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Real-time monitoring of S-adenosyl-l -homocysteine hydrolase using a chemodosimetric fluorescence “turn-on” sensor
Sensors and Actuators B 185 (2013) 663–668
Contents lists available at SciVerse ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Real-time monitoring of S-adenosyl-l-homocysteine hydrolase using a chemodosimetric fluorescence “turn-on” sensor Kyung-Sik Lee a,1 , Seung Hwan Lee b,1 , Jinrok Oh a , Ik-Soo Shin c,∗ , Tai Hyun Park b,∗ ∗ ∗ , Jong-In Hong a,∗∗ a b c
Department of Chemistry, Seoul National University, Seoul 151-747, Republic of Korea School of Chemical and Biological Engineering, Bio-MAX Institute, Seoul National University, Seoul 151-744, Republic of Korea Department of Chemistry, Soongsil University, Seoul 156-743, Republic of Korea
a r t i c l e
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Article history: Received 11 March 2013 Received in revised form 6 May 2013 Accepted 13 May 2013 Available online 25 May 2013 Keywords: S-adenosyl-l-homocysteine hydrolase Chemodosimeter Coumarin Fluorescent assay l-Homocysteine
a b s t r a c t Although various methods have been used to measure the activity of S-adenosyl-l-homocysteine hydrolase (SAHase), most of them are rather complex, expensive, and not suitable for routine analysis. Herein, we describe simple fluorescence monitoring for SAHase activity using a synthetic probe that undergoes chemical binding selectively with l-homocysteine (Hcy), an enzyme metabolite from SAHase catalysis. The ring formation between the probe and Hcy during the enzyme catalysis is quantified by fluorescent emission from the binding adduct, which provides facile, real-time monitoring for the SAHase activity. The chemodosimetric reaction between the probe and Hcy is ∼100-fold faster than that for the enzyme catalysis so that it is successfully applicable in SAHase assay. © 2013 Elsevier B.V. All rights reserved.
1. Introduction S-adenosyl-l-homocysteine hydrolase (SAHase, EC 3.3.1.1) is one of the most highly conserved and ubiquitous enzymes in existence and is present in various organisms, ranging from bacteria to mammals [1–3]. SAHase reversibly hydrolyzes S-adenosyl-lhomocysteine (SAH) into l-homocysteine (Hcy) and adenosine (Ade), and the inhibition of this cellular enzyme results in the intracellular accumulation of SAH. The accumulation of SAH leads to the feedback inhibition of S-adenosyl methionine-dependent methylation reactions (i.e., viral mRNA methylation), which are essential for viral replication [4–6]. Many pathological diseases (e.g., cardiovascular diseases) are related to altered SAHase function [7–9]. The monitoring of SAHase activity has a huge significance for disease diagnosis or for the development of drugs. Since the mechanism of catalysis was firstly elucidated by Palmer and Abeles [10], various methods, such as hydrolysis assays [11], radioactive labeling [12], and liquid chromatography
∗ Corresponding author at: Department of Chemistry, Soongsil University, 369 Sangdo-Ro, Dongjak-Gu, Seoul 156-743, Republic of Korea. Tel.: +82 2 880 4376; fax: +82 2 880 1568. ∗∗ Corresponding author. Tel.: +82 2 874 5902; fax: +82 2 889 1568. ∗ ∗ ∗Corresponding author. Tel: +82 2 880 8020; fax: +82 2 875 9348. E-mail addresses: [email protected] (I.-S. Shin), [email protected] (T.H. Park), [email protected] (J.-I. Hong). 1 These authors contributed equally to this work. 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.05.045
[13], have been employed to measure the enzymatic activity. In hydrolytic approaches, Ade, the enzymatic product of SAHase, is converted into inosine by adenosine deaminase (ADA), and the decrease in absorbance at 265 nm can be traced by spectrophotometric methods [14–22]. Radioactive labeling approaches are derived from hydrolytic methods, but they employ radiolabeled SAH ([8-14 C] SAH) coupled with ADA [23–28]. [8-14 C] inosine, which is the product of the deamination of [8-14 C] Ade formed by the hydrolysis of SAH, is poured into a column, and is eluted with acid into a scintillation vial, after which its radioactivity is measured. For chromatographic determinations, the first step is SAH hydrolysis, which is assayed by incubating SAH with SAHase for several minutes. The reaction is then stopped with perchloric acid to precipitate the protein, after which the Ade in the supernatant is analyzed through high-performance liquid chromatography [29–31]. Although these methods are efficient and accurate for use in SAHase activity assays, they are fastidious, expensive, and time-consuming for use in routine analysis. Herein, we demonstrate a fluorescent assay for SAHase activity using a synthetic probe, 8-formyl-7-hydroxycoumarin (1), which selectively recognizes Hcy resulting in a strong fluorescence. The approach is based on the titration of amino acids that contain a thiol group, which is produced when SAHase hydrolyzes SAH. The amino acid-containing thiol (Hcy), one of its metabolites, reacts with the aldehyde group of the probe 1 to form an adduct that possesses six-membered ring structures, thiazines, which results in the blue fluorescence at 450 nm. The sensitive character of the
