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ABri peptide aggregation quantification by fluorescamine and alpha imager assay
Journal of Industrial and Engineering Chemistry 17 (2011) 427–429
Contents lists available at ScienceDirect
Journal of Industrial and Engineering Chemistry journal homepage: www.elsevier.com/locate/jiec
ABri peptide aggregation quantification by fluorescamine and alpha imager assay Keunhong Jeong *, Woo Young Chung, Young Sik Kye, Dongwook Kim Department of Chemistry, Korea Military Academy, Seoul 139-799, South Korea
A R T I C L E I N F O
Article history: Received 12 January 2010 Accepted 2 September 2010 Available online 28 April 2011 Keywords: ABri Fluorescamine Alpha imager Aggregation rate
A B S T R A C T
Familial British Dementia (FBD) is the early-onset autosomal dominant disorders characterized by cognitive impairment. The ABri is a 34-mer peptide which is the main component of amyloid deposits in FBD. As a first step toward shedding light for the discovery of amyloid inhibition drugs for FBD, we set the optimal conditions for quantification ABri peptide by the fluorescamine and alpha imager assay, which can provide a precise peptide quantity for determination of amyloid inhibition rate at micromolecular level and estimate aggregation rate by measuring monomer and dimer quantity which are filtered through 10,000 Da MW cutoff filter. ß 2011 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
1. Introduction The Familial British Dementia (FBD) is an early-onset autosomal dominant disease. The average onset age of FBD is usually 60 years old and patients suffer from recognition impairment, progressive spastic all four limbs’ paralysis, and cerebellar ataxia [1]. Amyloid deposits on cerebral blood vessel and brain parenchyma and neurofibrillary tangles in limbic areas are the hallmark of FBD [2]. The 34-residue peptide ABri is the major component of the insoluble amyloid deposits that are likely to be involved in pathogenesis [3]. The ABri is created by the abolishment of the stop codon in its precursor, which is 266 residue protein codified by single gene (BRI) in chromosome 13 [4]. A nucleotide transition from TGA to AGA extends precursor protein to 277 residue protein, which is cleaved to form 34 C-terminal amino acid residues (EASNCFAIRHFENKFAVETLICSRTVKKNIIEEN) by furin-mediated processing [5,6]. Researches on developing drugs that are capable of inhibiting disease relating proteins to cure many amyloidogenic diseases have mainly focused on well-known amyloidogenic diseases including Alzheimer’s disease (AD), variant Creutzfeldt–Jakob Disease (vCJD), tuberculosis, diabetes, and rheumatoid arthritis. Total protein quantification by fluorescamine and alpha imager assay was established on several amyloid peptides which have been on the center of researchers’ interest: Ab 42-mer peptide [7], human islet amyloid polypeptide [8,9], and prion protein 106–126 [10]. Unlike Th-T assay [11], far UV-CD analysis [12], and immunoblotting assay [13] systems which have been convention-
* Corresponding author. Tel.: +82 2 2197 2845. E-mail address: [email protected] (K. Jeong).
ally used for identifying amyloid aggregation inhibitors, fluorescamine and alpha imager assay would be more optimum method for the researches of finding amyloid aggregation inhibitors, which can provide precise figures for the protein quantity. However, in the amyloid deposits, the ABri peptide forms aggregated b-pleated sheet structure, similar to those formed by the Ab peptide of AD and other amyloid forming proteins [6]. Therefore, in this study, we applied the fluorescamine and alpha imager assay on ABri peptide and quantification of total ABri peptides was successfully performed. 2. Experimental The fluorescamine and alpha imager assay was carried out by the published procedure [14]. Fluorescamine, methanesulfonic acid, hexafluoroisopropanol (HFIP), and phosphate buffer powder were purchased from Sigma–Aldrich and used without more purification. 96 well plate was purchased from F96 Cert. (Maxisorp, Nunc-Immuno Plate). Synthesized ABri 34-mer peptide 1 mg (more than 95% purity, purchased from Peptron Inc.) was dissolved in HFIP 1 mL and sonificated for 5 min. Then, 32 mL of aliquot was transferred to 31 microtubes to make 8 mM/mL final concentration for amino acid analysis. After HFIP was completely lyophilized, microtubes were stored in refrigerator ( 70 8C) before use. After 100 mL HFIP was added to a microtube, microtube was sonificated for 3 min. Then, 2, 4, 6, 8, and 10 mL of aliquot were added to two 500 mL microtubes to make 1.6, 3.2, 4.8, 6.4, and 8.0 mM/100 mL final concentration in phosphate buffer, respectively. After HFIP was completely evaporated in heating block (80 8C, 5 min), 100 mL phosphate buffer (50 mM, pH 7.5) was added, respectively.
