DSpace at VNU: Detection of protease inhibitors by a reverse zymography method, performed in a tris(hydroxymethyl)aminomethane - Tricine buffer system

ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 324 (2004) 237–240 www.elsevier.com/locate/yabio Detection of protease inhibitors by a reverse zymography method, performed in a tris(hydroxymethyl)aminomethane–Tricine buffer system Q.T Lea,b and N Katunumaa,* a Institute for Health Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima City, Tokushima 770-8514, Japan b Biotechnology Center, Vietnam National University, Hanoi 144 Xuan thuy-Cau giay, Hanoi, Vietnam Received 22 July 2003 Abstract A new detecting method for protease inhibitors, especially for low-molecular-weight inhibitors, is reported Inhibitor samples were separated on a protein substrate–SDS–polyacrylamide gel in a Tris–Tricine buffer system that improves the separation and identification of peptides and low-molecular-weight proteins After electrophoresis, the gel was incubated with the target proteases to hydrolyze the background protein substrate The inhibitor bands, which were protected from proteolysis by the target proteases, were stained Standard low-molecular-weight inhibitors, such as pepstatin A for pepsin or matrix metalloproteases inhibitor I for collagenase, as well as larger inhibitors, such as soybean trypsin inhibitor or aprotinin for tryspin and cystatin C for papain, were demonstrated by this method and showed clear blue inhibitor bands in the white background when the gels were treated with the target proteases Some significant applications of this method are introduced This method is an ideal system for discovering new protease inhibitors in small natural samples Ó 2003 Elsevier Inc All rights reserved Keywords: Reverse zymography; Protease inhibitors Endogenous protease inhibitors are potential key regulators of proteases in living organisms Reverse zymography techniques are effective tools for isolating and characterizing natural protease inhibitors, particularly in medical science [1–4] These methods are based on the separation of the inhibitor samples on an SDS–polyacrylamide gel containing protein substrate such as gelatin or casein copolymerized into the gel After electrophoresis, washing and incubating the gel with the target protease solution at 37 °C for an optimal incubation period allow the substrate to be digested by the target proteases The undigested protein substrate remains where the inhibitor molecules are located and can be stained as blue bands These methods, although useful in searching for numerous protease inhibitors, failed to identify low-molecular-weight inhibitors We developed a new reverse zymography method, performed in a Tris– Tricine buffer system [5], which allows better resolution of peptides and low-molecular-weight proteins than the Tris–glycine system [5,6] The new method offers several significant advantages (i) it is quite simple and the inhibitor bands can be visualized clearly in the gel, (ii) a wide range of different-molecular-weight inhibitors within protein pools could be selectively detected and (iii) it allows the simultaneous determination of protease inhibitory activity and molecular weight and could be useful for discovering new protease inhibitors in small amounts of crude material We have successfully discovered various inhibitors in physiological materials using this reverse zymography technique Materials and methods * Corresponding author Fax: +81-88-622-2503 E-mail address: katunuma@tokushima.bunri-u.ac.jp (N Katunuma) 0003-2697/$ - see front matter Ó 2003 Elsevier Inc All rights reserved doi:10.1016/j.ab.2003.09.033 Reagents required for gel preparation, gelatin, standard inhibitors such as cystatin C, pepstatin A, soybean 238 Q.T Le, N Katunuma / Analytical Biochemistry 324 (2004) 237–240 trypsin inhibitor (STI),1 aprotinin, matrix metalloproteases inhibitor I (MMPI), and other materials, all pure grade, were purchased from Sigma Chemical Co.(St, Louis, MO, USA) Molecular weight markers were obtained from Bio-Rad Chemical Co (Richmond, CA, USA) All other reagents used were of analytical grade Solutions for making gels were prepared according to Schagger and Von Jagow [5] with some modification The separating gel, consisting of 0.1% (w/v) gelatin, 15% (w/v) acrylamide, 0.4% (w/v) bis-acrylamide, 10% (v/v) glycerol, 0.75 M Tris–HCl (pH 8.45), and 0.1% SDS, was cast in a Hoefer gel cassette (Pharmacia Biosciences Co Upsala, Sweden) with gel dimensions of  10  0.1 cm In some experiments, to compare with the gelatin gel, 0.1% collagen or hemoglobin was used in the gels Then a stacking gel of a mixture of stock solution of 4% (w/v) acrylamide, 0.14% (w/v) bisacrylamide, and 330 mM Tris–HCl, pH 6.8 (no substrate), was cast with sample combs on top of the separating gel The authentic inhibitors or human tears were mixed with an equal volume of a treatment buffer (nonreducing reagent) of 4.0% SDS, 20% (v/v) glycerol, 0.25 M Tris– HCl, pH 6.8, and 0.02% bromophenol without heat treatment Samples of 15 ll were applied to the sample wells The electrophoresis was performed in a Tris–Tricine buffer system [0.2 M Tris (pH 8.9) as an anode buffer and 0.1 M Tris, 0.1 M Tricine, 0.1% SDS (pH 8.25) as a cathode buffer] as described by Schagger and Von Jagow [5] at °C at a constant current of 10–12 mA After the electrophoresis was completed, the gel was removed and shaken at room temperature for 45 in 2.5% Triton X-100 to remove SDS The gel was washed with distilled water several times and incubated at 37 °C in reverse zymography developing buffer containing a target enzyme for 9–10 h to digest out the background substrate The development buffer for soybean trypsin inhibitor or aprotinin consisted of 10 mM Tris–HCl buffer (pH 7.6), 200 mM NaCl, 10 mM CaCl2 , 0,02% Brij-35, and 450 lg /100 ml trypsin Cystatin C or human tears were developed in 10 mM sodium acetate buffer (pH 6.1) containing mg papain (31 unit/mg of papain), MMPI was in 20 mM phosphate-buffered saline, pH 7.2, containing mg /100 ml collagenase type 1, and pepstatin A was in 20 mM universal Britton buffer, pH 3.0, containing mg/100 ml pepsin A After proteolysis by incubation in the protease solutions, the gel was stained with staining solution (0.025% Coomassie brilliant blue R250; 40% methanol, 7% acetic acid) for h The gels were washed three times Abbreviations used: SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; CBB, Coomassie brilliant blue; STI, soybean trypsin inhibitor; MMPI, matrix metalloproteases inhibitor I for h with destaining solution (40% methanol, 10% acetic acid, 50% distilled water.) Result and discussion To select the most suitable substrate for our reverse zymography technique, we compared the staining intensities of gelatin, collagen, and hemoglobin gels before and after electrophoresis without inhibitor samples As seen in Fig 1, the collagen, gelatin and hemoglobin gels were stained weak violet, purple-blue, and dark blue (lanes 1a and 1b, 2a and 2b, and 3a and 3b), respectively Both before and after electrophoresis, the stained collagen gel lost its color very quickly after or h Fig Comparison of the CBB binding of collagen, hemoglobin, and gelatin gels before and after electrophoresis without inhibitor samples Dye-stained gels were destained 12 h Lane 1, collagen gel: (a) before running, (b) after running Lane 2, gelatin gel: (a) before running, (b) after running Lane 3, hemoglobin gel: (a) before running, (b) after running Fig Gelatin background intensity pattern and densitogram of soybean trypsin inhibitor after different incubation times (A) Gelatin background intensity during incubation period (B) Densitogram of soybean trypsin inhibitor after a 10-h incubation Q.T Le, N Katunuma / Analytical Biochemistry 324 (2004) 237–240 (lanes 1a and 1b), while the gelatin and hemoglobin gels maintained their color until 12 h (lanes 2a and 2b and 3a and 3b) Furthermore, the stained color was not homogeneous in the case of the hemoglobin gel, while the gelatin gel could retain its purple-blue color homogeneously throughout the gel (lane 2b) after electrophoresis We observed the strongest Coomassie blue staining intensity toward the end of the hemoglobin gel run, which might be due to the hemoglobin migrating out of the gel during electrophoresis (lane 3b) Based upon our observation above that gelatin was sensitive to dye staining and did not migrate out from the gel, we chose gelatin as the best substrate for our reverse zymography technique To extend the scope of this new reverse zymography technique, different incubation times (from 30 to 15 h) were evaluated using STI as a sample Fig shows that the optimal reverse zymography pattern giving the sharpest STI band on the background gel was obtained after 8–10 h After longer incubation times, the STI band gradually disappeared Fig Comparison of the reverse zymography pattern of standard inhibitors according to Hanspal et al.Õs method with those of our method Slab gels consisted of 15% acylamide, 0.1% SDS, and 0.1% gelatin Samples were subjected to electrophoresis: lane 1, cystatin C (10 ng); lane 2, soybean trypsin inhibitor (15 ng); lane 3, aprotinin (10 ng); lane 4, MMPI type (5  10À7 M); lane 5, pepstatin A (5  10À7 M) (A) Electrophoresis was performed in the Tris–glycine buffer (B) Electrophoresis was performed in the Tris–Tricine buffer After running, the gels from both procedures were developed under the same conditions as described under Materials and methods 239 To determine which types of inhibitors can be identified by our technique, we analyzed pepstatin A, MMPI, STI, aprotinin, and cystatin C on a 15% SDS– polyacrylamide gel containing 0.1% gelatin made by following the well- known Hanspal et al method [1] and our method running in Tris–glycine and Tris–Tricine system buffers, respectively After running, the gels from both procedures were treated under the same conditions as described under Materials and methods Surprisingly, the inhibitor activity of 10–20 ng of cystatin C, STI, or aprotinin was detected as a sharp single inhibitor band on the stained gels by both methods with an apparent molecular mass of 15, 25, or 12 kDa (Figs 3A and B, lanes 1–3), respectively Thus, these inhibitors showed similar molecular sizes using both methods; however, their migration was slightly slower by our method This result suggested that our method is suitable for detecting common protease inhibitors The previously reported method [1] based on the Laemmli buffer system [7] has been preferred for the study of common protease inhibitors However, as can be seen in Fig 3A, it afforded poor resolution for detecting small inhibitors under kDa such as pepstatin A (685 Da) or MMPI (490 Da) They could not be detected in the gel (lanes and 5, Fig 3A) In contrast, in our reverse zymography method running in the Tris–Tricine buffer system, the resolution was considerably improved compared with Hanspal et al.