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Blue native electrophoresis study on lipases
Analytical Biochemistry 377 (2008) 270–271
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Blue native electrophoresis study on lipases M. Saminathan, K. Thirumalai Muthukumaresan, Srinivas Rengarajan, Nandhini Muthukrishnan, Pennathur Gautam * Centre for Biotechnology, Anna University, Chennai 600025, India
a r t i c l e
i n f o
Article history: Received 16 January 2008 Available online 14 March 2008
a b s t r a c t We have developed a modified blue native polyacrylamide gel electrophoresis (PAGE) protocol that can overcome aggregation of lipases seen in native PAGE. We have shown that two lipases, Pseudomonas aeruginosa lipase and Candida rugosa lipase, which aggregate in the native gel, can be resolved using our protocol. Activity staining was done to test for the functionality of the two lipases. Ó 2008 Elsevier Inc. All rights reserved.
Lipases (EC 3.1.1.3) are enzymes that catalyze the hydrolysis of neutral lipids in biological systems. These catalyze three enzyme reactions: hydrolysis of ester in aqueous solutions, esterification in organic solvents, and transesterification between ester and acyl group donor. Their ability to accept a wide range of substrates (lipids, sugars, alcohols, acids, and esters) and their capability to maintain activity and selectivity in organic solvents has enabled their wide use as biocatalysts [1]. Because of their activities in both aqueous and nonaqueous solvent systems, they find applications in food, dairy, detergent, and pharmaceutical industries and hence need more efficient analytical techniques for their qualitative and quantitative analysis. Zymography is one of the widely used techniques to study the functionality of enzymes. Traditional native electrophoresis is limited in its applicability to native protein analysis because of high or low operative pH which may adversely affect proteins. Another drawback in native gel electrophoresis is the need for separate acidic and basic gels for resolution of enzymes. Traditional native gel electrophoresis is limited by its incompatibility with native samples that aggregate [2]. SDS–PAGE1 can be successfully used for the activity staining of lipase after removing SDS from the gel [3]. However, it works best for monomeric enzymes. Some SDS-sensitive proteins and enzymes with multisubunits can be denatured irreversibly. This may lead to the loss of activity of the enzyme. In this report, we demonstrate the use of a modified blue native polyacrylamide gel electrophoresis (BN–PAGE) that can overcome the above-mentioned limitations [4]. To our knowledge, this is the first report on the use of BN–PAGE for lipase studies. BN–PAGE is an electrophoretic method for resolution by native size designed for use with mild detergents to study hydrophobic proteins and
* Corresponding author. Fax: +91 44 22350299. E-mail address: [email protected] (P. Gautam). 1 Abbreviations used: BN, blue native; PAGE, polyacrylamide gel electrophoresis; CBB G-250, coomassie brilliant blue G-250; SDS, sodium dodecyl sulphate; CRL, Candida rugosa lipase; MTCC, Microbial Type Culture Collection- Chandigarh, India. 0003-2697/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.03.010
membrane protein complexes. BN–PAGE preserves the biochemical activity of the components separated after electrophoresis [5]. It is preferably used for isolating and studying protein complexes [6]. Its use can also be extended to the study of enzymes such as lipases and esterases. An added advantage is that the activity of the enzyme under study can be evaluated by zymography, which is a simple extension of the BN–PAGE method. The BN–PAGE technique uses Coomassie brilliant blue G-250 as a charge-shift molecule. The CBB G-250 binds to surface-positive residues of proteins and confers a negative charge without denaturing them, unlike SDS which completely denatures the protein. The binding of G-250 to protein molecules provides two main advantages. Proteins with acidic or basic isoelectric points [7] are converted to a net negative charge so that they migrate to the anode, and proteins with significant surface-exposed hydrophobic area are less prone to aggregation when G-250 binds nonspecifically to the hydrophobic sites, converting them to negatively charged sites [8]. The samples can be separated by electrophoresis depending on the amount of dye