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Walter Stocker
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Proteolytic Enzymes Tools and largest
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4
Detection of Proteolytic Enzymes Using Protein Substrates KEVIN K.W. WANG
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Introduction J(l
Short peptidic substrates are powerful tools for studying and detecting pro teases. However, the natural substrates for the majority of proteases are in fact proteins. The larger size of a protein substrate can allow additional in
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teraction with the protease of interest. In fact, some proteases are much more efficient in hydrolyzing proteins than small peptides. The calcium activated protease (calpain) present in all mammalian cells is one example
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(Murachi, 1983; Saido et al., 1994; Wang and Yuen, 1994). Thus, a protein substrate assay can be a sensitive and powerful assay for studying certain proteases. In this chapter, we will describe such an assay, using casein as a protein substrate (Buroker and Wang, 1993). Arguably, for certain protease systems, a protein substrate may better mimick the physiological processes
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in which the proteases participate. Another area in which a protein substrate is more useful than a peptide is protease zymography (Paech, et al., 1993a; Raser et al., 1995). In this chapter, we will describe two zymography pro tocols that call for co-polymerizing a protein substrate into an acrylamide gel. Both assays are useful tools in studying purified enzymes, .but in ana
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lyzing proteolytic activities in tissues or culture cell samples (Buroker et al., 1993; Raser et al., 1995).
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Kevin K.W. Wang, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Laboratory ofNeuro-biochemistry, Department ofNeuroscience Therapeu tics,
2800
Plymouth Road, Ann ArborMichigan,
Kevin.W
[email protected])
48105, USA fax (734) 622-7178; e-mail
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KEVIN K.W. WANG
Outline
Time-dependent hydrolysis of protein substrate with protease
Binding of remaining protein to Coomassie Blue G-250 at pH 1.2
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Detection of Coomassie Blue-protein complex by absorbance at 595 n m Fig. 1 . Schematic flow-shee t demonstrating the proteolytic hydrolysis o f protein substrate
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(e.g., casein) with a protease and the subsequent detection of remaining unhydrolyzed sub strate by Coomassie blue-binding
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Materials For Colorimetric Assay Reagents
- Bio-Rad protein dye reagent concentrate [0.05% (w/v) Coomassie Bril-
liant Blue G-250, 23.5% (w/v) ethanol, and 42.5% (w/v) phosphoric acid] (Bio-Rad Labor ato ries, #500-0006) - Bovine casein (sodium salt) (Sigma, #C8654) - Porcine m- calpai n (Calbiochem, #208715) and �L-calpain (Calbiochem,
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#208712)
CaCI2, dit h iothreitol (DTT), EGTA (Sigma Chemical Company, reagent grade)
- 96-well microtiter plates
Equipment
Thermomax microplate reader (Molecular Dev ices) w ith prin te r or com
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puter connection
- 8- or 12-channel multipipettor (Eppendorf)
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4 Detection
of Proteolytic
Enzymes Using Protein Subst rates
Incubation of protease samples with inhibitory agents (optional)
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for most proteases
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Electrophoresis in SD$ polyacrylamide gel with gelatin
Electrophoresis in non denaturing polyacrylamide gel with casein
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Renaturation or reactivation of embedded protease In an activation buffer (with several changes of buffer and long incubation)
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Staining of gels with Coomassie Blue G-250 In an acetic acid/methanol solution and destaining
Fig. 2. Schematic flow-chart showing the electrophoresis of protease into protein substrate cnntaining polyacrylamide gel, the reactivation oft he protease and the subsequent detection of proteoly'tic activity by Coomassie blue staining of the substrate gels
(zymograms).
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DTCC Soup 33.3 mM DTT 83.3 mM Tris-HCl (pH 7.4 at 2S''C) 0.83 mg/ml casein 10 J.lg/ml �t-calpain (unless otherwise stated) or 14 m�tg/ml m-calpain or 14 J.lg/ml trypsin or 60 ng/ml papain
Buffers and
solutions
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l(a,VIN K.W. WA!I:G
Coomassie Blue Solution Bio-Rad protein assay dye reagent: water (1:1)
For Zymography Reagents
- Bovine casein (sodium salt) (Sigma, #C8654) - Porcine m-calpain (Calbiochem, #208715) and �t-calpain (Calbiochem, #208712) - Collagenase (type Ill; Worthington, #CLS-3) - Polyacrylamide gel cassettes (1.5 mm thick) (Novcx, #NC2015) and 10weU comb (Novex, #NC3510).