Õs method In the new method,  10À7 M pepstatin A and MMPI were visualized as clear inhibitor bands on the gel against pepsin A or collagenase type 1, respectively (lanes and 5, Fig 3B), indicating the advantage of this technique for detecting low-molecular-weight inhibitors These results Fig Inhibitors in normal human tears using reverse zymography to papain Lane 1, Coomassie blue staining of all proteins in human tear Lane 2, new reverse zymography pattern of papain inhibitors in human tear 240 Q.T Le, N Katunuma / Analytical Biochemistry 324 (2004) 237–240 were in agreement with those of Shagger and Von Jagow [5], who pointed out that separation of peptides and small proteins are not possible in the Tris–glycine buffer because the comigration of SDS and smaller protein obscures the resolution Using the Tris–Tricine buffer system in which Tricine was substituted for glycine as the trailing ion in the running buffer of the new reverse zymography method reported here allows the separation of small inhibitors–SDS complexes away from the rapidly moving SDS micelles, providing distinct small inhibitor bands in the gel Fig shows the reverse zymography pattern for cysteine protease inhibitors in human tears There were three major inhibitor bands with molecular sizes of 78, 18, and 15 kDa, as shown in lane The high-molecular-weight inhibitor of 78 kDa was identified as a lactoferrin using intramolecular amino acid sequence analysis [8] The peptide sequences of 18 amino acids in the near C-terminals area of lactoferrin showed 89% homology and 61% identity with amino acid sequence of a common active site of the cystatin family [8] The finding of lactoferrin as a new cysteine protease inhibitor has not been reported in the literature Additionally, the 15 and 18-kDa of low-molecular-weight inhibitor bands were clearly separated and these inhibitors were identified as well-known cysteine proteases: a cystatin S [9] and von EbnerÕs gland protein [10], respectively In summary, this reverse zymography method is useful for identifying both low- and high-molecularweight inhibitors against a variety of proteolytic enzymes in small amounts of natural materials Furthermore, we can compare the changes in these inhibitor profiles in various biological and pathological conditions using this reverse zymography Acknowledgment We thank Dr H Tsuge for helpful discussions References [1] G.R Bushell, P Ghosh, J.S Hanspal, Detection of protease inhibitors using substrate-containing sodium dodecyl sulfatepolyacrylamide gel electrophoresis, Anal Biochem 132 (1983) 288–293 [2] G Herron, S Michael, J Banda, J Clark, J Gavrilovic, Z Werb, Secretion of metalloproteinases by stimulated capillary endothelial cells, J Biol Chem 261 (1986) 2814–2818 [3] D.S Seidl, W.G Jaffe, E Gonzalez, A Callejas, Microelectrophoretic method for the detection of proteinase inhibitors, Anal Biochem 88 (2) (1978) 417–424 [4] D Michcaud, D Cantin, A Raworth, Vrain, Assessing the stability of cystatin/cystein complex using denaturing gelatin-polyacrylamide gel electrophoresis, Electrophoresis 17 (1996) 74–79 [5] H Schagger, Von Jagow, Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of protein in the range from to 100 kDa, Anal Biochem 166 (1987) 368–379 [6] F Marcelo, L Claudia, Electrophoretic analysis (Tricine-SDSPAGE) of bovine caseins, Acta Farm Bonaerence 21 (2002) 57– 60 [7] U.K Laemmli, Cleavage of structural protein during the assembly of the head of bacteriophage T4 , Nature 227 (1970) 680–685 [8] A Ohashi, E Murata, K Yamamoto, E Majima, E Sano, Q.T Le, N Katununma, New functions of lactoferrin and beta-casein in mammalian milk as cysteine protease inhibitors, Biochem Biophys Res Commun 306 (2003) 98–103 [9] S Isemura, E Saitoh, E Ito, M Isemura, K Sanada, Cystatin S: a cysteine proteinase inhibitor of human saliva, J Biochem (Tokyo) 96 (1984) 1311–1314 [10] P Wojnar, W Vant Hof, P Merschak, B Redl, The N-terminal part of recombinant human tear lipocalin/von EbnerÕs gland protein confers cysteine proteinase inhibition depending on the presence of the entire cystatin-like sequence motifs, Biol Chem 382 (10) (2001) 1515–1520  ... trypsin inhibitor after different incubation times (A) Gelatin background intensity during incubation period (B) Densitogram of soybean trypsin inhibitor after a 10-h incubation Q.T Le, N Katunuma... types of inhibitors can be identified by our technique, we analyzed pepstatin A, MMPI, STI, aprotinin, and cystatin C on a 15% SDS– polyacrylamide gel containing 0.1% gelatin made by following the... the detection of proteinase inhibitors, Anal Biochem 88 (2) (1978) 417–424 [4] D Michcaud, D Cantin, A Raworth, Vrain, Assessing the stability of cystatin/cystein complex using denaturing gelatin-polyacrylamide