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probe 1 to Hcy can facilitate the real-time monitoring of SAHase through a fluorescent “turn-on” manner. The method provides a high-throughput, real-time analysis for SAHase activity, and also proposes a SAHase inhibitor test through a simple experimental procedure. Moreover, the reaction kinetics between probe 1 and Hcy was investigated using a droplet-based microfluidic system. Various conventional methods, such as stopped flow [32] and hydrodynamic focusing [33], have been developed to understand biological and chemical kinetics. These approaches, however, require large sample volumes for the performance of kinetic studies [34]. On the other hand, in droplet-based microfluidic systems, a large number of nanoliter-sized droplets are generated using immiscible phases [35]. Discrete nanoliter reactors can be continuously generated without increasing the device size, and these can be individually transported and analyzed without dispersion [36,37]. The droplet microfluidic devices can be used to monitor the progress of reactions at the desired positions and time scales, using fluorescence microscopy with requiring low sample consumption. Thus, the microfluidic droplet system is one of the most suitable methods for monitoring the fast reaction between the probe 1 and Hcy. 2. Materials and methods 2.1. Synthesis of probe 1 The chemodosimetric probe 1 was synthesized as described in these authors’ previous report [38]. 2.1.1. Acetylation Ac2 O (2 mL) and pyridine (1 drop) were added to a stirred solution of 7-hydroxycoumarin (3.1 g, 19.1 mmol) in 50 mL CH2 Cl2 . The solution was stirred overnight at room temperature and then evaporated under vacuum. The crude products were dissolved in water and then extracted with ethyl acetate. The combined organic layer was washed with brine and then dried with Na2 SO4 . Next, the solvent was removed under reduced pressure, and the residue was purified via column chromatography, using petroleum ether/ethyl acetate (v/v, 2:1, Rf = 0.50) as an eluent to afford 7-acetoxycoumarin as a white solid (3.8 g, 97% yield). 1 H NMR (300 MHz, CDCl ): 2.36 (s, 3H), 6.41 (d, J = 9.6 Hz, 1H), 3 7.07 (d, J = 8.4 Hz, 1H), 7.14 (s, 1H), 7.50 (d, J = 8.4 Hz, 1H), and 7.71 (d, J = 9.6 Hz, 1H). 2.1.2. Formylation 7-Acetoxycoumarin (1.5 g, 7.4 mmol) and hexamethylenetetramine (1.5 g, 10.7 mmol) were added to the stirred solution of trifluoracetic acid (TFA; 10 mL) on ice. The solution was allowed to warm to room temperature and was then refluxed for 8 h. The excess TFA was removed under vacuum, and 30 mL water was added to the remaining solution. The mixture was then warmed at 60 ◦ C while being stirred for another 30 min. Upon cooling on ice, a pale-yellow solid precipitated from the solution. It was collected by filtration and washed several times with water to obtain the desired product, which was a pale-yellow solid. The solid was purified via column chromatography, using dichloromethane/MeOH (20:1 v/v, Rf = 0.80) as an eluent. Evaporation and drying under a vacuum produced the desired product at a yield of 45%. 1 H NMR (300 MHz, CDCl ): 6.34 (d, J = 9.6 Hz, 1H), 6.90 (d, 3 J = 8.8 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 7.67 (d, J = 9.6 Hz, 1H), 10.62 (s, 1H), and 12.23 (s, 1H). 13 C NMR (75 MHz, CDCl ): 108.69, 110.87, 113.42, 114.70, 3 136.01, 143.37, 156.76, 159.10, 165.50, and 192.93. HR-MS (EI+): m/z calculated to be 190.0266 and found to be 190.0265
2.2. SAHase hydrolytic activity assay using the probe 1 The activity of the reaction mixtures (1 mL) at 37 ◦ C containing 100 M SAH, 100 M the probe 1, and various amounts of SAHase (0.0016 g L−1 , 0.00432 g L−1 , and 0.0108 g L−1 ) were measured at 450 nm (maximum for fluorescence emission of the reaction complex between the probe 1 and Hcy), using a Jasco FP-6500 spectrofluorometer. 