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Real-Time Zymography and Reverse Zymography: A Method for Detecting Activities of Matrix Metalloproteinases and Their Inhibitors Using FITC-Labeled Collagen and Casein as Substrates
Analytical Biochemistry 301, 27–34 (2002) doi:10.1006/abio.2001.5479, available online at http://www.idealibrary.com on
Real-Time Zymography and Reverse Zymography: A Method for Detecting Activities of Matrix Metalloproteinases and Their Inhibitors Using FITCLabeled Collagen and Casein as Substrates Shunji Hattori,* ,† ,1 Hitomi Fujisaki,* Tomomi Kiriyama,* Tsukao Yokoyama,‡ and Shinkichi Irie* ,† *Nippi Research Institute of Biomatrix and †Japan Institute of Leather Research, Adachi-ku, Tokyo 120-8601, Japan; and ‡Collagen Research Center, Kiyose, Tokyo 204-0013, Japan
Received July 30, 2001; published online December 19, 2001
Zymography and reverse zymography are widely used techniques for identifying the proteolytic activity of enzymes and the presence of protease inhibitors in polyacrylamide gels. In the current studies, we utilized a fluorescein-isothiocyanate-labeled substrate to develop novel zymographic and reverse zymographic methods for detecting matrix metalloproteinases and tissue inhibitors of the metalloproteinases, respectively. Using a transilluminator, the results can be observed visually without stopping the enzymatic reaction. For this reason, we have named these methods real-time zymography and real-time reverse zymography. These methods have the following advantages compared with conventional protocols: (1) because the reaction can be repeatedly monitored on the polyacrylamide gels, optimization of the incubation time can be achieved without preliminary analyses; (2) higher sensitivity is achieved with a lower amount of substrate than with conventional methods; (3) a semiquantitative analysis of matrix metalloproteinases is possible. An additional advantage of the real-time reverse zymography is that, because the fluorescence detection is specific for substrate digestion, the inhibitor bands can be easily distinguished from contaminating proteins. © 2002 Elsevier Science Key Words: zymography; matrix metalloproteinase; tissue inhibitor of metalloproteinase; FITC; real-time; collagen.
MMPs 2 are thought to be involved in the metastasis and growth of tumors (1–3), the progression of inflammatory diseases such as rheumatoid arthritis (4), and the differentiation of skin keratinocytes (5, 6). The inhibitors of MMPs are also of interest as blockers of tumor growth and metastasis (7). Several synthetic inhibitors are now in clinical trials (2, 7), and TIMPs are thought to suppress the metastasis of carcinoma cells (8). Zymography, which uses an SDS-substrate gel, is a convenient method for detecting proteolytic enzymes like MMPs. The first forms of zymography utilized a gelatin substrate slab gel to detect plasminogen activators (9). Since then, zymography with gelatin or casein has been used to detect MMPs (10). TIMPs can be detected by reverse zymography (10, 11) using an acrylamide gel containing copolymerized MMP and gelatin. Fluorescein-labeled synthetic peptides have been utilized to detect the enzymatic activity of gelatinase or MMPs (12, 13). FITC-labeled denatured collagen has also been used to assay gelatinolitic activity in the solution method (14, 15). Furthermore, an assay with sensitivity almost as high as that using radioisotopelabeled polymeric gelatin has been developed using a fluorescent-labeled substrate (16, 17). In this report, we describe novel zymographic and reverse zymographic methods using FITC-labeled denatured collagen as the substrate. These methods are 2
1
To whom correspondence should be addressed at Nippi Research Institute of Biomatrix, 1-1-1 Senjumidori-cho, Adachi-ku, Tokyo 1208601, Japan. Fax: ⫹81-33870-9631. E-mail: [email protected]. 0003-2697/02 $35.00 © 2002 Elsevier Science All rights reserved.
Abbreviations used: APMA, 4-aminophenylmercuric acetate; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; hr, human recombinant; MMP, matrix metalloproteinase; SDS, sodium dodecyl sulfate; TIMP, tissue inhibitor of metalloproteinase; CBB, coomassie brilliant blue. 27
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more sensitive than conventional ones, and they allow for real-time analyses of enzymatic activity. MATERIAL AND METHODS
Materials hrMMP-2 and hrMMP-9 were from Genzyme Techne Co. hrTIMP-1 and hrTIMP-2 were from Fuji Chemical Co. (Toyama, Japan), and purified MMP-2 (from human fibrosarcoma cell) and MMP-3 (from human tumor cell) were from the Yagai Research Center (Yamagata, Japan). FITC-labeled BSA and FITC-labeled casein were from Sigma (St. Louis, MO).
(Fuji Film Co., Tokyo) which had a light cutoff under 520-nm wavelength, after which it was photographed by a Polaroid camera using FB-3000B black and white film (ASA3000) (Fuji Film Co., Tokyo). The SC52 filter showed the best result for our purpose and the No. 12 filter (Eastman Kodak, NY) also could be used substitutively. The exposure time varied with the intensity of illumination, but an f-stop of 5.6 for 1 s was typical for our system. After being photographed, the gel was returned to the chamber, and the incubation was continued. The gel was photographed at intervals of several hours. The pictures were scanned using Adobe Photoshop software (Adobe Systems, Inc., CA), and densitometric quantification using an NIH image was performed.