1226-086X/$ – see front matter ß 2011 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jiec.2010.09.017
[(Fig._1)TD$IG]
428
[(Fig._3)TD$IG]
K. Jeong et al. / Journal of Industrial and Engineering Chemistry 17 (2011) 427–429
Fig. 1. Fluorescence intensity linear to concentration. (Raw Ref. well contains nothing, pH 9 Ref. well contains pH 9 phosphate buffer (100 mL) alkali hydrolysis + neutralization + fluorescamine mixing).
Fig. 3. Fluorescence intensity picture after incubated peptide solution filtration and MALDI-TOF mass spectrum by incubation of ABri 34-mer peptide for 4 h. The peak with m/z values of 3935.3 corresponds to ABri monomer.
For alkaline hydrolysis of solution, to a microbute of 100 mL buffer solution 10 mL of 13.5 M NaOH was added. Then, microtubes were vortexed and centrifuged briefly. After 2 h autoclaving at 120 8C, the mixtures were cooled to room temperature. After 7.5 mL methanesulfonic acid was added for neutralization, samples were vortexed and centrifuged. After boric acid (0.7 M, 48 mL) was added to make pH 9.0 solution, samples were vortexed and centrifuged again. The amount of hydrolyzed amino acids is linearly proportional to initial amount of ABri 34-mer peptides. At pH 9.0, fluorescamine reacts with primary amines within a second at room temperature [15,16]. After solution of fluorescamine in acetonitrile (3.0 mg/mL, 10 mL) was loaded to a 96 well plate, 165.5 mL of the hydrolyzed mixture was added and mixed in plate, respectively. The relative value of fluorescence of the resulting solution was measured with an imaging instrument (Alpha Imager Model 1220 INT) (Fig. 1). The rate of ABri aggregation of greater sizes than dimer after 4 h was estimated by quantifying by same approach. The two samples of 8 mM/100 mL were incubated for 4 h in 37 8C. After incubation, 100 mL aliquot form each samples was filtered through 10,000 Da MW membrane filter (Micropore Microcon centrifugal filter device YM-10) at 10,000 g for 30 min (Hanil Model Micro 12) to obtain monomer and dimer of ABri peptide, and then filtrate quantity was estimated by fluorescamine and alpha imager assay.
aggregation inhibitor finding researches. After alkaline hydrolysis of the ABri, samples are neutralized into pH 9.0 with acid and buffer. The concentration of hydrolyzed amino acids is linearly correlated with initial amount of protein. The mixture reacted with fluorescamine at room temperature followed by fluorescence intensity measurement. Fluorescence intensity in 96 well plate was measured by alpha imager and the relative intensities are compared. Meaningful correlation between ABri 34-mer peptide concentration and fluorescence intensity upon the reaction of fluorescamine with cleaved primary amines from ABri peptide was observed (Fig. 2). The aggregation rate of ABri 34-mer peptide after 4 h incubation in 37 8C was determined by fluorescamine and alpha imager assay. Fluorescence intensity rate of filtrate was estimated 28.2% by comparing the standard curve. Therefore, the aggregation rate was determined by 71.8%. Solution in sample was checked by MALDI-TOF MS after 4 h incubation. The monomer spectrum peak was very weak, the intensity is 70–200 (Fig. 3). However, monomer peak intensity was more than 1000–10,000 (data not shown). Although the intensities of MALDI-TOF MS peak do not precisely correlated with the relative concentration, it can be concluded that monomers of ABri peptide remain in low concentration. Therefore, the major component of filtrate after 4 h incubation would be ABri peptide dimer.