2.3. Fabrication of a droplet-based microfluidic device The microfluidic device was fabricated based on polydimethylsiloxane (PDMS; Sylgard 184, Dow-Corning, MI, USA), using standard soft lithography and replica-molding techniques. A master mold was made by spin-coating a negative photoresist (SU-8 2075; Microchem, MA, USA) onto a silicon wafer. Through softbaking followed by UV exposure and development, a pattern was formed on the wafer. The pattern was in the form of a channel for droplet generation and monitoring kinetics (300 m wide and 110 m deep). The PDMS prepolymer was mixed with its curing agent at a ratio of 10:1. The mixture was then poured onto the master mold and was baked at 80 ◦ C for 5 h. Next, the cured PDMS was peeled off the master mold and was punched to create reservoirs. After being cleaned with acetone and isopropyl alcohol, it was treated with oxygen plasma and was bound to a glass slide. The channel was 300 m wide and 110 m deep. The system consisted of an inlet reservoir for injecting carrier fluid and a Y-shaped channel for injecting Hcy and the probe 1. To determine the kinetic properties using the droplet system, Hcy (50 mM) and the probe 1 (10 mM) were separately diluted in a 50 mM HEPES buffer. The carrier fluid was prepared as mineral oil that contained 2% span 80. The microfluidic device was operated using precision syringe pumps (Kd Scientific, Holliston, MA). The carrier fluid was introduced using an inlet reservoir at the rate of 0.3 L min−1 , and Hcy and the probe 1 were injected through the Y-shaped channel at 0.8 L min−1 , respectively. The fluorescence intensities of the droplets were observed using a fluorescent microscope (IX71; Olympus, Tokyo, Japan) equipped with a charge-coupled device (CCD) camera. 2.4. Calculation of the reaction kinetics between the probe 1 and Hcy 2.4.1. Pseudo-first-order reaction kinetics of SAHase activity When assuming the concentration of SAH is much less than the SAHase, pseudo-first-order reaction kinetics can be used to determine the kinetic properties of SAH hydrolysis reaction; −
d[SAH] = kobs [SAH] dt
(1)
where the reaction constant, kobs , will be linearly related to the SAHase concentration. Therefore, we can write the pseudo-firstorder reaction of SAHase hydrolysis as:
[SAH] = [SAH]0 e−kobs t or [SAH] = [SAH]0 e−k [SAHase]0 t
(2)
where [SAH]0 is the initial concentration of SAH, [SAHase]0 is the initial concentration of the enzyme SAHase, and k is the actual second-order-reaction rate constant between SAH and SAHase. Using natural log to both sides, we can get: ln[SAH] = ln [SAH]0 − kobs t
(3)
2.4.2. Second-order reaction kinetics between the probe 1 and Hcy A rate constant was obtained from the SigmaPlotTM program using the following equations for regression analysis. Assumed that
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Fig. 1. Chemical structure of probe 1 and its use in fluorescence real-time monitoring for S-adenosyl-l-homocysteine hydrolase activity.
the reaction kinetics followed second-order rate kinetics between Hcy and the probe 1, Eq. (4) was obtained as follows: (the initial concentration of the probe 1 is symbolized as [1]0 , and the initial concentration of Hcy can be expressed as [Hcy]0 = [1]0 + ˛, where ˛ = [Hcy]0 − [1]0 ) [1]t =
˛ · [1]0 · e−˛·k·t
(4)
˛ + [1]0 · {1 − e−˛·k·t }
Second, when the instant concentration of the probe 1 is denoted as [1]t , the reaction product between the probe 1 and Hcy can be expressed as [1]0 − [1]t . To obtain the rate constant k, the timedependent change in fluorescence intensity was derived and fitted as follows: I = · [1]t + ı · ([1]0 − [1]t ) = ı · [1]0 − (ı − ) · [1]t = c − (ı − ) · [1]t = c − ε ·
˛ · [1]0 · e−˛·k·t ˛ + [1]0 · {1 − e−˛·k·t }
,
(5)
where , ı, and ε are arbitrarily defined constants of proportionality. 3. Results and discussion The technique proposed in this paper is based on the titration of amino acids that contain a thiol group, which is produced when SAHase hydrolyzes SAH to yield Hcy. The thiol (Hcy), one of its metabolites, reacts with the aldehyde group of the probe 1 to form an adduct that possesses six-membered ring structures, thiazines (Fig. 1), which generates the blue fluorescence of 1 at approximately 450 nm [38]. As shown in Fig. 2A, 1 exhibited the enhanced fluorescence after the addition of SAHase to its substrate (SAH) solution. This finding demonstrated that 1 could be used to successfully perform real-time monitoring of SAHase, and that the large increase in fluorescence could be perceived by the naked eye (Fig. 1). Hcy was expected to be released from SAH during SAHase hydrolysis, and the fluorescence intensity increased over time because of the presence of the reaction adduct formed from 1 and Hcy. The enzymatic reaction rate gradually increased with the amount of SAHase from 1.6 to 10.8 ng L−1 , as shown in Fig. 2B. The probe 1 was then directly applied for the real-time monitoring of SAHase, which was derived from the erythrocytes of a rabbit. The time-dependent change in the emission intensity indicated the concentration of SAH at an individual point, and from this, the rate constant for SAHase catalysis could be estimated. As shown in
Fig. 2. (A) Time-dependent changes in the fluorescence spectra of probe 1 after the addition of S-adenosyl-l-homocysteine hydrolase (SAHase) at 37 ◦ C ([1]0 = 100 M, [SAH]0 = 100 M, [SAHase] = 10.8 ng L, ex = 365 nm, and em = 450 nm). (B) Timedependent increase in fluorescence intensity at 450 nm with increasing amounts of SAHase (1.6 ng L−1 , 4.3 ng L−1 , and 10.8 ng L−1 ).