Fluorescein Labeling of Type I Collagen Collagen was extracted from newborn calf skin by pepsin digestion and purified by sequential salt precipitation (18). Collagen was labeled with FITC in a 0.5% sodium carbonate–sodium hydrogen carbonate buffer (pH 9.5) as previously described (16). FITC– collagen was purified by salt precipitation (2 M NaCl) under acidic conditions and ethanol precipitation (30% ethanol). The absorbance at 495 nm for the purified 0.2% FITC– collagen in 1% of sodium carbonate solution was 0.9390. Calculation from the molecular extinction coefficient of FITC (77,000 at 495 nm) indicated 1.83 molecules of FITC bound to each molecule of type I collagen (300,000 Da). Real-Time Zymography Gelatin zymography was performed on a 12% polyacrylamide gel in an 8 ⫻ 7 cm slab gel cast (Bio-Rad Lab., Hercules, CA) under nonreducing conditions. The thickness of the gel was 0.75 mm. Heat-denatured (in boiling water for 2 min) FITC-labeled collagen was copolymerized (final concentration, 0.5 mg/ml gel) in the polyacrylamide gel. In some cases, FITC– casein or FITC–BSA was used as the substrate. A standard stacking gel (5%) was used. Samples were loaded without heat denaturation. The samples were run at a constant voltage (110 V) for approximately 1.5 h with cooling in a water bath (at approximately 10°C). After electrophoresis, the gel was washed four times (15 min each) in 20 ml of 2.5% Triton X-100 containing buffer (50 mM Tris–HCl, 200 mM NaCl, pH 7.6) and incubated in 20 ml of digestion buffer (50 mM Tris–HCl, 200 mM NaCl, 5 mM CaCl 2, 0.02% NaN 3, pH 7.6) at 37°C with shaking (19). The use of a bacterial incubation chamber was convenient for this purpose. The gel container was placed in a light-shielded box to avoid decreasing the fluorescence intensity during the incubation. After an appropriate period of incubation, the gel was placed directly on the transilluminator (Spectroline Co., Model No. TC312A, 312 nm ultraviolet). The gel was observed through a sharp cut filter SC52
Real-Time Reverse Zymography SDS–PAGE (15% polyacrylamide gel) was performed in the same manner as the real-time zymography described above except that the gel contained hrMMP-2, hrMMP-9 (11), or the conditioned medium of HT1080 cells activated with 12-0-tetra-decanoylphorbol-13acetate (TPA) in the absence of fetal bovine serum. A total of 50 ng of hrMMP-2 or hrMMP9 or 2 ml of the conditioned medium was added to 5 ml of the gel. The polyacrylamide gel must be prepared the same day of the electrophoresis; otherwise MMPs in the gel will be irreversibly inactivated by SDS. If the polyacrylamide gel for reverse zymography (containing hrMMP-9) in the absence of SDS was kept in the dark at 4°C, the gel was usable for at least 2 weeks and the electrophoresis pattern was not affected by the absence of SDS in the gel. Samples were loaded without heat denaturation. The conditions for the electrophoresis were same as for the real-time zymography. After electrophoresis, the gel was washed four times (15 min each) in 20 ml of 2.5% Triton X-100 containing buffer and incubated in 20 ml of digestion buffer at 37°C with shaking (70 – 80 rpm). The gel container was shielded from light. After an appropriate period of incubation, the gel was placed directly on the UV transilluminator. The gel was photographed through the SC52 filter. The typical exposure time was 1 or 2 s with an f-stop of 4.5–5.6. After being photographed, the gel was returned to the chamber, and the incubation was continued and photographed at intervals of several hours. Occasionally, the gel was incubated for up to 1 week. After incubation, the gel was stained with Coomassie brilliant blue. Assay of MMPs The assay for MMP-2 and MMP-9 was carried out in the presence of 1 mM APMA according to Harris and Krane (20), and heat-denatured FITC-type I collagen was used as the substrate (14, 15) instead of the 3Hlabeled collagen (17). One unit of MMP degraded 1 g of denatured collagen per minute at 37°C.
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lagen. However, our results showed that the FITC– collagen could be used for zymographic analyses. Stability of MMPs in the SDS–Polyacrylamide Gel
FIG. 1. Digestion pattern of FITC– collagen cleaved by MMPs. FITC-labeled collagen (50 g) was denatured for 2 min at 100°C and then incubated with 200 pg of hrMMP-2 or MMP-9 for 20 h at 37°C. An aliquot of 5 g collagen was subjected to 15% SDS–PAGE. After electrophoresis, the gel was photographed on a transilluminator using an SC52 filter (FITC, lanes 2 and 4) to detect the FITC-labeled collagen fragments. The illuminated band is shown in white. The same gel was stained with Coomassie brilliant blue (CBB, lanes 1 and 3).
RESULTS
Digestion of Denatured FITC–Collagen by MMPs The digestion patterns and the susceptibility of the FITC– collagen to cleavage by the MMPs were examined before zymographic analysis. The heat-denatured FITC– collagen was digested by hrMMP-2 or hrMMP-9 that had been activated by APMA for 20 h at 37°C. The digestion of FITC– collagen was analyzed by 15% SDS– PAGE and visualized on the transilluminator. Figure 1 shows the cleavage of FITC– collagen by MMP-2 (lane 2) and MMP-9 (lane 4). The same gel was stained with Coomassie brilliant blue (lanes 1 and 3). The staining patterns of the collagen fragments in the Coomassiestained gel were very similar to those identified on the transilluminator. The results show that the FITC binding positions on the collagen molecule were random even though there were fewer than two molecules of FITC per collagen molecule. Also, these results show that the bands observed by transillumination correspond to the presence of collagen fragments. Next, we compared the ability of MMPs to cleave collagen and FITC– collagen. Following a 20-h activation with AMPA, hrMMP-2, hrMMP-9, and MMP-3 were incubated with heat-denatured FITC– collagen or unlabeled collagen. After a 20-h incubation, the collagen cleavage was analyzed by 12% (MMP-2 and MMP-9 digestion) or 15% (MMP-3 digestion) SDS– PAGE, and the gels were stained with Coomassie brilliant blue (CBB) (Fig. 2). The digestion patterns of FITC– collagen (Fig. 2, lane 3) and unlabeled collagen (Fig. 2, lane 2) were nearly identical. MMP-2 was somewhat more effective at cleaving FITC– collagen than unlabeled collagen, and MMP-9 was slightly less effective at cleaving FITC– collagen than unlabeled col-
MMPs were gradually inactivated in SDS-containing gels. This restricts the zymographic protocols in two ways. First, the washing time of gel in the Triton X-100 containing buffer should be minimized. When a 0.75-mm thickness was used, a 1-h washing (exchange the buffer every 15 min) was enough to obtain the maximum sensitivity. When a gel with a 1-mm thickness was used, a 3- to 4-h washing was desirable. Therefore, to minimize the loss of MMP activity, a thinner gel is recommended. Second, the loss of MMP activity in the SDS-containing gel affects the conditions for preparing gels for reverse zymography. In fact, we recommend preparing gels without SDS if one wants to make a stock of running gels for reverse zymography. We next examined the effect of several other factors on MMP stability, such as pH and the presence of calcium. The presence or absence of calcium ion in the gel had no effect on the stability of the MMPs, but EDTA irreversibly inactivated the MMPs even after the addition of calcium (data not shown). To examine the effects of pH on MMP stability in the gel, FITC– collagen and MMP-9 were copolymerized with acrylamide in Tris–HCl buffer at pH 8.8 or 7.4. After incubation at 4°C for several days, the gel was transferred to digestion buffer at 37°C and the digestion activity of MMP-9 was examined by transillumination. Regardless of the pH for the gel stock, the activity of MMP-9 did not decrease even after 2 weeks (data not shown).
