A Phos-Tag-Based Fluorescence Quenching System for Activity 2014-08-29¢  acceptor, resulting in FRET

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  • American Journal of Analytical Chemistry, 2014, 5, 796-804 Published Online September 2014 in SciRes. http://www.scirp.org/journal/ajac http://dx.doi.org/10.4236/ajac.2014.512088

    How to 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. http://dx.doi.org/10.4236/ajac.2014.512088

    A Phos-Tag-Based Fluorescence Quenching System for Activity Assay and Inhibitor Screening for Alkaline Phosphatase Emiko Kinoshita-Kikuta1, Hiromasa Kurosaki2, Natsumi Kunisada1, Eiji Kinoshita1*, Tohru Koike1* 1Department of Functional Molecular Science, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan 2Department of Structure-Function Physical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan Email: *kinoeiji@hiroshima-u.ac.jp, *tkoike@hiroshima-u.ac.jp Received 23 June 2014; revised 12 August 2014; accepted 28 August 2014

    Copyright © 2014 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/

    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 fluores- cence 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 di- zinc(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 sys- tem, we succeeded in detecting time- and dose-dependent dephosphorylation of FMN by AP under near-physiological conditions.

    Keywords Fluorescence Resonance Energy Transfer, Enzyme Assays, Enzyme Inhibitors, Phos-Tag, Alkaline Phosphatase

    *Corresponding authors.

    http://www.scirp.org/journal/ajac http://dx.doi.org/10.4236/ajac.2014.512088 http://dx.doi.org/10.4236/ajac.2014.512088 http://www.scirp.org mailto:kinoeiji@hiroshima-u.ac.jp mailto:tkoike@hiroshima-u.ac.jp http://creativecommons.org/licenses/by/4.0/

  • E. Kinoshita-Kikuta et al.


    1. Introduction Alkaline phosphatases (APs, EC are found in many organisms, from bacteria to mammals [1]. The en- zymes catalyze hydrolyses of various phosphoric monoesters and transphosphorylations of phosphorylated spe- cies to other molecules [2]. As one of the most commonly assayed enzymes, serum AP is widely used as a clin- ical indicator for several diseases, including liver dysfunction, several kinds of cancer, and diabetes [3] [4]. Sev- eral assays for the activity of AP have been reported; these generally involve colorimetric, chromatographic, ra- dioactive, or electrochemical approaches. Unlike many of these assays, fluorescence-based methods do not nec- essarily require the use of radioactive or immunoactive labels, and they are therefore attractive as convenient and reliable procedures for the analysis of AP activity [5]-[7].

    We previously reported that the dinuclear metal complex 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2- olato dizinc(II) (Phos-tag) acted as a phosphate-binding tag molecule under near-physiological conditions in aqueous solution at neutral pH values [8]. As a result, a number of original analytical methods that use various Phos-tag derivatives have been developed for research on the phosphoproteome [9]-[12]. In 2009, the phos- phate-capturing ability of an aminocoumarin-attached Phos-tag molecule was utilized in the development of a fluorescence resonance energy transfer (FRET) system for the analysis of the dephosphorylation of a fluoresce- in-labeled phosphopeptide substrate by bovine intestinal AP [13]. The assay is based on the principle that the Phos-tag derivative captures the fluorogenic phosphopeptide in preference to its nonphosphorylated counterpart. The formation of a 1:1 complex between the Phos-tag moiety and the phosphopeptide brings the donor near the acceptor, resulting in FRET with an efficiency that varies from 47% to 86%, depending on the type of peptide sequence. Furthermore, we applied a similar FRET system to an examination of the reverse reaction, phospho- rylation, of a fluorescein-labeled peptide substrate by a certain kinase [14].

    To date, several fluorescence-quenching systems that use a fluorophore-quencher (donor-acceptor) pair have been developed for the analysis of biomolecules such as nucleic acids or peptides [15] [16]. If the donor and ac- ceptor molecules approach one another closely, the fluorescence from the donor group is efficiently reduced by the acceptor. In the case, the acceptor is referred to as a “dark quencher” or “black-hole quencher”. One of the most commonly used quenchers is the 4-([4-(dimethylamino)phenyl]diazenyl)benzoyl group (Dabcyl), which has a strong absorption in the visible region of the spectrum [17]. In this study, we introduce a novel quencher, Dabcyl-labeled Phos-tag (Dabcyl-Phos-tag, λmax = 475 nm), which preferentially captures the fluorogenic AP- substrate riboflavin 5’-phosphate {1-deoxy-1-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)- 5-O-phosphono-D-ribitol}, also known as flavin mononucleotide (FMN) [18], at micromolar concentrations. The emission spectrum of FMN (λem = 525 nm) efficiently overlaps the absorption spectrum of the Dabcyl group.