bound to it, which is proportional to their size. Native polyacrylamide gel electrophoresis was performed using the discontinuous gel system of Davis [9]. For BN–PAGE, the stacking and separating gels were prepared with 4% (w/v) and 10% (w/v) acrylamide, respectively, using 50 mM Tris–Cl, pH 7.5. Cathode buffer was prepared at pH 7.5 with 50 mM Tricine and 15 mM Tris and anode buffer was prepared with 50 mM Tris–Cl at pH 7.5. The cathode buffer was supplemented with 0.002% (w/v) CBB G-250 dye. The 2X sample solubilizing buffer contained 1% (v/v) Triton X-100, 50 mM Tris–Cl, pH 7.5, 1% (w/v) CBB G-250, and 20% (v/v) glycerol. Protein samples were mixed with equal volume of sample solubilizing buffer and incubated for 0.5 h before loading; 5 ll of the sample was loaded in each well. The gel was run at 20 °C and 60 V. Two protein samples, commercially obtained Candida rugosa lipase (CRL) and a lipase secreted by Pseudomonas aeruginosa MTCC- 2297 were loaded in the gel in duplicate (Twelve-hourgrown 1% P. aeruginosa culture was inoculated into 50 ml of nutrient broth and incubated for 16 h at 30 °C at 160 rpm. Cells were
Notes & Tips / Anal. Biochem. 377 (2008) 270–271
pelleted at 5000g for 10 min and the cell-free culture supernatant was precipitated with 4 volumes of ice-cold acetone). After half the run time, the cathode buffer without CBB G-250 was applied. This allowed most of the unbound dye to leave the gel and hence gave a colorless background. The gel was run for a period of about 6 h until the dye front reached the end of the gel. One half of the gel was used for activity staining while the other half was stained for locating the corresponding enzyme bands. The gel was first equilibrated in 25 mM Tris–Cl, pH 7.5, for 0.5 h for activity staining. Subsequently, the gel was sandwiched between a substrate–agar emulsion for the detection of lipase activity. The substrate–agar emulsion was prepared using 1.3% (w/v) agar, 1% (v/v) tributyrin, 25 mM Tris–Cl, pH 7.5, 10 mM CaCl2, and 0.1% (v/v) Triton X-100. The plate was incubated at room temperature until a clear zone was observed (See Figs. 1 and 2). Both the lipases were successfully resolved by BN–PAGE, whereas aggregation of both was seen in the native gel. Zymography of the enzyme showed that the biochemical activities of the resolved enzymes were still preserved after electrophoresis. Buffers used for electrophoresis were near neutral (pH 7.5) which is well within the buffering range of both Tris and Tricine buffers. This provides gentle conditions for proteins and helps to keep the proteins intact upon solubilization and through electrophoresis. We have developed an activity staining protocol to study aggregating lipases. Highly hydrophobic enzymes such as lipases and esterases can be easily resolved using BN–PAGE. Another possible application of BN–PAGE in the study of lipases is the use of second-dimension analysis to study the subunits of the enzymes in a manner analogous to the way BN–PAGE is currently used to study protein complexes [10]. Because complexes separated by BN–PAGE are still functionally active, activity staining can be done.
271
Fig. 2. Lipase activity staining after BN–PAGE. Lanes: 1, Coomassie-stained CRL (commercial lipase; Sigma; 500 ng); 2, activity-stained CRL (500 ng); 3, Coomassiestained P. aeruginosa extracellular protein (15 lg total protein); 4, activity-stained P. aeruginosa extracellular protein (15 lg total protein). Arrows indicate stacking separating interface. Aggregation and nonentry of both the lipases into the separating gel were seen in case of native PAGE (Fig. 1). On the other hand, the BN–PAGE (Fig. 2) shows entry of both the lipases into the separating gel. Clear zones indicating activity of the lipases were seen in both the BN–PAGE and the native PAGE gels.
Acknowledgment P.G. thanks Department of Science and Technology and Department of Biotechnology, Government of India, for its financial support. M.S. thanks CSIR (Council of Scientific and Industrial Research), Government of India, for research fellowship. References
Fig. 1. Lipase activity staining after native gel electrophoresis. Lanes: 1, Coomassiestained CRL (commercial lipase; Sigma; 2 lg); 2, activity-stained CRL (2 lg); 3, Coomassie-stained P. aeruginosa extracellular protein (15 lg total protein); 4, activity-stained P. aeruginosa extracellular protein (15 lg total protein). Arrows indicate stacking separating interface.