3. Result and discussion
4. Conclusions
ABri peptide quantity was estimated by using fluorescamine and alpha imager assay which is optimum system for amyloid
There have been a lot of studies to develop drugs that can inhibit the aggregation of prion protein. The inhibition of pathogenic oligomers of amyloidogenic proteins have been widely studied as a therapeutic option for amyloidogenic diseases [17– 20]. To enhance our knowledge for the discovery of ABri aggregation inhibiting drug, it is critical to quantify protein accurately and aggregation rate. In this study, quantification for ABri aggregation rate at micromolecular level was successfully carried out. This study will contribute to identify ABri aggregation inhibitor and develop new FBD drug.
[(Fig._2)TD$IG]
Acknowledgments This study was supported by the Korea Military Academy Hwarang-dae Research Institute research program. I appreciate to Professor Suh, Junghun of Seoul National University for valuable advice and allowing us to use their research facilities during the course of this research. References Fig. 2. Linear plot for ABri 34-mer peptide concentrations with fluorescence intensities.
[1] S. Mead, M. James-Galton, T. Revesz, R. Doshi, G. Harwood, E. Pan, J. Ghiso, B. Frangione, G. Plant, Brain 123 (2000) 975. [2] G. Plant, T. Revesz, R. Barnard, A. Harding, P. Gautier-Smith, Brain 113 (1990) 721.
K. Jeong et al. / Journal of Industrial and Engineering Chemistry 17 (2011) 427–429 [3] R. Vidal, B. Frangione, A. Rostagno, S. Mead, T. Revesz, G. Plant, J. Ghiso, Nature 399 (1999) 776. [4] J. Ghiso, J. Holton, L. Miravalle, M. Calero, T. Lashley, R. Vidal, H. Houlden, N. Wood, T. Neubert, A. Rostagno, Journal of Biological Chemistry 276 (2001) 43909. [5] S. Kim, R. Wang, D. Gordon, J. Bass, D. Steiner, D. Lynn, G. Thinakaran, S. Meredith, S. Sisodia, Nature Neuroscience 2 (1999) 984. [6] R. Srinivasan, E. Jones, K. Liu, J. Ghiso, R. Marchant, M. Zagorski, Journal of Molecular Biology 333 (2003) 1003. [7] J. Suh, S. Yoo, M. Kim, K. Jeong, J. Ahn, M. Kim, P. Chae, T. Lee, J. Lee, Angewandte Chemie-International Edition 46 (2007) 7064. [8] J. Suh, W. Chei, T. Lee, M. Kim, S. Yoo, K. Jeong, J. Ahn, Journal of Biological Inorganic Chemistry 13 (2008) 693. [9] K. Jeong, W.Y. Chung, Y.-S. Kye, D. Kim, Bioorganic & Medicinal Chemistry 18 (2010) 2598. [10] K. Jeong, J. Suh, W. Chung, Y. Kye, D. Kim, T. Lee, Bulletin of Korean Chemical Society 31 (2010) 1029.
429
[11] T. Florio, D. Paludi, V. Villa, D. Principe, A. Corsaro, E. Millo, G. Damonte, C. D’Arrigo, C. Russo, G. Schettini, Journal of Neurochemistry 85 (2003) 62. [12] G. Bohm, R. Muhr, R. Jaenicke, Protein Engineering Design and Selection 5 (1992) 191. [13] K. Chung, H. Hwang, K. Lee, Journal of Industrial and Engineering Chemistry 16 (2010) 115. [14] K. Chung, H. Hwang, K. Lee, Journal of the Korean Industrial and Engineering Chemistry 20 (2009) 628. [15] M. McCaman, E. Robins, Journal of Laboratory and Clinical Medicine 59 (1962) 885. [16] S. Udenfriend, S. Stein, P. Bohlen, W. Dairman, W. Leimgruber, M. Weigele, Science 178 (1972) 871. [17] B. Austen, K. Paleologou, S. Ali, M. Qureshi, D. Allsop, O. El-Agnaf, Biochemistry 47 (2008) 1984. [18] C. Haass, D. Selkoe, Nature Reviews Molecular Cell Biology 8 (2007) 101. [19] J. Sacchettini, J. Kelly, Nature Reviews Drug Discovery 1 (2002) 267. [20] K. Jeong, M. Kim, W. Chung, Y. Kye, J. Kim, J. Suh, Journal of Industrial and Engineering Chemistry 15 (2009) 342.
