker
40 mL methanol
80 mL acetic acid
880 mL ddH
2
O

XII.Homogenization Buffer

MATERIALS: 500 ml ddH
2
O
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 H
2
O.
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.
21

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) 117; Acute actions and novel targets of matrix
metalloproteinases in the heart and vasculature

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