[1] A.M. Klibanov, Improving enzymes by using them in organic solvents, Nature 409 (2001) 241–246. [2] Shuen-Fuh Lin, Chien-Ming Chiou, Chuan-Mei Yeh, Ying-Chieh Tsai, Purification and partial characterization of an alkaline lipase from Pseudomonas pseudoalcaligenes F-111, Appl. Environ. Microbiol. 62 (1996) 1093–1095. [3] P. Sommer, C. Bormann, F. Gotz, Genetic and Biochemical Characterization of a New Extracellular Lipase from Streptomyces cinnamomeus, Appl. Environ. Microbiol. 63 (1997) 3553–3560. [4] H. Schägger, G. von Jagow, Blue Native Electrophoresis for isolation of membrane protein complexes in enzymatically active form, Anal. Biochem. 199 (1991) 223–231. [5] H. Schägger, W.A. Cramer, G. von Jagow, Analysis of molecular masses and oligomeric states of protein complexes by Blue Native Electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis, Anal. Biochem. 217 (1994) 220–230. [6] L.G.J. Nijtmans, N.S. Henderson, I.J. Holt, Blue Native electrophoresis to study mitochondrial and other protein complexes, Methods 26 (2002) 327–334. [7] B. Meyer, I. Wittig, E. Trifilieff, M. Karas, H. Schägger, Identification of two proteins associated with mammalian ATP synthase, Mol. Cell. Proteomics 6 (2007) 1690–1699. [8] J. Kjell, Blue Native Polyacrylamide Gel Electrophoresis – A Functional Approach To Plant Plasma Membrane Proteome Studies, Thesis (2004), Department of Cell and Organism Biology, Lund University, Sweden. [9] B.J. Davis, Disc Electrophoresis II: Method and application to human serum proteins, Ann. N. Y. Acad. Sci. 121 (1964) 321–349. [10] P.S. Brookes, A. Pinner, A. Ramachandran, L. Coward, S. Barnes, H. Kim, V.M. Darley-Usmar, High throughput two-dimensional blue-native electrophoresis: A tool for functional proteomics of mitochondria and signaling complexes, Proteomics 2 (2002) 969–977.
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Blue native electrophoresis study on lipases M. Saminathan, K. Thirumalai Muthukumaresan, Srinivas Rengarajan, Nandhini Muthukrishnan, Pennathur Gautam * Centre for Biotechnology, Anna University, Chennai 600025, India
a r t i c l e
i n f o
Article history: Received 16 January 2008 Available online 14 March 2008
a b s t r a c t We have developed a modified blue native polyacrylamide gel electrophoresis (PAGE) protocol that can overcome aggregation of lipases seen in native PAGE. We have shown that two lipases, Pseudomonas aeruginosa lipase and Candida rugosa lipase, which aggregate in the native gel, can be resolved using our protocol. Activity staining was done to test for the functionality of the two lipases. Ó 2008 Elsevier Inc. All rights reserved.
Lipases (EC 3.1.1.3) are enzymes that catalyze the hydrolysis of neutral lipids in biological systems. These catalyze three enzyme reactions: hydrolysis of ester in aqueous solutions, esterification in organic solvents, and transesterification between ester and acyl group donor. Their ability to accept a wide range of substrates (lipids, sugars, alcohols, acids, and esters) and their capability to maintain activity and selectivity in organic solvents has enabled their wide use as biocatalysts [1]. Because of their activities in both aqueous and nonaqueous solvent systems, they find applications in food, dairy, detergent, and pharmaceutical industries and hence need more efficient analytical techniques for their qualitative and quantitative analysis. Zymography is one of the widely used techniques to study the functionality of enzymes. Traditional native electrophoresis is limited in its applicability to native protein analysis because of high or low operative pH which may adversely affect proteins. Another drawback in native gel electrophoresis is the need for separate acidic and basic gels for resolution of enzymes. Traditional native gel electrophoresis is limited by its incompatibility with native samples that aggregate [2]. SDS–PAGE1 can be successfully used for the activity staining of lipase after removing SDS from the gel [3]. However, it works best for monomeric enzymes. Some SDS-sensitive proteins and enzymes with multisubunits can be denatured irreversibly. This may lead to the loss of activity of the enzyme. In this report, we demonstrate the use of a modified blue native polyacrylamide gel electrophoresis (BN–PAGE) that can overcome the above-mentioned limitations [4]. To our knowledge, this is the first report on the use of BN–PAGE for lipase studies. BN–PAGE is an electrophoretic method for resolution by native size designed for use with mild detergents to study hydrophobic proteins and
