AJAC  Vol.5 No.12 , September 2014
A Phos-Tag-Based Fluorescence Quenching System for Activity Assay and Inhibitor Screening for Alkaline Phosphatase
ABSTRACT
Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules. Here we introduce a novel FRET-based fluorescence quenching system for assaying the activity of alkaline phosphatase (AP) by using a phos-phate-binding tag molecule, Phos-tag {1,3-bis[bis(pyridine-2-ylmethyl)amino]propan-2-olato dizinc(II) complex}, attached to a nonfluorescent 4-{[4-(dimethylamino)phenyl]diazenyl}benzoyl (Dabcyl: λmax 475 nm) dye group. The fluorogenic biomolecule riboflavin 5’-phosphate (FMN: λem 525 nm) was used as an AP substrate. The Dabcyl-labeled Phos-tag specifically captured FMN to form a stable 1:1 complex, resulting in efficient fluorescence quenching. The quenching efficiency was more than 95% for a mixture of 12 μM FMN and 13.5 μM Dabcyl-labeled Phos-tag in aqueous solution at pH 7.4 and 25°C. When FMN was dephosphorylated with AP, riboflavin was released into the solution and fluorescence from the flavin moiety appeared. By using this quenching system, we succeeded in detecting time- and dose-dependent dephosphorylation of FMN by AP under near-physiological conditions.

Cite this paper
Kinoshita-Kikuta, E. , Kurosaki, H. , Kunisada, N. , Kinoshita, E. and Koike, T. (2014) A Phos-Tag-Based Fluorescence Quenching System for Activity Assay and Inhibitor Screening for Alkaline Phosphatase. American Journal of Analytical Chemistry, 5, 796-804. doi: 10.4236/ajac.2014.512088.
References
[1]   Coleman, J.E. (1992) Structure and Mechanism of Alkaline Phosphatase. Annual Review of Biophysics and Biomolecular Structure, 21, 441-483.
http://dx.doi.org/10.1146/annurev.bb.21.060192.002301

[2]   Millán, J.L. (2006) Alkaline Phosphatases: Structure, Substrate Specificity and Functional Relatedness to Other Members of a Large Superfamily of Enzymes. Purinergic Signaling, 2, 335-341.
http://dx.doi.org/10.1007/s11302-005-5435-6

[3]   Stinghen, S.T., Moura, J.F., Zancanella, P., Rodrigues, G.A., Pianovski, M.A., Lalli, E., Arnold, D.L., Minozzo, J.C., Callefe, L.G., Ribeiro, R.C. and Figueiredo, B.C. (2006) Specific Immunoassays for Placental Alkaline Phosphatase as a Tumor Marker. Journal of Biomedicine and Biotechnology, 2006, Article ID: 56087.
http://dx.doi.org/10.1155/JBB/2006/56087

[4]   Peake, M.J., Pejakovic, M. and White, G.H. (1988) Quantitative Method for Determining Serum Alkaline Phosphatase Isoenzyme Activity: Estimation of Intestinal Component. Journal of Clinical Pathology, 41, 202-206.
http://dx.doi.org/10.1136/jcp.41.2.202

[5]   Schrenkhammer, P., Rosnizeck, I.C., Duerkop, A., Wolfbeis, O.S. and Schaferling, M. (2008) Time-Resolved Fluorescence-Based Assay for the Determination of Alkaline Phosphatase Activity and Application to the Screening of Its Inhibitors. Journal of Biomolecular Screening, 13, 9-16.
http://dx.doi.org/10.1177/1087057107312031

[6]   Liu, Y. and Schanze, K.S. (2008) Conjugated Polyelectrolyte-Based Real-Time Fluorescence Assay for Alkaline Phosphatase with Pyrophosphate as Substrate. Analytical Chemistry, 80, 8605-8612.
http://dx.doi.org/10.1021/ac801508y

[7]   Gu, X., Zhang, G., Wang, Z., Liu, W., Xiao, L. and Zhang, D. (2013) A New Fluorometric Turn-On Assay for Alkaline Phosphatase and Inhibitor Screening Based on Aggregation and Deaggregation of Tetraphenylethylene Molecules. Analyst, 138, 2427-2431.
http://dx.doi.org/10.1039/c3an36784c

[8]   Kinoshita, E., Takahashi, M., Takeda, H., Shiro, M. and Koike, T. (2004) Recognition of Phosphate Monoester Dianion by an Alkoxide-Bridged Dinuclear Zinc(II) Complex. Dalton Transactions, 1189-1193.
http://dx.doi.org/10.1039/b400269e