FIG. 2. Comparison of digestion profiles of unlabeled and FITClabeled collagen cleaved with MMPs. A total of 50 g of unlabeled (lane 2) and FITC-labeled collagen (lane 3) was incubated for 20 h with 200 pg of hrMMP-2 or -9 or with 5 mU of purified MMP-3. The MMP-2 and MMP-9 digested samples were subjected to 12% SDS– PAGE and the MMP-3 digested sample was subjected to 15% SDS– PAGE. Lane 1 shows SDS–PAGE of unlabeled collagen used as the starting material, lane 2 shows digested unlabeled collagen, and lane 3 shows digested FITC-labeled collagen. The positions of the ␣1, ␣2, and  chains of type I collagen are shown in the left margin.
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FIG. 3. Comparison of conventional zymography and real-time zymography. Purified MMP-2 (active form, 62 kDa) was analyzed using a 12% gel containing 0.05% heat-denatured FITC-labeled collagen. A, Real-time zymography after a 20-h incubation at 37°C. The faint band at 50 kDa was a contaminant of MMP-3 in the sample. B, Conventional zymography after 20 h of incubation. The gel contained 0.1% heat-denatured collagen. Lanes 1, 2: 500 U MMP-2; lanes 3, 4: 100 U; lanes 5, 6: 20 U; lanes 7, 8: 4 U; lanes 9, 10: 0.8 U. C and D, Sensitivity and linearity of the real-time zymography after 4- and 20-h incubations. Densities of the 62-kDa bands were analyzed by NIH Image. Each plot was the average of three independent measurements.
Real-Time Zymography Using FITC–Collagen Because our protocol does not require the use of gel staining agents, we were able to follow zymography in real time. For this real-time zymography, MMPs were detected as dark bands against a green fluorescent background (Fig. 3A). We compared conventional and real-time zymographies using a commercial source of MMP-2. For conventional zymography, 1 mg/ml of denatured nonlabeled collagen was used. After a 4-h incubation, 20 U of MMP-2 activity was detected by real-time zymography, whereas at least
100 U was needed to detect MMP-2 with the conventional method. After a 20-h incubation, the lower limit of detection was 0.8 U for our fluorescent method and 20 U for the conventional method (Figs. 3A and 3B). According to the estimation of the specific activity of MMP-2 by Okada et al. (4), 0.8 U of activity was equivalent to 64 pg of commercial MMP-2. These results show that our method is 5–25 times more sensitive than the conventional method. Further analysis showed that the detection limits for rhMMP-2 and -9 were even lower than for commercial MMP-2. After a
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Real-Time Reverse Zymography
FIG. 4. Detection of MMP-3 using FITC– casein as a substrate. MMP-3 (50 kDa) was detected using a 12% polyacrylamide gel containing 0.05% FITC-labeled casein. FITC, real-time zymography after 4 h incubation. CBB, conventional casein zymography after 4 h of incubation. Lane 1: 500 U MMP-3; lane 2: 100 U; lane 3: 20 U; lane 4: 4 U; lane 5: 0.8 U; lane 6: 0.16 U.
4-h incubation the detection limit was 80 pg for both rhMMP-2 and rhMMP-9, and after a 24-h incubation, it was 16 pg for rhMMP-2 and less than 3 pg for rhMMP-9. Usually, 1–5 mg/ml of gelatin is used in conventional zymography (5, 21), while 0.5 mg/ml of gelatin is used in our real-time method. The reason for this difference in substrate concentration is that fluorescence detection is much more sensitive than protein staining with Coomassie brilliant blue or amido black. This difference between gel staining and fluorescent detection may also contribute to the ability to detect lower amounts of enzyme activity by real-time zymography. Furthermore, densitometric quantitation showed linearity between 20 and 500 U at 4 h and between 0.8 and 20 U at 24 h (Figs. 3C and 3D) for real-time zymography. To obtain linearity in the same range by conventional zymography, more than two gel slabs must be prepared for each time point.
Next, we examined the use of FITC– collagen for real-time reverse zymography using hrTIMP-1 and hrTIMP-2. When denatured FITC– collagen was used as the substrate and hrMMP-2 or hrMMP-9 as the digesting enzyme, we could monitor substrate digestion without stopping the reaction. During the first few hours, the gel was homogeneously illuminated. As the background of the luminescence decreased later in the incubation, the TIMP bands could be seen as an illuminated spot (Figs. 5 and 6, FITC). Figure 5 shows the typical real-time reverse zymography pattern of TIMP-1 and TIMP-2 after 20 h when using MMP-2 as the digestion enzyme. Figure 6 compares the detection limits for TIMPs by Coomassie brilliant blue staining and FITC detection after a 20-h incubation in the presence of MMP-9. We found that the detection limits were 800 and 6 pg for TIMP-1 and TIMP-2, respectively. The detection limit of the fluorescent method and CBB staining was comparable, although fluorescent detection was better for TIMP-2 than it was for TIMP-1. The results also show that TIMP-1 was more readily detected when MMP-2 was used as the digestion enzyme than MMP-9, while the detection of TIMP-2 by MMP-2 and MMP-9 was equivalent. However, when MMP-2 was used, the incubation time to get the best results varied between experiments. This may be due to irreversible inactivation of MMP-2 in the SDS containing buffer during electrophoresis. MMP-2 was more labile by SDS. The detection limit of MMP activity was approximately 5 times lower with conditioned medium of HT1080 cells than when recombinant MMP was used (data not shown). As described by Oliver et al. (11), this reduced sensitivity may be due to the TIMP-1 found in the
Real-Time Zymography Using FITC–Casein or FITC–BSA We examined the detection of MMP-3 using FITC– casein in place of FITC– collagen. After a 4-h incubation, an amount as low as 0.8 U of MMP-3 could be detected by real-time zymography (Fig. 4, FITC). The sensitivity using FITC– casein was 5 times better than the conventional method (Fig. 4, CBB). We were unable to determine the identity of the upper (75 kDa) and lower (smaller than 30 kDa) bands found in both the real-time and the conventional methods. These results indicate that other FITC-labeled substrates could be used in our detection system. However, we were unable to detect MMP-2, -9 or -3 with FITC–BSA.