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Fig. 4. Real-time measurements of S-adenosyl-l-homocysteine hydrolase (SAHase; 0.0016 g L−1 ) activity in the presence of adenosine ([1] = 100 M, SAH = 1 mM, SAHase = 0.0043 g L−1 , ex = 365 nm, and em = 450 nm).
Fig. 3. (A) Pseudo-first-order rate kinetics of each enzyme reaction. (B) Observed rate constant (kobs ) values depending on the amount of S-adenosyl-l-homocysteine hydrolase.
Fig. 3A, the instantaneous amount of SAH showed logarithmic linearity with the reaction time, which suggested that the hydrolysis of SAH was a pseudo-first-order reaction. The slope of the timedependent curve corresponded to the observed rate constant of the reaction (kobs ), and kobs showed good linearity with the amount of SAHase enzyme added (Fig. 3B). The observed reaction rates, kobs , for the addition of the consecutive amount of SAHase (1.6, 4.3, and 10.8 ng L−1 ) were 2.3 × 10−3 , 4.8 × 10−3 , and 1.1 × 10−2 s−1 , respectively; the values were comparable with other reported values [39]. Furthermore, it was observed that the efficiency of SAHase became attenuated upon the addition of Ade (Fig. 4). As Ade analogs have been previously used as inhibitors of SAHase activity [40], inhibition kinetics were observed for the SAHase assay when an excess amount of Ade was present in the solution. The proposed approach assumed that the reaction rate for the ring formation between the probe 1 and Hcy was so fast that hydrolysis by SAHase could be considered only as an estimation for the enzyme kinetics. Although the pseudo-first-order behavior of SAHase catalysis (Fig. 2B) corroborated this assumption, the reaction kinetics of the ring formation between 1 and Hcy were investigated using a droplet-based microfluidic channel, to confirm it. The design of the proposed droplet microfluidic device is outlined in Fig. 5A. The system consists of an inlet reservoir for injecting a carrier fluid and a Y-shaped channel for Hcy (10, 20, 30, and 40 mM) and 1 (2 mM). To determine the kinetic rate constant
Fig. 5. (A) Real-time measurement of the reaction kinetics of probe 1 when forming the enzyme product (Hcy) based on the microfluidic droplet. Left: schematic design of the microfluidic droplet; right: one-shot fluorescent images of probe 1 (2 mM) with (i) 10 mM, (ii) 20 mM, (iii) 30 mM, and (iv) 40 mM Hcy. (B) Timedependent increase in emission intensity according to various concentrations of Hcy as observed from one-shot fluorescent images.
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using the droplet system, Hcy and 1 were injected through the Y-shaped channel at 0.8 L min−1 whereas the carrier fluid was introduced into the inlet reservoir at 0.3 L min−1 . After the segmentation of the droplets, the concentration gradient profile of the binding product could be preserved in the droplets with high fidelity. Increased fluorescence was obtained from the droplet array owing to the time-dependent reaction process between Hcy and 1. The fluorescence intensities from the droplet were recorded in a one-shot image, using a CCD microscope (Fig. 5B). Assumed that the reaction followed second-order rate kinetics with respect to Hcy and 1, a rate constant (k) of 0.6 mol L−1 s−1 was estimated by fitting the intensity profiles. Given that the estimated value of kobs was approximately 100-fold lower than the value for k, the ring formation of Hcy and 1 occurred almost instantaneously and could be ignored in the SAHase assay. 4. Conclusions The SAHase activity was monitored using a simple syntheticprobe-based fluorescence real-time monitoring system. The synthetic probe 1 undergoes ring formation with Hcy, which is a product of the SAHase catalysis, and strongly fluoresces such that it is clearly visible to the naked eye. The reaction kinetics of SAHase and the inhibition effect of Ade on SAHase were observed using 1. Furthermore, the droplet-based microfluidic system was used to investigate the kinetics of 1 and Hcy. The reaction kinetics between 1 and Hcy followed a fast, second-order reaction. The method showed that the activity of SAHase can be successfully monitored real-time using the simple fluorescent approach. Acknowledgements This work was supported by the Basic Science Research Program, the Pioneer Research Program, and the Bio & Medical Technology Development Program through National Research Foundation of Korea (NRF) grants funded by the Ministry of Education, Science, and Technology (MEST) of South Korea (Grant Nos. 2012-0008314, 2012-0080734, and 2012-050100).