* Corresponding author. Fax: +91 44 22350299. E-mail address: [email protected] (P. Gautam). 1 Abbreviations used: BN, blue native; PAGE, polyacrylamide gel electrophoresis; CBB G-250, coomassie brilliant blue G-250; SDS, sodium dodecyl sulphate; CRL, Candida rugosa lipase; MTCC, Microbial Type Culture Collection- Chandigarh, India. 0003-2697/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.03.010
membrane protein complexes. BN–PAGE preserves the biochemical activity of the components separated after electrophoresis [5]. It is preferably used for isolating and studying protein complexes [6]. Its use can also be extended to the study of enzymes such as lipases and esterases. An added advantage is that the activity of the enzyme under study can be evaluated by zymography, which is a simple extension of the BN–PAGE method. The BN–PAGE technique uses Coomassie brilliant blue G-250 as a charge-shift molecule. The CBB G-250 binds to surface-positive residues of proteins and confers a negative charge without denaturing them, unlike SDS which completely denatures the protein. The binding of G-250 to protein molecules provides two main advantages. Proteins with acidic or basic isoelectric points [7] are converted to a net negative charge so that they migrate to the anode, and proteins with significant surface-exposed hydrophobic area are less prone to aggregation when G-250 binds nonspecifically to the hydrophobic sites, converting them to negatively charged sites [8]. The samples can be separated by electrophoresis depending on the amount of dye bound to it, which is proportional to their size. Native polyacrylamide gel electrophoresis was performed using the discontinuous gel system of Davis [9]. For BN–PAGE, the stacking and separating gels were prepared with 4% (w/v) and 10% (w/v) acrylamide, respectively, using 50 mM Tris–Cl, pH 7.5. Cathode buffer was prepared at pH 7.5 with 50 mM Tricine and 15 mM Tris and anode buffer was prepared with 50 mM Tris–Cl at pH 7.5. The cathode buffer was supplemented with 0.002% (w/v) CBB G-250 dye. The 2X sample solubilizing buffer contained 1% (v/v) Triton X-100, 50 mM Tris–Cl, pH 7.5, 1% (w/v) CBB G-250, and 20% (v/v) glycerol. Protein samples were mixed with equal volume of sample solubilizing buffer and incubated for 0.5 h before loading; 5 ll of the sample was loaded in each well. The gel was run at 20 °C and 60 V. Two protein samples, commercially obtained Candida rugosa lipase (CRL) and a lipase secreted by Pseudomonas aeruginosa MTCC- 2297 were loaded in the gel in duplicate (Twelve-hourgrown 1% P. aeruginosa culture was inoculated into 50 ml of nutrient broth and incubated for 16 h at 30 °C at 160 rpm. Cells were
Notes & Tips / Anal. Biochem. 377 (2008) 270–271
pelleted at 5000g for 10 min and the cell-free culture supernatant was precipitated with 4 volumes of ice-cold acetone). After half the run time, the cathode buffer without CBB G-250 was applied. This allowed most of the unbound dye to leave the gel and hence gave a colorless background. The gel was run for a period of about 6 h until the dye front reached the end of the gel. One half of the gel was used for activity staining while the other half was stained for locating the corresponding enzyme bands. The gel was first equilibrated in 25 mM Tris–Cl, pH 7.5, for 0.5 h for activity staining. Subsequently, the gel was sandwiched between a substrate–agar emulsion for the detection of lipase activity. The substrate–agar emulsion was prepared using 1.3% (w/v) agar, 1% (v/v) tributyrin, 25 mM Tris–Cl, pH 7.5, 10 mM CaCl2, and 0.1% (v/v) Triton X-100. The plate was incubated at room temperature until a clear zone was observed (See Figs. 1 and 2). Both the lipases were successfully resolved by BN–PAGE, whereas aggregation of both was seen in the native gel. Zymography of the enzyme showed that the biochemical activities of the resolved enzymes were still preserved after electrophoresis. Buffers used for electrophoresis were near neutral (pH 7.5) which is well within the buffering range of both Tris and Tricine buffers. This provides gentle conditions for proteins and helps to keep the proteins intact upon solubilization and through electrophoresis. We have developed an activity staining protocol to study aggregating lipases. Highly hydrophobic enzymes such as lipases and esterases can be easily resolved using BN–PAGE. Another possible application of BN–PAGE in the study of lipases is the use of second-dimension analysis to study the subunits of the enzymes in a manner analogous to the way BN–PAGE is currently used to study protein complexes [10]. Because complexes separated by BN–PAGE are still functionally active, activity staining can be done.