FIG. 5. Detection of TIMP-1 and TIMP-2 by real-time reverse zymography. hrTIMP-1 and hrTIMP-2 were subjected to 15% polyacrylamide gel containing denatured FITC– collagen (0.05%) and hrMMP-2 (50 ng/5 ml gel). After a 20-h incubation and electrophoresis, the gel was photographed. Lane 1; molecular weight marker; lane 2: 20 ng of TIMP-1; lane 3: 4 ng of TIMP-1; lane 4: 20 ng of TIMP-2; lane 5: 4 ng of TIMP-2. TIMP-1 was approximately 30 kDa and TIMP-2 was 20 kDa.
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DISCUSSION
FIG. 6. Comparison of conventional reverse zymography and realtime reverse zymography. A polyacrylamide 15% gel containing 0.05% FITC-labeled denatured collagen and hrMMP-9 (50 ng/5 ml gel) was used for analyses. FITC indicates the result of real-time zymography after a 20-h incubation. After being imaged with the transilluminator, the gel was stained with Coomassie brilliant blue (CBB). The amounts of TIMPs subjected to electrophoresis are shown.
conditioned medium of HT1080 cells (Fig. 7, lanes 2 and 4). We compared the conventional reverse zymography and real-time reverse zymography using conditioned media from HT1080 and fibroblast cells cultured in the presence or absence of fetal bovine serum. In addition to TIMP-1, there were several serum-associated protein bands in the 30- to 90-kDa range present on the Coomassie-stained gel (Fig. 7, CBB, lanes 1 and 2). In contrast, using real-time reverse zymography, only the fluorescein-labeled substrate was detected on the transilluminator (Fig. 7, FITC, lanes 1 and 2). Therefore, we were able to distinguish between real TIMP bands and false ones by comparing the fluorescent and conventional reverse zymographies. Additionally, realtime reverse zymography can be adapted to the detection of other protease inhibitors. For example, using this system coupled with trypsin, we were able to detect less than 3 ng of soybean trypsin inhibitor (data not shown).
There are many methods for determining enzymatic activity after gel electrophoresis, including dye formation, chemiluminescence, fluorescence, and radioisotopic methods (22). Among these methods, fluorescence is one of the most inexpensive and sensitive. For example, insoluble fluorescein-labeled collagen II (23) and FITC-labeled type I collagen (14 –16) have been used to assay gelatinase and collagenase activity. Zymography is a technique widely used for the detection of MMPs. Although this typically involves staining of gels with Coomassie brilliant blue, there were previous reports that this method may be adapted to fluorescent systems. For example, 2-methoxy-2,4diphenyl-3(2H)-furanone-labeled collagen was used to detect collagenase or gelatinase in a solution (24) or in zymography (25, 26). In this report, we showed that FITC, a more popular and readily available fluorescent dye, is a good labeling reagent for zymography. By using FITC– collagen, we could repeatedly analyze the enzymatic reaction in the same gel, making it possible to perform real-time analyses of enzyme activity. Because we can easily select the best incubation time, the sensitivity of MMP detection was improved. In fact, the detection limits of MMP-2 and MMP-9 were 16 and 3 pg, respectively. This sensitivity was almost equal to that of the careful measurements by Kleiner and Stetler-Stevenson (19). In addition, real-time zymography makes it easier to perform quantitative analyses of enzyme activity, and by combining the data taken from different incubation times, a wider range of linearity can be achieved using a single gel.
FIG. 7. Real-time reverse zymography of serum containing samples. A polyacrylamide 15% gel containing denatured 0.05% FITClabeled collagen and hrMMP-9 was used for analyses. A, Real-time zymography of conditioned media from HT1080 and human dermal fibroblast cells. The gel was incubated for 20 h. B, The same gel in A stained with Coomassie brilliant blue. Each lane contains 10 l of conditioned medium. Lane 1: Fibroblast-conditioned medium incubated in the presence of bovine serum; lane 2: HT1080-conditioned medium incubated in the presence of bovine serum; lane 3: fibroblast-conditioned medium incubated in the absence of bovine serum; lane 4: HT1080-conditioned medium incubated in the absence of bovine serum. The band at 30 kDa is TIMP-1.
REAL-TIME ZYMOGRAPHY AND REVERSE ZYMOGRAPHY
Reverse zymography allows concurrent semi-quantitative analysis of inhibitor levels and molecular weight (11). The sensitivity and resolution of this method have been improved by incorporating a proteinase source into the gel (7) and by using recombinant MMPs (11). However, this technique is still problematic. For example, it is very hard to predict the proper incubation time for a sample containing an unknown concentration of TIMPs because the gel staining procedure terminates the reaction. As a result, multiple gel samples are typically required to obtain the best reaction time. In contrast, with FITC– collagen as a substrate, the proper incubation time is determined simply by examining a single gel at multiple time points. This new real-time reverse zymography is not only easier, but also more sensitive than standard zymography. In fact, picogram levels of TIMP-1 and TIMP-2 were detected using hrMMP-2 and hrMMP-9 as the digestion enzymes. Furthermore, the detection limits of TIMP-1 and TIMP-2 were in the same range as that reported by Oliver et al. (11) and by an ELISA system (27), and only real TIMP bands are visualized by realtime reverse zymographs. This is much simpler and more accurate than the method of trying to distinguish real TIMPs from artifacts by the color of Coomassiestained collagen-derived peptides. The staining color of the collagen-derived peptide (reddish and to purple) with Coomassie brilliant blue was different from most other proteins which stained in blue (29, 30). In this report, we showed that FITC-labeled proteins were good substrates for both zymography and reverse zymography. By using a FITC-labeled substrate, we developed a procedure that was simpler and more sensitive than standard zymography. The real-time result could be recorded as a photograph or as a digital file if the data were collected using a CCD camera. These simplifications achieved by real-time zymography should help allow its use for diagnostic analyses.
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ACKNOWLEDGMENTS We are grateful to Mr. Masashi Kusubata for his very helpful discussions and also to Dr. Osamu Hayashida for his technical advice on the photography.