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Biographies
Kyung-Sik Lee obtained B.S. (2003) and M.S. (2005) in chemistry from Andong National University. He received his Ph.D. (2011), and was a postdoctoral fellow at Seoul National University under the supervision of Prof. J.-I. Hong (2011–2012). Currently he is working as a senior research engineer at the Hansol Chemical Co., Ltd. His research interests include the development of precursors for semiconductor manufacturing and the development of self-assembled superstructures based on metal-ligand.
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Seung Hwan Lee received his B.S. degree in chemical engineering at Sungkyunkwan University, Korea, in 2004. He obtained his Ph.D. degree in chemical and biological engineering from Seoul National University, Korea, in 2011. He has been working with professor Tai Hyun Park at Seoul National University as a postdoctoral fellow since 2011. His research area includes microfabrication and development of microfluidic platform for nanobiotechnology.
Jinrok Oh was born in Seoul, Korea in 1988. He obtained B.S. degree in Chemistry at KAIST in 2010 and is currently a Ph.D. candidate at Department of Chemistry, Seoul National University under the supervision of Prof. Jong-In Hong. His research focuses on the design and synthesis of new receptors for biomolecules.
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Ik-Soo Shin was born in Korea in 1975, and obtained Ph.D. degree from Seoul National University (SNU) in 2007. He was a postdoctoral fellow at the University of Texas at Austin, and SNU. Currently he is an assistant professor at Soongsil University, in Korea. His research interests include the design and development of biosensors for biological targets.
Tai Hyun Park is professor at the School of Chemical and Biological Engineering, Seoul National University. He was Director of Bio-MAX Institute, Director of Institute of Bioengineering, and Founding Chair of Engineering Biotechnology Program at Seoul National University. He is currently Executive board member of AFOB (Asian Federation of Biotechnology), Member of the National Academy of Engineering of Korea, Editor and Editorial board member of several journals. His research interests include olfactory biosensor, cellular engineering, and nanobiotechnology.
Jong-In Hong obtained his Ph.D. from Columbia University, and did his postdoctoral work at MIT. He has been a professor at Seoul National University since 1993. He currently serves a vice chairman of Department of Chemistry at SNU. He has published over 70 scientific papers. His research interests include development of optical and/or electrochemical sensors for biologically or clinically important ions and molecules.
Contents lists available at SciVerse ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Real-time monitoring of S-adenosyl-l-homocysteine hydrolase using a chemodosimetric fluorescence “turn-on” sensor Kyung-Sik Lee a,1 , Seung Hwan Lee b,1 , Jinrok Oh a , Ik-Soo Shin c,∗ , Tai Hyun Park b,∗ ∗ ∗ , Jong-In Hong a,∗∗ a b c
Department of Chemistry, Seoul National University, Seoul 151-747, Republic of Korea School of Chemical and Biological Engineering, Bio-MAX Institute, Seoul National University, Seoul 151-744, Republic of Korea Department of Chemistry, Soongsil University, Seoul 156-743, Republic of Korea
a r t i c l e
i n f o
Article history: Received 11 March 2013 Received in revised form 6 May 2013 Accepted 13 May 2013 Available online 25 May 2013 Keywords: S-adenosyl-l-homocysteine hydrolase Chemodosimeter Coumarin Fluorescent assay l-Homocysteine
a b s t r a c t Although various methods have been used to measure the activity of S-adenosyl-l-homocysteine hydrolase (SAHase), most of them are rather complex, expensive, and not suitable for routine analysis. Herein, we describe simple fluorescence monitoring for SAHase activity using a synthetic probe that undergoes chemical binding selectively with l-homocysteine (Hcy), an enzyme metabolite from SAHase catalysis. The ring formation between the probe and Hcy during the enzyme catalysis is quantified by fluorescent emission from the binding adduct, which provides facile, real-time monitoring for the SAHase activity. The chemodosimetric reaction between the probe and Hcy is ∼100-fold faster than that for the enzyme catalysis so that it is successfully applicable in SAHase assay. © 2013 Elsevier B.V. All rights reserved.