Contents lists available at ScienceDirect
Journal of Industrial and Engineering Chemistry journal homepage: www.elsevier.com/locate/jiec
ABri peptide aggregation quantification by fluorescamine and alpha imager assay Keunhong Jeong *, Woo Young Chung, Young Sik Kye, Dongwook Kim Department of Chemistry, Korea Military Academy, Seoul 139-799, South Korea
A R T I C L E I N F O
Article history: Received 12 January 2010 Accepted 2 September 2010 Available online 28 April 2011 Keywords: ABri Fluorescamine Alpha imager Aggregation rate
A B S T R A C T
Familial British Dementia (FBD) is the early-onset autosomal dominant disorders characterized by cognitive impairment. The ABri is a 34-mer peptide which is the main component of amyloid deposits in FBD. As a first step toward shedding light for the discovery of amyloid inhibition drugs for FBD, we set the optimal conditions for quantification ABri peptide by the fluorescamine and alpha imager assay, which can provide a precise peptide quantity for determination of amyloid inhibition rate at micromolecular level and estimate aggregation rate by measuring monomer and dimer quantity which are filtered through 10,000 Da MW cutoff filter. ß 2011 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
1. Introduction The Familial British Dementia (FBD) is an early-onset autosomal dominant disease. The average onset age of FBD is usually 60 years old and patients suffer from recognition impairment, progressive spastic all four limbs’ paralysis, and cerebellar ataxia [1]. Amyloid deposits on cerebral blood vessel and brain parenchyma and neurofibrillary tangles in limbic areas are the hallmark of FBD [2]. The 34-residue peptide ABri is the major component of the insoluble amyloid deposits that are likely to be involved in pathogenesis [3]. The ABri is created by the abolishment of the stop codon in its precursor, which is 266 residue protein codified by single gene (BRI) in chromosome 13 [4]. A nucleotide transition from TGA to AGA extends precursor protein to 277 residue protein, which is cleaved to form 34 C-terminal amino acid residues (EASNCFAIRHFENKFAVETLICSRTVKKNIIEEN) by furin-mediated processing [5,6]. Researches on developing drugs that are capable of inhibiting disease relating proteins to cure many amyloidogenic diseases have mainly focused on well-known amyloidogenic diseases including Alzheimer’s disease (AD), variant Creutzfeldt–Jakob Disease (vCJD), tuberculosis, diabetes, and rheumatoid arthritis. Total protein quantification by fluorescamine and alpha imager assay was established on several amyloid peptides which have been on the center of researchers’ interest: Ab 42-mer peptide [7], human islet amyloid polypeptide [8,9], and prion protein 106–126 [10]. Unlike Th-T assay [11], far UV-CD analysis [12], and immunoblotting assay [13] systems which have been convention-
* Corresponding author. Tel.: +82 2 2197 2845. E-mail address: [email protected] (K. Jeong).
ally used for identifying amyloid aggregation inhibitors, fluorescamine and alpha imager assay would be more optimum method for the researches of finding amyloid aggregation inhibitors, which can provide precise figures for the protein quantity. However, in the amyloid deposits, the ABri peptide forms aggregated b-pleated sheet structure, similar to those formed by the Ab peptide of AD and other amyloid forming proteins [6]. Therefore, in this study, we applied the fluorescamine and alpha imager assay on ABri peptide and quantification of total ABri peptides was successfully performed. 2. Experimental The fluorescamine and alpha imager assay was carried out by the published procedure [14]. Fluorescamine, methanesulfonic acid, hexafluoroisopropanol (HFIP), and phosphate buffer powder were purchased from Sigma–Aldrich and used without more purification. 96 well plate was purchased from F96 Cert. (Maxisorp, Nunc-Immuno Plate). Synthesized ABri 34-mer peptide 1 mg (more than 95% purity, purchased from Peptron Inc.) was dissolved in HFIP 1 mL and sonificated for 5 min. Then, 32 mL of aliquot was transferred to 31 microtubes to make 8 mM/mL final concentration for amino acid analysis. After HFIP was completely lyophilized, microtubes were stored in refrigerator ( 70 8C) before use. After 100 mL HFIP was added to a microtube, microtube was sonificated for 3 min. Then, 2, 4, 6, 8, and 10 mL of aliquot were added to two 500 mL microtubes to make 1.6, 3.2, 4.8, 6.4, and 8.0 mM/100 mL final concentration in phosphate buffer, respectively. After HFIP was completely evaporated in heating block (80 8C, 5 min), 100 mL phosphate buffer (50 mM, pH 7.5) was added, respectively.