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26. Ma¨kela¨, M., Salo, T., Uitto, V-J., and Larjava, H. (1994) Matrix metalloproteinases (MMP-2 and MMP-9) of the oral cavity: Cellular origin and relationship to periodontal status. J. Dent. Res. 73, 1397–1406. 27. Fujimoto, N., Zhang, J., Iwata, K., Shinya, T., Okada, Y., and Hayakawa, T. (1993) A one-step sandwich enzyme immunoassay for tissue inhibitor of metalloproteinase-2 using monoclonal antibodies. Clin. Chim. Acta 220, 31– 45. 28. Rodgers, K. J., Melrose, J., and Ghosh, P. (1995) Biotin-labeled potato chymotrypsin inhibitor-1: A useful probe for the detection and quantitation of chymotrypsin-like serine proteinases on Western blots and its application in the detection of a serine proteinase synthesised by articular chondrocytes. Anal. Biochem. 227, 129–134. 29. Micko, S., and Schlaepfer, W. W. (1978) Metachromasy of peripheral nerve collagen on polyacrylamide gels stained with Coomassie brilliant blue R-250. Anal. Biochem. 88, 566 –572. 30. Hattori, S., Sakai, K., Watanabe, K., and Fujii, T. (1996) The induction of metachromasia and circular dichroism of Coomassie brilliant blue R-250 with collagen and histone H1 is due to the low content of hydrophobic amino acid residues in these proteins. J. Biochem. 119, 400 – 406.
Real-Time Zymography and Reverse Zymography: A Method for Detecting Activities of Matrix Metalloproteinases and Their Inhibitors Using FITCLabeled Collagen and Casein as Substrates Shunji Hattori,* ,† ,1 Hitomi Fujisaki,* Tomomi Kiriyama,* Tsukao Yokoyama,‡ and Shinkichi Irie* ,† *Nippi Research Institute of Biomatrix and †Japan Institute of Leather Research, Adachi-ku, Tokyo 120-8601, Japan; and ‡Collagen Research Center, Kiyose, Tokyo 204-0013, Japan
Received July 30, 2001; published online December 19, 2001
Zymography and reverse zymography are widely used techniques for identifying the proteolytic activity of enzymes and the presence of protease inhibitors in polyacrylamide gels. In the current studies, we utilized a fluorescein-isothiocyanate-labeled substrate to develop novel zymographic and reverse zymographic methods for detecting matrix metalloproteinases and tissue inhibitors of the metalloproteinases, respectively. Using a transilluminator, the results can be observed visually without stopping the enzymatic reaction. For this reason, we have named these methods real-time zymography and real-time reverse zymography. These methods have the following advantages compared with conventional protocols: (1) because the reaction can be repeatedly monitored on the polyacrylamide gels, optimization of the incubation time can be achieved without preliminary analyses; (2) higher sensitivity is achieved with a lower amount of substrate than with conventional methods; (3) a semiquantitative analysis of matrix metalloproteinases is possible. An additional advantage of the real-time reverse zymography is that, because the fluorescence detection is specific for substrate digestion, the inhibitor bands can be easily distinguished from contaminating proteins. © 2002 Elsevier Science Key Words: zymography; matrix metalloproteinase; tissue inhibitor of metalloproteinase; FITC; real-time; collagen.
MMPs 2 are thought to be involved in the metastasis and growth of tumors (1–3), the progression of inflammatory diseases such as rheumatoid arthritis (4), and the differentiation of skin keratinocytes (5, 6). The inhibitors of MMPs are also of interest as blockers of tumor growth and metastasis (7). Several synthetic inhibitors are now in clinical trials (2, 7), and TIMPs are thought to suppress the metastasis of carcinoma cells (8). Zymography, which uses an SDS-substrate gel, is a convenient method for detecting proteolytic enzymes like MMPs. The first forms of zymography utilized a gelatin substrate slab gel to detect plasminogen activators (9). Since then, zymography with gelatin or casein has been used to detect MMPs (10). TIMPs can be detected by reverse zymography (10, 11) using an acrylamide gel containing copolymerized MMP and gelatin. Fluorescein-labeled synthetic peptides have been utilized to detect the enzymatic activity of gelatinase or MMPs (12, 13). FITC-labeled denatured collagen has also been used to assay gelatinolitic activity in the solution method (14, 15). Furthermore, an assay with sensitivity almost as high as that using radioisotopelabeled polymeric gelatin has been developed using a fluorescent-labeled substrate (16, 17). In this report, we describe novel zymographic and reverse zymographic methods using FITC-labeled denatured collagen as the substrate. These methods are 2
1
To whom correspondence should be addressed at Nippi Research Institute of Biomatrix, 1-1-1 Senjumidori-cho, Adachi-ku, Tokyo 1208601, Japan. Fax: ⫹81-33870-9631. E-mail: [email protected]. 0003-2697/02 $35.00 © 2002 Elsevier Science All rights reserved.
Abbreviations used: APMA, 4-aminophenylmercuric acetate; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; hr, human recombinant; MMP, matrix metalloproteinase; SDS, sodium dodecyl sulfate; TIMP, tissue inhibitor of metalloproteinase; CBB, coomassie brilliant blue. 27
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more sensitive than conventional ones, and they allow for real-time analyses of enzymatic activity. MATERIAL AND METHODS
Materials hrMMP-2 and hrMMP-9 were from Genzyme Techne Co. hrTIMP-1 and hrTIMP-2 were from Fuji Chemical Co. (Toyama, Japan), and purified MMP-2 (from human fibrosarcoma cell) and MMP-3 (from human tumor cell) were from the Yagai Research Center (Yamagata, Japan). FITC-labeled BSA and FITC-labeled casein were from Sigma (St. Louis, MO).
(Fuji Film Co., Tokyo) which had a light cutoff under 520-nm wavelength, after which it was photographed by a Polaroid camera using FB-3000B black and white film (ASA3000) (Fuji Film Co., Tokyo). The SC52 filter showed the best result for our purpose and the No. 12 filter (Eastman Kodak, NY) also could be used substitutively. The exposure time varied with the intensity of illumination, but an f-stop of 5.6 for 1 s was typical for our system. After being photographed, the gel was returned to the chamber, and the incubation was continued. The gel was photographed at intervals of several hours. The pictures were scanned using Adobe Photoshop software (Adobe Systems, Inc., CA), and densitometric quantification using an NIH image was performed.