1. Introduction S-adenosyl-l-homocysteine hydrolase (SAHase, EC 3.3.1.1) is one of the most highly conserved and ubiquitous enzymes in existence and is present in various organisms, ranging from bacteria to mammals [1–3]. SAHase reversibly hydrolyzes S-adenosyl-lhomocysteine (SAH) into l-homocysteine (Hcy) and adenosine (Ade), and the inhibition of this cellular enzyme results in the intracellular accumulation of SAH. The accumulation of SAH leads to the feedback inhibition of S-adenosyl methionine-dependent methylation reactions (i.e., viral mRNA methylation), which are essential for viral replication [4–6]. Many pathological diseases (e.g., cardiovascular diseases) are related to altered SAHase function [7–9]. The monitoring of SAHase activity has a huge significance for disease diagnosis or for the development of drugs. Since the mechanism of catalysis was firstly elucidated by Palmer and Abeles [10], various methods, such as hydrolysis assays [11], radioactive labeling [12], and liquid chromatography
∗ Corresponding author at: Department of Chemistry, Soongsil University, 369 Sangdo-Ro, Dongjak-Gu, Seoul 156-743, Republic of Korea. Tel.: +82 2 880 4376; fax: +82 2 880 1568. ∗∗ Corresponding author. Tel.: +82 2 874 5902; fax: +82 2 889 1568. ∗ ∗ ∗Corresponding author. Tel: +82 2 880 8020; fax: +82 2 875 9348. E-mail addresses: [email protected] (I.-S. Shin), [email protected] (T.H. Park), [email protected] (J.-I. Hong). 1 These authors contributed equally to this work. 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.05.045
[13], have been employed to measure the enzymatic activity. In hydrolytic approaches, Ade, the enzymatic product of SAHase, is converted into inosine by adenosine deaminase (ADA), and the decrease in absorbance at 265 nm can be traced by spectrophotometric methods [14–22]. Radioactive labeling approaches are derived from hydrolytic methods, but they employ radiolabeled SAH ([8-14 C] SAH) coupled with ADA [23–28]. [8-14 C] inosine, which is the product of the deamination of [8-14 C] Ade formed by the hydrolysis of SAH, is poured into a column, and is eluted with acid into a scintillation vial, after which its radioactivity is measured. For chromatographic determinations, the first step is SAH hydrolysis, which is assayed by incubating SAH with SAHase for several minutes. The reaction is then stopped with perchloric acid to precipitate the protein, after which the Ade in the supernatant is analyzed through high-performance liquid chromatography [29–31]. Although these methods are efficient and accurate for use in SAHase activity assays, they are fastidious, expensive, and time-consuming for use in routine analysis. Herein, we demonstrate a fluorescent assay for SAHase activity using a synthetic probe, 8-formyl-7-hydroxycoumarin (1), which selectively recognizes Hcy resulting in a strong fluorescence. The approach is based on the titration of amino acids that contain a thiol group, which is produced when SAHase hydrolyzes SAH. The amino acid-containing thiol (Hcy), one of its metabolites, reacts with the aldehyde group of the probe 1 to form an adduct that possesses six-membered ring structures, thiazines, which results in the blue fluorescence at 450 nm. The sensitive character of the
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probe 1 to Hcy can facilitate the real-time monitoring of SAHase through a fluorescent “turn-on” manner. The method provides a high-throughput, real-time analysis for SAHase activity, and also proposes a SAHase inhibitor test through a simple experimental procedure. Moreover, the reaction kinetics between probe 1 and Hcy was investigated using a droplet-based microfluidic system. Various conventional methods, such as stopped flow [32] and hydrodynamic focusing [33], have been developed to understand biological and chemical kinetics. These approaches, however, require large sample volumes for the performance of kinetic studies [34]. On the other hand, in droplet-based microfluidic systems, a large number of nanoliter-sized droplets are generated using immiscible phases [35]. Discrete nanoliter reactors can be continuously generated without increasing the device size, and these can be individually transported and analyzed without dispersion [36,37]. The droplet microfluidic devices can be used to monitor the progress of reactions at the desired positions and time scales, using fluorescence microscopy with requiring low sample consumption. Thus, the microfluidic droplet system is one of the most suitable methods for monitoring the fast reaction between the probe 1 and Hcy. 2. Materials and methods 2.1. Synthesis of probe 1 The chemodosimetric probe 1 was synthesized as described in these authors’ previous report [38]. 