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Precasted 0.1% gelatin Tris-glycine 10% polyacrylamide gel (1.0 mm thick, 10 wells) (zymogram gel, Novex, #EC6175) - N-acetyl-Leu-Leu-Met-H (Calbiochem, #208721) - E64c, calcium chloride (CaC12), dithiothreitol (DTT), casein (sodium salt), gelatin, EGTA, Tris-basc and glycerol (Sigma Chemical Company, reagent grade) - Glycine, Coomassie Brilliant Blue G-250, bromophenol blue, 2-mercap toethanol and sodium dodecyl sulfate (SDS) (aU from BioRad, electro phoresis grade) I
Equipment
- Xcell l1 mini-cell electrophoresis unit (Novex, #£19001) - Power supply unit (BioRad, model 200/2.0)
Buffers and solutions
- Separating substrate gel solution (25 ml for 4 gels) -12% (w/v) acrylamide -0.32% (w/v) N,N'-methylene-bisacrylamide -375 mM Tris-HCI (pH 8.8) -0.2% (w/v) casein or gelatin -3.5% glycerol - Stacking gel solution (5 ml for 4 gels) -4% (w/v) acrylarnide -0.10% (w/v) N,N' -methylenc-bisacrylamide -330 mM Tris-HCl (pH 6.8) - 4X Sample buffer -20% (v/v) glycerol -2 mM 2-mercaptoethanol (not used for metalloprotcases) -0.004% (w/v) bromophenol blue -200 mM Tris-HCI (pH 7.0)
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Detection of
Proteolytic
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Substrates
- 4X SDS-sample buffer -10% (w/v) SDS -20% (v/v) glycerol -2 mM mercaptoethanol -0.004% (w/v) bromophenol blue -200 mM Tris-HCl (pH 6.8) SDS-Gel running buffer -0.2% (w/v) SDS -25 mM Tris-base -192 mM glycine (pH 8.3) Non-SDS running buffer -25 mM Tris-base -192 mM glycine -1 mM EGTA and 1 mM DTT (pH 8.3) - Reactivation buffer C (for calpain) -20 mM Tris-llCI (pH 7.4 at room temperature) -10 mM DTT -1-4 mM CaCl2 - Renaturation buffer Ml (for metalloproteasc) -2.5% (v/v) triton X-100 -100 mM glycine (pll 8.3) - Proteolysis buffer M2 (for metalloprotease) -100 mM glycine (pH 8.3) Fixing/Destaining solution -methanol/water/acetic acid (5:4:1) - Staining solution -0.25% (w/v) Coomassie blue R-250 in flxing/destaining solution
Procedure Colorimetric Assay 1.
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Pipette 2.5 �tl of calpain inhibitor as DMSO or DMF stock into a 96-well polyproprolene microliter plate, to achieve a desirable final concentra tion in a total volume of 250 �tl (optional).
2. Add 47.5 ).!1 (or 50 J..tl if no compound is added in step 1 of water. 3. Add 150 ).!1 of freshly made DTCC soup and shake briefly.
Proteolysis reaction
plates
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KEVIN K.W. WANG
4. Start the proteolytic reaction by adding either SO J!l of CaCh to positive
J Colorimetric development
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control wells and wells with inhibitors or 1 mM EDTA to negative control wells.
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5. After shaking, the plate is covered with a lid and is incubated at 2S"'C for 60 min.
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1. Add 50 1!1 of water to a separate polystrylene 96-well plate.
2. Transfer two 100 J!l-aliquots from each used well of the reaction plate to two separate wells of a new plate (to create duplicates). 3. Add 100 f!} of Coomassie Blue solution and shake briefly; burst any bub bles in the wells using a needle. 4. Incubate at room temperature for 10-15 min. 5. Read absorbance at S9S nm in a microplate reader.
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Zymogram
PAGE in non-de
naturing
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To initiate polymerization, added ammonium persulfate (0.04%, w/v) and TEMED (0.028%, v/v) to the separating casein gel solution.
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Immediately pour the separating casein gel solution into the empty gel cassettes and allow to polymerize for 30-60 min.
(native)
substrate gels
3. After the well-forming combs are inserted into the top of the cassettes, again, ammonium persulfate (0.08%, w/v) and TEMED (0.028%, v/v) are added to the stacking gel solution. 4. Fill the top part of the cassettes with the mixture. Allow the gel to poly merize for 15-30 min. Alternatively, precated casein (O.Sh) polyacryl amide gels are now commercially available (Novax). S.