Fluorescein Labeling of Type I Collagen Collagen was extracted from newborn calf skin by pepsin digestion and purified by sequential salt precipitation (18). Collagen was labeled with FITC in a 0.5% sodium carbonate–sodium hydrogen carbonate buffer (pH 9.5) as previously described (16). FITC– collagen was purified by salt precipitation (2 M NaCl) under acidic conditions and ethanol precipitation (30% ethanol). The absorbance at 495 nm for the purified 0.2% FITC– collagen in 1% of sodium carbonate solution was 0.9390. Calculation from the molecular extinction coefficient of FITC (77,000 at 495 nm) indicated 1.83 molecules of FITC bound to each molecule of type I collagen (300,000 Da). Real-Time Zymography Gelatin zymography was performed on a 12% polyacrylamide gel in an 8 ⫻ 7 cm slab gel cast (Bio-Rad Lab., Hercules, CA) under nonreducing conditions. The thickness of the gel was 0.75 mm. Heat-denatured (in boiling water for 2 min) FITC-labeled collagen was copolymerized (final concentration, 0.5 mg/ml gel) in the polyacrylamide gel. In some cases, FITC– casein or FITC–BSA was used as the substrate. A standard stacking gel (5%) was used. Samples were loaded without heat denaturation. The samples were run at a constant voltage (110 V) for approximately 1.5 h with cooling in a water bath (at approximately 10°C). After electrophoresis, the gel was washed four times (15 min each) in 20 ml of 2.5% Triton X-100 containing buffer (50 mM Tris–HCl, 200 mM NaCl, pH 7.6) and incubated in 20 ml of digestion buffer (50 mM Tris–HCl, 200 mM NaCl, 5 mM CaCl 2, 0.02% NaN 3, pH 7.6) at 37°C with shaking (19). The use of a bacterial incubation chamber was convenient for this purpose. The gel container was placed in a light-shielded box to avoid decreasing the fluorescence intensity during the incubation. After an appropriate period of incubation, the gel was placed directly on the transilluminator (Spectroline Co., Model No. TC312A, 312 nm ultraviolet). The gel was observed through a sharp cut filter SC52
Real-Time Reverse Zymography SDS–PAGE (15% polyacrylamide gel) was performed in the same manner as the real-time zymography described above except that the gel contained hrMMP-2, hrMMP-9 (11), or the conditioned medium of HT1080 cells activated with 12-0-tetra-decanoylphorbol-13acetate (TPA) in the absence of fetal bovine serum. A total of 50 ng of hrMMP-2 or hrMMP9 or 2 ml of the conditioned medium was added to 5 ml of the gel. The polyacrylamide gel must be prepared the same day of the electrophoresis; otherwise MMPs in the gel will be irreversibly inactivated by SDS. If the polyacrylamide gel for reverse zymography (containing hrMMP-9) in the absence of SDS was kept in the dark at 4°C, the gel was usable for at least 2 weeks and the electrophoresis pattern was not affected by the absence of SDS in the gel. Samples were loaded without heat denaturation. The conditions for the electrophoresis were same as for the real-time zymography. After electrophoresis, the gel was washed four times (15 min each) in 20 ml of 2.5% Triton X-100 containing buffer and incubated in 20 ml of digestion buffer at 37°C with shaking (70 – 80 rpm). The gel container was shielded from light. After an appropriate period of incubation, the gel was placed directly on the UV transilluminator. The gel was photographed through the SC52 filter. The typical exposure time was 1 or 2 s with an f-stop of 4.5–5.6. After being photographed, the gel was returned to the chamber, and the incubation was continued and photographed at intervals of several hours. Occasionally, the gel was incubated for up to 1 week. After incubation, the gel was stained with Coomassie brilliant blue. Assay of MMPs The assay for MMP-2 and MMP-9 was carried out in the presence of 1 mM APMA according to Harris and Krane (20), and heat-denatured FITC-type I collagen was used as the substrate (14, 15) instead of the 3Hlabeled collagen (17). One unit of MMP degraded 1 g of denatured collagen per minute at 37°C.
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lagen. However, our results showed that the FITC– collagen could be used for zymographic analyses. Stability of MMPs in the SDS–Polyacrylamide Gel
FIG. 1. Digestion pattern of FITC– collagen cleaved by MMPs. FITC-labeled collagen (50 g) was denatured for 2 min at 100°C and then incubated with 200 pg of hrMMP-2 or MMP-9 for 20 h at 37°C. An aliquot of 5 g collagen was subjected to 15% SDS–PAGE. After electrophoresis, the gel was photographed on a transilluminator using an SC52 filter (FITC, lanes 2 and 4) to detect the FITC-labeled collagen fragments. The illuminated band is shown in white. The same gel was stained with Coomassie brilliant blue (CBB, lanes 1 and 3).
RESULTS
Digestion of Denatured FITC–Collagen by MMPs The digestion patterns and the susceptibility of the FITC– collagen to cleavage by the MMPs were examined before zymographic analysis. The heat-denatured FITC– collagen was digested by hrMMP-2 or hrMMP-9 that had been activated by APMA for 20 h at 37°C. The digestion of FITC– collagen was analyzed by 15% SDS– PAGE and visualized on the transilluminator. Figure 1 shows the cleavage of FITC– collagen by MMP-2 (lane 2) and MMP-9 (lane 4). The same gel was stained with Coomassie brilliant blue (lanes 1 and 3). The staining patterns of the collagen fragments in the Coomassiestained gel were very similar to those identified on the transilluminator. The results show that the FITC binding positions on the collagen molecule were random even though there were fewer than two molecules of FITC per collagen molecule. Also, these results show that the bands observed by transillumination correspond to the presence of collagen fragments. Next, we compared the ability of MMPs to cleave collagen and FITC– collagen. Following a 20-h activation with AMPA, hrMMP-2, hrMMP-9, and MMP-3 were incubated with heat-denatured FITC– collagen or unlabeled collagen. After a 20-h incubation, the collagen cleavage was analyzed by 12% (MMP-2 and MMP-9 digestion) or 15% (MMP-3 digestion) SDS– PAGE, and the gels were stained with Coomassie brilliant blue (CBB) (Fig. 2). The digestion patterns of FITC– collagen (Fig. 2, lane 3) and unlabeled collagen (Fig. 2, lane 2) were nearly identical. MMP-2 was somewhat more effective at cleaving FITC– collagen than unlabeled collagen, and MMP-9 was slightly less effective at cleaving FITC– collagen than unlabeled col-
MMPs were gradually inactivated in SDS-containing gels. This restricts the zymographic protocols in two ways. First, the washing time of gel in the Triton X-100 containing buffer should be minimized. When a 0.75-mm thickness was used, a 1-h washing (exchange the buffer every 15 min) was enough to obtain the maximum sensitivity. When a gel with a 1-mm thickness was used, a 3- to 4-h washing was desirable. Therefore, to minimize the loss of MMP activity, a thinner gel is recommended. Second, the loss of MMP activity in the SDS-containing gel affects the conditions for preparing gels for reverse zymography. In fact, we recommend preparing gels without SDS if one wants to make a stock of running gels for reverse zymography. We next examined the effect of several other factors on MMP stability, such as pH and the presence of calcium. The presence or absence of calcium ion in the gel had no effect on the stability of the MMPs, but EDTA irreversibly inactivated the MMPs even after the addition of calcium (data not shown). To examine the effects of pH on MMP stability in the gel, FITC– collagen and MMP-9 were copolymerized with acrylamide in Tris–HCl buffer at pH 8.8 or 7.4. After incubation at 4°C for several days, the gel was transferred to digestion buffer at 37°C and the digestion activity of MMP-9 was examined by transillumination. Regardless of the pH for the gel stock, the activity of MMP-9 did not decrease even after 2 weeks (data not shown).