2.1.1. Acetylation Ac2 O (2 mL) and pyridine (1 drop) were added to a stirred solution of 7-hydroxycoumarin (3.1 g, 19.1 mmol) in 50 mL CH2 Cl2 . The solution was stirred overnight at room temperature and then evaporated under vacuum. The crude products were dissolved in water and then extracted with ethyl acetate. The combined organic layer was washed with brine and then dried with Na2 SO4 . Next, the solvent was removed under reduced pressure, and the residue was purified via column chromatography, using petroleum ether/ethyl acetate (v/v, 2:1, Rf = 0.50) as an eluent to afford 7-acetoxycoumarin as a white solid (3.8 g, 97% yield). 1 H NMR (300 MHz, CDCl ): 2.36 (s, 3H), 6.41 (d, J = 9.6 Hz, 1H), 3 7.07 (d, J = 8.4 Hz, 1H), 7.14 (s, 1H), 7.50 (d, J = 8.4 Hz, 1H), and 7.71 (d, J = 9.6 Hz, 1H). 2.1.2. Formylation 7-Acetoxycoumarin (1.5 g, 7.4 mmol) and hexamethylenetetramine (1.5 g, 10.7 mmol) were added to the stirred solution of trifluoracetic acid (TFA; 10 mL) on ice. The solution was allowed to warm to room temperature and was then refluxed for 8 h. The excess TFA was removed under vacuum, and 30 mL water was added to the remaining solution. The mixture was then warmed at 60 ◦ C while being stirred for another 30 min. Upon cooling on ice, a pale-yellow solid precipitated from the solution. It was collected by filtration and washed several times with water to obtain the desired product, which was a pale-yellow solid. The solid was purified via column chromatography, using dichloromethane/MeOH (20:1 v/v, Rf = 0.80) as an eluent. Evaporation and drying under a vacuum produced the desired product at a yield of 45%. 1 H NMR (300 MHz, CDCl ): 6.34 (d, J = 9.6 Hz, 1H), 6.90 (d, 3 J = 8.8 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 7.67 (d, J = 9.6 Hz, 1H), 10.62 (s, 1H), and 12.23 (s, 1H). 13 C NMR (75 MHz, CDCl ): 108.69, 110.87, 113.42, 114.70, 3 136.01, 143.37, 156.76, 159.10, 165.50, and 192.93. HR-MS (EI+): m/z calculated to be 190.0266 and found to be 190.0265
2.2. SAHase hydrolytic activity assay using the probe 1 The activity of the reaction mixtures (1 mL) at 37 ◦ C containing 100 M SAH, 100 M the probe 1, and various amounts of SAHase (0.0016 g L−1 , 0.00432 g L−1 , and 0.0108 g L−1 ) were measured at 450 nm (maximum for fluorescence emission of the reaction complex between the probe 1 and Hcy), using a Jasco FP-6500 spectrofluorometer. 2.3. Fabrication of a droplet-based microfluidic device The microfluidic device was fabricated based on polydimethylsiloxane (PDMS; Sylgard 184, Dow-Corning, MI, USA), using standard soft lithography and replica-molding techniques. A master mold was made by spin-coating a negative photoresist (SU-8 2075; Microchem, MA, USA) onto a silicon wafer. Through softbaking followed by UV exposure and development, a pattern was formed on the wafer. The pattern was in the form of a channel for droplet generation and monitoring kinetics (300 m wide and 110 m deep). The PDMS prepolymer was mixed with its curing agent at a ratio of 10:1. The mixture was then poured onto the master mold and was baked at 80 ◦ C for 5 h. Next, the cured PDMS was peeled off the master mold and was punched to create reservoirs. After being cleaned with acetone and isopropyl alcohol, it was treated with oxygen plasma and was bound to a glass slide. The channel was 300 m wide and 110 m deep. The system consisted of an inlet reservoir for injecting carrier fluid and a Y-shaped channel for injecting Hcy and the probe 1. To determine the kinetic properties using the droplet system, Hcy (50 mM) and the probe 1 (10 mM) were separately diluted in a 50 mM HEPES buffer. The carrier fluid was prepared as mineral oil that contained 2% span 80. The microfluidic device was operated using precision syringe pumps (Kd Scientific, Holliston, MA). The carrier fluid was introduced using an inlet reservoir at the rate of 0.3 L min−1 , and Hcy and the probe 1 were injected through the Y-shaped channel at 0.8 L min−1 , respectively. The fluorescence intensities of the droplets were observed using a fluorescent microscope (IX71; Olympus, Tokyo, Japan) equipped with a charge-coupled device (CCD) camera. 2.4. Calculation of the reaction kinetics between the probe 1 and Hcy 2.4.1. Pseudo-first-order reaction kinetics of SAHase activity When assuming the concentration of SAH is much less than the SAHase, pseudo-first-order reaction kinetics can be used to determine the kinetic properties of SAH hydrolysis reaction; −
d[SAH] = kobs [SAH] dt
(1)
where the reaction constant, kobs , will be linearly related to the SAHase concentration. Therefore, we can write the pseudo-firstorder reaction of SAHase hydrolysis as:
[SAH] = [SAH]0 e−kobs t or [SAH] = [SAH]0 e−k [SAHase]0 t
(2)
where [SAH]0 is the initial concentration of SAH, [SAHase]0 is the initial concentration of the enzyme SAHase, and k is the actual second-order-reaction rate constant between SAH and SAHase. Using natural log to both sides, we can get: ln[SAH] = ln [SAH]0 − kobs t
(3)
2.4.2. Second-order reaction kinetics between the probe 1 and Hcy A rate constant was obtained from the SigmaPlotTM program using the following equations for regression analysis. Assumed that
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Fig. 1. Chemical structure of probe 1 and its use in fluorescence real-time monitoring for S-adenosyl-l-homocysteine hydrolase activity.