271
Fig. 2. Lipase activity staining after BN–PAGE. Lanes: 1, Coomassie-stained CRL (commercial lipase; Sigma; 500 ng); 2, activity-stained CRL (500 ng); 3, Coomassiestained P. aeruginosa extracellular protein (15 lg total protein); 4, activity-stained P. aeruginosa extracellular protein (15 lg total protein). Arrows indicate stacking separating interface. Aggregation and nonentry of both the lipases into the separating gel were seen in case of native PAGE (Fig. 1). On the other hand, the BN–PAGE (Fig. 2) shows entry of both the lipases into the separating gel. Clear zones indicating activity of the lipases were seen in both the BN–PAGE and the native PAGE gels.
Acknowledgment P.G. thanks Department of Science and Technology and Department of Biotechnology, Government of India, for its financial support. M.S. thanks CSIR (Council of Scientific and Industrial Research), Government of India, for research fellowship. References
Fig. 1. Lipase activity staining after native gel electrophoresis. Lanes: 1, Coomassiestained CRL (commercial lipase; Sigma; 2 lg); 2, activity-stained CRL (2 lg); 3, Coomassie-stained P. aeruginosa extracellular protein (15 lg total protein); 4, activity-stained P. aeruginosa extracellular protein (15 lg total protein). Arrows indicate stacking separating interface.
[1] A.M. Klibanov, Improving enzymes by using them in organic solvents, Nature 409 (2001) 241–246. [2] Shuen-Fuh Lin, Chien-Ming Chiou, Chuan-Mei Yeh, Ying-Chieh Tsai, Purification and partial characterization of an alkaline lipase from Pseudomonas pseudoalcaligenes F-111, Appl. Environ. Microbiol. 62 (1996) 1093–1095. [3] P. Sommer, C. Bormann, F. Gotz, Genetic and Biochemical Characterization of a New Extracellular Lipase from Streptomyces cinnamomeus, Appl. Environ. Microbiol. 63 (1997) 3553–3560. [4] H. Schägger, G. von Jagow, Blue Native Electrophoresis for isolation of membrane protein complexes in enzymatically active form, Anal. Biochem. 199 (1991) 223–231. [5] H. Schägger, W.A. Cramer, G. von Jagow, Analysis of molecular masses and oligomeric states of protein complexes by Blue Native Electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis, Anal. Biochem. 217 (1994) 220–230. [6] L.G.J. Nijtmans, N.S. Henderson, I.J. Holt, Blue Native electrophoresis to study mitochondrial and other protein complexes, Methods 26 (2002) 327–334. [7] B. Meyer, I. Wittig, E. Trifilieff, M. Karas, H. Schägger, Identification of two proteins associated with mammalian ATP synthase, Mol. Cell. Proteomics 6 (2007) 1690–1699. [8] J. Kjell, Blue Native Polyacrylamide Gel Electrophoresis – A Functional Approach To Plant Plasma Membrane Proteome Studies, Thesis (2004), Department of Cell and Organism Biology, Lund University, Sweden. [9] B.J. Davis, Disc Electrophoresis II: Method and application to human serum proteins, Ann. N. Y. Acad. Sci. 121 (1964) 321–349. [10] P.S. Brookes, A. Pinner, A. Ramachandran, L. Coward, S. Barnes, H. Kim, V.M. Darley-Usmar, High throughput two-dimensional blue-native electrophoresis: A tool for functional proteomics of mitochondria and signaling complexes, Proteomics 2 (2002) 969–977.