    As a first practical example of the use of Dabcyl-Phos-tag, we demonstrate the detection of the time-depen- dent dephosphorylation of FMN by AP under near-physiological conditions. Furthermore, dose-dependent inhi- bitions of AP activity by the well-known inhibitors vanadate [19] [20] and (6S)-6-phenyl-2,3,5,6-tetrahydroi- midazo[2,1-b][1,3]thiazole (levamisole) [21] [22] were also examined by using the Phos-tag-based fluorescence quenching system.

    2. Materials and Methods 2.1. Materials 1-[(4-{[4-(Dimethylamino)phenyl]diazenyl}benzoyl)oxy]pyrrolidine-2,5-dione (Dabcyl NHS ester) was pur- chased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Disodium 2,2’,2’’,2’’’-(1,2-ethanediyldiammonio) tetraacetate (disodium EDTA) and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid (Hepes) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan).Sodium orthovanadate (Na3VO4) was purchased from Cal- biochem (La Jolla, CA, USA). Levamisole was purchased from Sigma-Aldrich (St. Louis, MO, USA).FMN monosodium salt (purity >95%) and riboflavin, both of which have the same λmax value of 445 nm (ε = 1.25 × 104 M−1∙cm−1) in H2O [18], were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan): FMN was used after purification (>99%) by column chromatography on ODS silica gel, Cosmosil 140C18-OPN (Na- calai Tesque). Bovine intestinal mucosa AP Type VII-S and bovine kidney AP were purchased from Sigma-Al- drich. One unit of the phosphatase hydrolyzed 1 µmol of 4-nitrophenylphosphate per minute at pH 9.8 and 37˚C.

    All aqueous solutions were prepared by using distilled water. All chemical reagents and solvents were of the highest commercial quality and were used without further purification. TLC was performed on TLC silica gel

  • E. Kinoshita-Kikuta et al.


    plates 60 NH2 F254s (No. 5533; Merck, Darmstadt, Germany) and silica gel column chromatography was per- formed on silica gel NH-DM 1020 (Fuji Silysia Chemical, Ltd.; Kasugai, Japan).

    2.2. Apparatus UV and visible spectra were recorded on a V-630 spectrophotometer (JASCO Corp.; Tokyo, Japan) at 25.0˚C ± 0.1˚C. Fluorescence spectra were obtained with an F-2500 fluorescence spectrophotometer (Hitachi High-Tech- nologies Corp.; Tokyo, Japan) equipped with a 1-cm quartz cell at 25.0˚C ± 0.1˚C. IR spectra were recorded on an FT-710 IR spectrophotometer (Horiba Ltd.; Kyoto, Japan) at room temperature. 1H (500 MHz) and 13C (125 MHz) NMR spectra were recorded on a JEOL LA500 spectrometer with a field-gradient unit (JEOL Ltd.; Tokyo, Japan) at 25˚C. Tetramethylsilane (in CDCl3) was used as an internal reference for the NMR measurements. The 1H and 13C signals were assigned by means of 1D and 2D (1H COSY, HMQC and HMBC) NMR experiments. The pH measurements were conducted with an F-53 pH meter (Horiba Ltd.; Kyoto, Japan) equipped with a combination pH electrode (Horiba-6378) calibrated by using pH standard buffers (pH 4.01 and 6.86) at 25˚C. HPLC was performed by using an HPLC system (JASCO Corp.) consisting of a column oven (CO-2060), a UV detector (UV-2070), a degasser (DG-2080-53), and two pumps (PU-2080 plus). The flow rate was 1.0 ml/min and the column temperature was 40˚C. Purities of FMN and riboflavin were determined by HPLC with a re- verse-phase column (Shiseido CAPCELL PAK C18 UG80, 4.6 × 150 mm; Yokohama, Japan) eluted by a 7:3 mixture of 10 mM phosphate buffer (pH 5.5) and methanol. The retention times for FMN and riboflavin were 2.8 min and 4.2 min, respectively.