FIG. 2. Comparison of digestion profiles of unlabeled and FITClabeled collagen cleaved with MMPs. A total of 50 g of unlabeled (lane 2) and FITC-labeled collagen (lane 3) was incubated for 20 h with 200 pg of hrMMP-2 or -9 or with 5 mU of purified MMP-3. The MMP-2 and MMP-9 digested samples were subjected to 12% SDS– PAGE and the MMP-3 digested sample was subjected to 15% SDS– PAGE. Lane 1 shows SDS–PAGE of unlabeled collagen used as the starting material, lane 2 shows digested unlabeled collagen, and lane 3 shows digested FITC-labeled collagen. The positions of the ␣1, ␣2, and  chains of type I collagen are shown in the left margin.
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FIG. 3. Comparison of conventional zymography and real-time zymography. Purified MMP-2 (active form, 62 kDa) was analyzed using a 12% gel containing 0.05% heat-denatured FITC-labeled collagen. A, Real-time zymography after a 20-h incubation at 37°C. The faint band at 50 kDa was a contaminant of MMP-3 in the sample. B, Conventional zymography after 20 h of incubation. The gel contained 0.1% heat-denatured collagen. Lanes 1, 2: 500 U MMP-2; lanes 3, 4: 100 U; lanes 5, 6: 20 U; lanes 7, 8: 4 U; lanes 9, 10: 0.8 U. C and D, Sensitivity and linearity of the real-time zymography after 4- and 20-h incubations. Densities of the 62-kDa bands were analyzed by NIH Image. Each plot was the average of three independent measurements.
Real-Time Zymography Using FITC–Collagen Because our protocol does not require the use of gel staining agents, we were able to follow zymography in real time. For this real-time zymography, MMPs were detected as dark bands against a green fluorescent background (Fig. 3A). We compared conventional and real-time zymographies using a commercial source of MMP-2. For conventional zymography, 1 mg/ml of denatured nonlabeled collagen was used. After a 4-h incubation, 20 U of MMP-2 activity was detected by real-time zymography, whereas at least
100 U was needed to detect MMP-2 with the conventional method. After a 20-h incubation, the lower limit of detection was 0.8 U for our fluorescent method and 20 U for the conventional method (Figs. 3A and 3B). According to the estimation of the specific activity of MMP-2 by Okada et al. (4), 0.8 U of activity was equivalent to 64 pg of commercial MMP-2. These results show that our method is 5–25 times more sensitive than the conventional method. Further analysis showed that the detection limits for rhMMP-2 and -9 were even lower than for commercial MMP-2. After a
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Real-Time Reverse Zymography
FIG. 4. Detection of MMP-3 using FITC– casein as a substrate. MMP-3 (50 kDa) was detected using a 12% polyacrylamide gel containing 0.05% FITC-labeled casein. FITC, real-time zymography after 4 h incubation. CBB, conventional casein zymography after 4 h of incubation. Lane 1: 500 U MMP-3; lane 2: 100 U; lane 3: 20 U; lane 4: 4 U; lane 5: 0.8 U; lane 6: 0.16 U.
4-h incubation the detection limit was 80 pg for both rhMMP-2 and rhMMP-9, and after a 24-h incubation, it was 16 pg for rhMMP-2 and less than 3 pg for rhMMP-9. Usually, 1–5 mg/ml of gelatin is used in conventional zymography (5, 21), while 0.5 mg/ml of gelatin is used in our real-time method. The reason for this difference in substrate concentration is that fluorescence detection is much more sensitive than protein staining with Coomassie brilliant blue or amido black. This difference between gel staining and fluorescent detection may also contribute to the ability to detect lower amounts of enzyme activity by real-time zymography. Furthermore, densitometric quantitation showed linearity between 20 and 500 U at 4 h and between 0.8 and 20 U at 24 h (Figs. 3C and 3D) for real-time zymography. To obtain linearity in the same range by conventional zymography, more than two gel slabs must be prepared for each time point.
Next, we examined the use of FITC– collagen for real-time reverse zymography using hrTIMP-1 and hrTIMP-2. When denatured FITC– collagen was used as the substrate and hrMMP-2 or hrMMP-9 as the digesting enzyme, we could monitor substrate digestion without stopping the reaction. During the first few hours, the gel was homogeneously illuminated. As the background of the luminescence decreased later in the incubation, the TIMP bands could be seen as an illuminated spot (Figs. 5 and 6, FITC). Figure 5 shows the typical real-time reverse zymography pattern of TIMP-1 and TIMP-2 after 20 h when using MMP-2 as the digestion enzyme. Figure 6 compares the detection limits for TIMPs by Coomassie brilliant blue staining and FITC detection after a 20-h incubation in the presence of MMP-9. We found that the detection limits were 800 and 6 pg for TIMP-1 and TIMP-2, respectively. The detection limit of the fluorescent method and CBB staining was comparable, although fluorescent detection was better for TIMP-2 than it was for TIMP-1. The results also show that TIMP-1 was more readily detected when MMP-2 was used as the digestion enzyme than MMP-9, while the detection of TIMP-2 by MMP-2 and MMP-9 was equivalent. However, when MMP-2 was used, the incubation time to get the best results varied between experiments. This may be due to irreversible inactivation of MMP-2 in the SDS containing buffer during electrophoresis. MMP-2 was more labile by SDS. The detection limit of MMP activity was approximately 5 times lower with conditioned medium of HT1080 cells than when recombinant MMP was used (data not shown). As described by Oliver et al. (11), this reduced sensitivity may be due to the TIMP-1 found in the
Real-Time Zymography Using FITC–Casein or FITC–BSA We examined the detection of MMP-3 using FITC– casein in place of FITC– collagen. After a 4-h incubation, an amount as low as 0.8 U of MMP-3 could be detected by real-time zymography (Fig. 4, FITC). The sensitivity using FITC– casein was 5 times better than the conventional method (Fig. 4, CBB). We were unable to determine the identity of the upper (75 kDa) and lower (smaller than 30 kDa) bands found in both the real-time and the conventional methods. These results indicate that other FITC-labeled substrates could be used in our detection system. However, we were unable to detect MMP-2, -9 or -3 with FITC–BSA.