1226-086X/$ – see front matter ß 2011 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jiec.2010.09.017
[(Fig._1)TD$IG]
428
[(Fig._3)TD$IG]
K. Jeong et al. / Journal of Industrial and Engineering Chemistry 17 (2011) 427–429
Fig. 1. Fluorescence intensity linear to concentration. (Raw Ref. well contains nothing, pH 9 Ref. well contains pH 9 phosphate buffer (100 mL) alkali hydrolysis + neutralization + fluorescamine mixing).
Fig. 3. Fluorescence intensity picture after incubated peptide solution filtration and MALDI-TOF mass spectrum by incubation of ABri 34-mer peptide for 4 h. The peak with m/z values of 3935.3 corresponds to ABri monomer.
For alkaline hydrolysis of solution, to a microbute of 100 mL buffer solution 10 mL of 13.5 M NaOH was added. Then, microtubes were vortexed and centrifuged briefly. After 2 h autoclaving at 120 8C, the mixtures were cooled to room temperature. After 7.5 mL methanesulfonic acid was added for neutralization, samples were vortexed and centrifuged. After boric acid (0.7 M, 48 mL) was added to make pH 9.0 solution, samples were vortexed and centrifuged again. The amount of hydrolyzed amino acids is linearly proportional to initial amount of ABri 34-mer peptides. At pH 9.0, fluorescamine reacts with primary amines within a second at room temperature [15,16]. After solution of fluorescamine in acetonitrile (3.0 mg/mL, 10 mL) was loaded to a 96 well plate, 165.5 mL of the hydrolyzed mixture was added and mixed in plate, respectively. The relative value of fluorescence of the resulting solution was measured with an imaging instrument (Alpha Imager Model 1220 INT) (Fig. 1). The rate of ABri aggregation of greater sizes than dimer after 4 h was estimated by quantifying by same approach. The two samples of 8 mM/100 mL were incubated for 4 h in 37 8C. After incubation, 100 mL aliquot form each samples was filtered through 10,000 Da MW membrane filter (Micropore Microcon centrifugal filter device YM-10) at 10,000 g for 30 min (Hanil Model Micro 12) to obtain monomer and dimer of ABri peptide, and then filtrate quantity was estimated by fluorescamine and alpha imager assay.
aggregation inhibitor finding researches. After alkaline hydrolysis of the ABri, samples are neutralized into pH 9.0 with acid and buffer. The concentration of hydrolyzed amino acids is linearly correlated with initial amount of protein. The mixture reacted with fluorescamine at room temperature followed by fluorescence intensity measurement. Fluorescence intensity in 96 well plate was measured by alpha imager and the relative intensities are compared. Meaningful correlation between ABri 34-mer peptide concentration and fluorescence intensity upon the reaction of fluorescamine with cleaved primary amines from ABri peptide was observed (Fig. 2). The aggregation rate of ABri 34-mer peptide after 4 h incubation in 37 8C was determined by fluorescamine and alpha imager assay. Fluorescence intensity rate of filtrate was estimated 28.2% by comparing the standard curve. Therefore, the aggregation rate was determined by 71.8%. Solution in sample was checked by MALDI-TOF MS after 4 h incubation. The monomer spectrum peak was very weak, the intensity is 70–200 (Fig. 3). However, monomer peak intensity was more than 1000–10,000 (data not shown). Although the intensities of MALDI-TOF MS peak do not precisely correlated with the relative concentration, it can be concluded that monomers of ABri peptide remain in low concentration. Therefore, the major component of filtrate after 4 h incubation would be ABri peptide dimer.