[9]   Kinoshita, E., Kinoshita-Kikuta, E., Takiyama, K. and Koike, T. (2006) Phosphate-Binding Tag, a New Tool to Visualize Phosphorylated Proteins. Molecular and Cellular Proteomics, 5, 749-757.
http://dx.doi.org/10.1074/mcp.T500024-MCP200

[10]   Kinoshita-Kikuta, E., Aoki, Y., Kinoshita, E. and Koike, T. (2007) Label-Free Kinase Profiling Using Phosphate Affinity Polyacrylamide Gel Electrophoresis. Molecular and Cellular Proteomics, 6, 356-366.
http://dx.doi.org/10.1074/mcp.T600044-MCP200

[11]   Kinoshita, E. and Kinoshita-Kikuta, E. (2011) Improved Phos-Tag SDS-PAGE under Neutral pH Conditions for Advanced Protein Phosphorylation Profiling. Proteomics, 11, 319-323.
http://dx.doi.org/10.1002/pmic.201000472

[12]   Tsunehiro, M., Meki, Y., Matsuoka, K., Kinoshita-Kikuta, E., Kinoshita, E. and Koike, T. (2013) A Phos-Tag-Based Magnetic-Bead Method for Rapid and Selective Separation of Phosphorylated Biomolecules. Journal of Chromatography B, 925, 86-94.
http://dx.doi.org/10.1016/j.jchromb.2013.02.039

[13]   Takiyama, K., Kinoshita, E., Kinoshita-Kikuta, E., Fujioka, Y., Kubo, Y. and Koike T. (2009) A Phos-Tag-Based Fluorescence Resonance Energy Transfer System for the Analysis of the Dephosphorylation of Phosphopeptides. Analytical Biochemistry, 388, 235-241.
http://dx.doi.org/10.1016/j.ab.2009.02.039

[14]   Somura, M., Takiyama, K., Kinoshita-Kikuta, E., Kinoshita, E. and Koike, T. (2011) A Phos-Tag-Based Fluorescence Energy Transfer System for the Analysis of the Kinase Reaction of a Substrate Peptide. Analytical Methods, 3, 1303-1309.
http://dx.doi.org/10.1039/C1AY05016H

[15]   Sapsford, K.E., Berti, L. and Medintz, I.L. (2006) Materials for Fluorescence Resonance Energy Transfer Analysis: Beyond Traditional Donor—Acceptor Combinations. Angewandte Chemie International Edition, 45, 4562-4588.
http://dx.doi.org/10.1002/anie.200503873

[16]   Kolpashchikov, D.M. (2012) An Elegant Biosensor Molecular Beacon Probe: Challenges and Recent Solutions. Scientifica, 2012, Article ID: 928783.
http://dx.doi.org/10.6064/2012/928783

[17]   Johansson, M.K. (2006) Choosing Reporter—Quencher Pairs for Efficient Quenching through Formation of Intramolecular Dimers. Methods in Molecular Biology, 335, 17-29.
http://dx.doi.org/10.1385/1-59745-069-3:17

[18]   Nielsen, P., Rauschenbach, P. and Bacher, A. (1983) Phosphates of Riboflavin and Riboflavin Analogs: A Reinvestigation by High-Performance Liquid Chromatography. Analytical Biochemistry, 130, 359-368.
http://dx.doi.org/10.1016/0003-2697(83)90600-0

[19]   Lopez, V., Stevens, T. and Lindquist, R.N. (1976) Vanadium Ion Inhibition of Alkaline Phosphatase-Catalyzed Phosphate Ester Hydrolysis. Archives of Biochemistry and Biophysics, 175, 31-38.
http://dx.doi.org/10.1016/0003-9861(76)90482-3

[20]   Seargeant, L.E. and Stinson, R.A. (1979) Inhibition of Human Alkaline Phosphatase by Vanadate. Biochemical Journal, 181, 247-250.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1161148/

[21]   Van Belle, H. (1976) Alkaline Phosphatase. I. Kinetics and Inhibition by Levamisole of Purified Isoenzymes from Humans. Clinical Chemistry, 22, 972-976.
http://www.clinchem.org/content/22/7/972.abstract

[22]   Kozlenkov, A., Le Du, M.H., Cuniasse, P., Ny, T., Hoylaerts, M.F. and Millán, J.L. (2004) Residues Determining the Binding Specificity of Uncompetitive Inhibitors to Tissue-Nonspecific Alkaline Phosphatase. Journal of Bone and Mineral Research, 19, 1862-1871.
http://dx.doi.org/10.1359/JBMR.040608

[23]   Wu, P. and Brand, L. (1994) Resonance Energy Transfer: Methods and Applications. Analytical Biochemistry, 218, 1-13.
http://dx.doi.org/10.1006/abio.1994.1134

 
 
Top