FIG. 5. Detection of TIMP-1 and TIMP-2 by real-time reverse zymography. hrTIMP-1 and hrTIMP-2 were subjected to 15% polyacrylamide gel containing denatured FITC– collagen (0.05%) and hrMMP-2 (50 ng/5 ml gel). After a 20-h incubation and electrophoresis, the gel was photographed. Lane 1; molecular weight marker; lane 2: 20 ng of TIMP-1; lane 3: 4 ng of TIMP-1; lane 4: 20 ng of TIMP-2; lane 5: 4 ng of TIMP-2. TIMP-1 was approximately 30 kDa and TIMP-2 was 20 kDa.
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DISCUSSION
FIG. 6. Comparison of conventional reverse zymography and realtime reverse zymography. A polyacrylamide 15% gel containing 0.05% FITC-labeled denatured collagen and hrMMP-9 (50 ng/5 ml gel) was used for analyses. FITC indicates the result of real-time zymography after a 20-h incubation. After being imaged with the transilluminator, the gel was stained with Coomassie brilliant blue (CBB). The amounts of TIMPs subjected to electrophoresis are shown.
conditioned medium of HT1080 cells (Fig. 7, lanes 2 and 4). We compared the conventional reverse zymography and real-time reverse zymography using conditioned media from HT1080 and fibroblast cells cultured in the presence or absence of fetal bovine serum. In addition to TIMP-1, there were several serum-associated protein bands in the 30- to 90-kDa range present on the Coomassie-stained gel (Fig. 7, CBB, lanes 1 and 2). In contrast, using real-time reverse zymography, only the fluorescein-labeled substrate was detected on the transilluminator (Fig. 7, FITC, lanes 1 and 2). Therefore, we were able to distinguish between real TIMP bands and false ones by comparing the fluorescent and conventional reverse zymographies. Additionally, realtime reverse zymography can be adapted to the detection of other protease inhibitors. For example, using this system coupled with trypsin, we were able to detect less than 3 ng of soybean trypsin inhibitor (data not shown).
There are many methods for determining enzymatic activity after gel electrophoresis, including dye formation, chemiluminescence, fluorescence, and radioisotopic methods (22). Among these methods, fluorescence is one of the most inexpensive and sensitive. For example, insoluble fluorescein-labeled collagen II (23) and FITC-labeled type I collagen (14 –16) have been used to assay gelatinase and collagenase activity. Zymography is a technique widely used for the detection of MMPs. Although this typically involves staining of gels with Coomassie brilliant blue, there were previous reports that this method may be adapted to fluorescent systems. For example, 2-methoxy-2,4diphenyl-3(2H)-furanone-labeled collagen was used to detect collagenase or gelatinase in a solution (24) or in zymography (25, 26). In this report, we showed that FITC, a more popular and readily available fluorescent dye, is a good labeling reagent for zymography. By using FITC– collagen, we could repeatedly analyze the enzymatic reaction in the same gel, making it possible to perform real-time analyses of enzyme activity. Because we can easily select the best incubation time, the sensitivity of MMP detection was improved. In fact, the detection limits of MMP-2 and MMP-9 were 16 and 3 pg, respectively. This sensitivity was almost equal to that of the careful measurements by Kleiner and Stetler-Stevenson (19). In addition, real-time zymography makes it easier to perform quantitative analyses of enzyme activity, and by combining the data taken from different incubation times, a wider range of linearity can be achieved using a single gel.
FIG. 7. Real-time reverse zymography of serum containing samples. A polyacrylamide 15% gel containing denatured 0.05% FITClabeled collagen and hrMMP-9 was used for analyses. A, Real-time zymography of conditioned media from HT1080 and human dermal fibroblast cells. The gel was incubated for 20 h. B, The same gel in A stained with Coomassie brilliant blue. Each lane contains 10 l of conditioned medium. Lane 1: Fibroblast-conditioned medium incubated in the presence of bovine serum; lane 2: HT1080-conditioned medium incubated in the presence of bovine serum; lane 3: fibroblast-conditioned medium incubated in the absence of bovine serum; lane 4: HT1080-conditioned medium incubated in the absence of bovine serum. The band at 30 kDa is TIMP-1.
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Reverse zymography allows concurrent semi-quantitative analysis of inhibitor levels and molecular weight (11). The sensitivity and resolution of this method have been improved by incorporating a proteinase source into the gel (7) and by using recombinant MMPs (11). However, this technique is still problematic. For example, it is very hard to predict the proper incubation time for a sample containing an unknown concentration of TIMPs because the gel staining procedure terminates the reaction. As a result, multiple gel samples are typically required to obtain the best reaction time. In contrast, with FITC– collagen as a substrate, the proper incubation time is determined simply by examining a single gel at multiple time points. This new real-time reverse zymography is not only easier, but also more sensitive than standard zymography. In fact, picogram levels of TIMP-1 and TIMP-2 were detected using hrMMP-2 and hrMMP-9 as the digestion enzymes. Furthermore, the detection limits of TIMP-1 and TIMP-2 were in the same range as that reported by Oliver et al. (11) and by an ELISA system (27), and only real TIMP bands are visualized by realtime reverse zymographs. This is much simpler and more accurate than the method of trying to distinguish real TIMPs from artifacts by the color of Coomassiestained collagen-derived peptides. The staining color of the collagen-derived peptide (reddish and to purple) with Coomassie brilliant blue was different from most other proteins which stained in blue (29, 30). In this report, we showed that FITC-labeled proteins were good substrates for both zymography and reverse zymography. By using a FITC-labeled substrate, we developed a procedure that was simpler and more sensitive than standard zymography. The real-time result could be recorded as a photograph or as a digital file if the data were collected using a CCD camera. These simplifications achieved by real-time zymography should help allow its use for diagnostic analyses.
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ACKNOWLEDGMENTS We are grateful to Mr. Masashi Kusubata for his very helpful discussions and also to Dr. Osamu Hayashida for his technical advice on the photography.
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