3. Result and discussion
4. Conclusions
ABri peptide quantity was estimated by using fluorescamine and alpha imager assay which is optimum system for amyloid
There have been a lot of studies to develop drugs that can inhibit the aggregation of prion protein. The inhibition of pathogenic oligomers of amyloidogenic proteins have been widely studied as a therapeutic option for amyloidogenic diseases [17– 20]. To enhance our knowledge for the discovery of ABri aggregation inhibiting drug, it is critical to quantify protein accurately and aggregation rate. In this study, quantification for ABri aggregation rate at micromolecular level was successfully carried out. This study will contribute to identify ABri aggregation inhibitor and develop new FBD drug.
[(Fig._2)TD$IG]
Acknowledgments This study was supported by the Korea Military Academy Hwarang-dae Research Institute research program. I appreciate to Professor Suh, Junghun of Seoul National University for valuable advice and allowing us to use their research facilities during the course of this research. References Fig. 2. Linear plot for ABri 34-mer peptide concentrations with fluorescence intensities.
[1] S. Mead, M. James-Galton, T. Revesz, R. Doshi, G. Harwood, E. Pan, J. Ghiso, B. Frangione, G. Plant, Brain 123 (2000) 975. [2] G. Plant, T. Revesz, R. Barnard, A. Harding, P. Gautier-Smith, Brain 113 (1990) 721.
K. Jeong et al. / Journal of Industrial and Engineering Chemistry 17 (2011) 427–429 [3] R. Vidal, B. Frangione, A. Rostagno, S. Mead, T. Revesz, G. Plant, J. Ghiso, Nature 399 (1999) 776. [4] J. Ghiso, J. Holton, L. Miravalle, M. Calero, T. Lashley, R. Vidal, H. Houlden, N. Wood, T. Neubert, A. Rostagno, Journal of Biological Chemistry 276 (2001) 43909. [5] S. Kim, R. Wang, D. Gordon, J. Bass, D. Steiner, D. Lynn, G. Thinakaran, S. Meredith, S. Sisodia, Nature Neuroscience 2 (1999) 984. [6] R. Srinivasan, E. Jones, K. Liu, J. Ghiso, R. Marchant, M. Zagorski, Journal of Molecular Biology 333 (2003) 1003. [7] J. Suh, S. Yoo, M. Kim, K. Jeong, J. Ahn, M. Kim, P. Chae, T. Lee, J. Lee, Angewandte Chemie-International Edition 46 (2007) 7064. [8] J. Suh, W. Chei, T. Lee, M. Kim, S. Yoo, K. Jeong, J. Ahn, Journal of Biological Inorganic Chemistry 13 (2008) 693. [9] K. Jeong, W.Y. Chung, Y.-S. Kye, D. Kim, Bioorganic & Medicinal Chemistry 18 (2010) 2598. [10] K. Jeong, J. Suh, W. Chung, Y. Kye, D. Kim, T. Lee, Bulletin of Korean Chemical Society 31 (2010) 1029.
429
[11] T. Florio, D. Paludi, V. Villa, D. Principe, A. Corsaro, E. Millo, G. Damonte, C. D’Arrigo, C. Russo, G. Schettini, Journal of Neurochemistry 85 (2003) 62. [12] G. Bohm, R. Muhr, R. Jaenicke, Protein Engineering Design and Selection 5 (1992) 191. [13] K. Chung, H. Hwang, K. Lee, Journal of Industrial and Engineering Chemistry 16 (2010) 115. [14] K. Chung, H. Hwang, K. Lee, Journal of the Korean Industrial and Engineering Chemistry 20 (2009) 628. [15] M. McCaman, E. Robins, Journal of Laboratory and Clinical Medicine 59 (1962) 885. [16] S. Udenfriend, S. Stein, P. Bohlen, W. Dairman, W. Leimgruber, M. Weigele, Science 178 (1972) 871. [17] B. Austen, K. Paleologou, S. Ali, M. Qureshi, D. Allsop, O. El-Agnaf, Biochemistry 47 (2008) 1984. [18] C. Haass, D. Selkoe, Nature Reviews Molecular Cell Biology 8 (2007) 101. [19] J. Sacchettini, J. Kelly, Nature Reviews Drug Discovery 1 (2002) 267. [20] K. Jeong, M. Kim, W. Chung, Y. Kye, J. Kim, J. Suh, Journal of Industrial and Engineering Chemistry 15 (2009) 342.