Calpain samples in SO mM Tris-HCl, 1 mM DTT, l mM EGTA is either untreated or incubated in 50 mM Tris-HCl (pH 7.4), 3 mM DTT, 2 mM CaC12 in the presence of a calpain inhibitor for S min on ice (volume 32 ).!1).
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6. Three J!l of 100 mM EGTA is added. 7. Five J!l of the non-SDS-sample buffer is added (total volume 40 )11) and keep on ice.
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Detection
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8. During step 5, The casein gels were pre-run with the non-SDS running
buffer for 15 min in an ice-water bath. 9. Protease samples are then loaded into the wells. Rainbow molecular
weight markers (Arnersham; RPN756) are also run alongside to monitor the progress of electrophoresis. 10. Electrophoresis is run at 125
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for 3 h in an ice-water bath.
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4. Five !-11 of the SDS sample buffer is added (total volume 40 f..ll) and the samples are kept on ice.
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Note: For metalloprotease the presence of 2-mercaptoethanol should be avoided.
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11. The gel is then removed and incubated in reactivation buffer C with two changes within 2 h. 12. The gel is then further incubated overnight (20 to 24 h) at ambient ternperature in the same buffer. 1. Commercially prccasted gelatin gels are used wherever possible; otherwise, follow the previous section to make your own gelatin gels. 2. Collagenase samples in 50 mM Tris-HCl are either untreated or incubated in 50 mM Tris-HCl (pH 7.4), 2 mM CaCh, 1 pM ZnCI2 in the presence of an inhibitor for 5 min on ice (volume 32 pl). 3. Three pi of 100 mM EGTA is added.
5. During step 5, the gels are pre-run with the SDS-gel running buffer for 15 min at room temperature. 6. Protease samples are then loaded into the wells. SDS-PAGE molecular weight markers (BioRad; # 161-0317) are also run alongside for subsequcnt molecular weight estimation. 7. Electrophoresis is run at 125 V for 2.2 h at room temperature. 8. The gel is then removed and incubated in reactivation buffer M I with two changes within 1 h. 9. The gel is then incubated in renaturing buffer M1 for 3 h with three
changes. 10. Further incubate the gels overnight in the proteolysis buffer M2for 16 to 20 h at ambient temperature.
PAGE in denaturing substrate gels
56
KEVI:-1
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Staining and destainutg of zymograms
1. At the end of the proteolysis reaction, incubate the gels in water for 1 h with two changes, followed by 30 min in the fixing solution.
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2. Stain the gel with the staining solution for 30 min, followed by the de staining solution with several changes in 2-5 h.
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3. Store the gel in 2.5% (v/v) acetic acid. ·'
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Results II
Colorimetric Assay This protocol is relatively simple to execute. It involves two major steps: the
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proteolysis incubation during which protein substrate casein is hydrolyzed by the added protease and the subsequent binding or unhydrolyzed casein to Coomassie blue at very low pH (see Fig. 1). This assay relies on the ability
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of a protease to cleave the substrate protein at multiple sites. Under these conditions, only large proteins, but not its proteolytic products (e.g. short peptides and amino acids), would bind to Coomassie blue (Bradford, 1976;
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Buroker et al., 1993). The dye-protein complex is blue while the free dye is brownish yellow. Thus, an absorbance at 595 nm essentially monitors the disappearance of substrate. For example, Absorbance of 0.850 and 1.900 for
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wells with and without protease activity, respectively. It should be noted that before using the assay for routine experiments, an initial run should be done to first determine the optimal concentration of the protease of in
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terest (Fig. 3). As expected, certain proteases are more readily detected than others. We found that papain, trypsin, chymotrypsin and calpains can be
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detected with this assay. Proteases that are more restrictive on their cleavage sequence would not be well suited for this assay. This assay can also be used to detect or monitor activity of a protease in tissues, cells (using homoge nate) or biological fluids. We extended the usage of this assay to evaluate the potency of a protease inhibitor. Using �l-calpain and its inhibitor Ac-Leu-Leu-Met-H as an exam ple, the calpain activity was determined at various concentrations of this inhibitor spanning three orders of magnitude ( 10 nM to 10 pM). Using cal pain activities (without inhibitor added) in the absence (-Ca) or the pre sence of calcium ( +Ca) as standards, we calculate calpain activity at x 11M of the inhibitor as: A595(X)- A595(+Ca). Thus, the % cal pain inhibition at x �tM of Ac-Leu-Leu-Met-H is:
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