the reaction kinetics followed second-order rate kinetics between Hcy and the probe 1, Eq. (4) was obtained as follows: (the initial concentration of the probe 1 is symbolized as [1]0 , and the initial concentration of Hcy can be expressed as [Hcy]0 = [1]0 + ˛, where ˛ = [Hcy]0 − [1]0 ) [1]t =
˛ · [1]0 · e−˛·k·t
(4)
˛ + [1]0 · {1 − e−˛·k·t }
Second, when the instant concentration of the probe 1 is denoted as [1]t , the reaction product between the probe 1 and Hcy can be expressed as [1]0 − [1]t . To obtain the rate constant k, the timedependent change in fluorescence intensity was derived and fitted as follows: I = · [1]t + ı · ([1]0 − [1]t ) = ı · [1]0 − (ı − ) · [1]t = c − (ı − ) · [1]t = c − ε ·
˛ · [1]0 · e−˛·k·t ˛ + [1]0 · {1 − e−˛·k·t }
,
(5)
where , ı, and ε are arbitrarily defined constants of proportionality. 3. Results and discussion The technique proposed in this paper is based on the titration of amino acids that contain a thiol group, which is produced when SAHase hydrolyzes SAH to yield Hcy. The thiol (Hcy), one of its metabolites, reacts with the aldehyde group of the probe 1 to form an adduct that possesses six-membered ring structures, thiazines (Fig. 1), which generates the blue fluorescence of 1 at approximately 450 nm [38]. As shown in Fig. 2A, 1 exhibited the enhanced fluorescence after the addition of SAHase to its substrate (SAH) solution. This finding demonstrated that 1 could be used to successfully perform real-time monitoring of SAHase, and that the large increase in fluorescence could be perceived by the naked eye (Fig. 1). Hcy was expected to be released from SAH during SAHase hydrolysis, and the fluorescence intensity increased over time because of the presence of the reaction adduct formed from 1 and Hcy. The enzymatic reaction rate gradually increased with the amount of SAHase from 1.6 to 10.8 ng L−1 , as shown in Fig. 2B. The probe 1 was then directly applied for the real-time monitoring of SAHase, which was derived from the erythrocytes of a rabbit. The time-dependent change in the emission intensity indicated the concentration of SAH at an individual point, and from this, the rate constant for SAHase catalysis could be estimated. As shown in
Fig. 2. (A) Time-dependent changes in the fluorescence spectra of probe 1 after the addition of S-adenosyl-l-homocysteine hydrolase (SAHase) at 37 ◦ C ([1]0 = 100 M, [SAH]0 = 100 M, [SAHase] = 10.8 ng L, ex = 365 nm, and em = 450 nm). (B) Timedependent increase in fluorescence intensity at 450 nm with increasing amounts of SAHase (1.6 ng L−1 , 4.3 ng L−1 , and 10.8 ng L−1 ).
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Fig. 4. Real-time measurements of S-adenosyl-l-homocysteine hydrolase (SAHase; 0.0016 g L−1 ) activity in the presence of adenosine ([1] = 100 M, SAH = 1 mM, SAHase = 0.0043 g L−1 , ex = 365 nm, and em = 450 nm).
Fig. 3. (A) Pseudo-first-order rate kinetics of each enzyme reaction. (B) Observed rate constant (kobs ) values depending on the amount of S-adenosyl-l-homocysteine hydrolase.
Fig. 3A, the instantaneous amount of SAH showed logarithmic linearity with the reaction time, which suggested that the hydrolysis of SAH was a pseudo-first-order reaction. The slope of the timedependent curve corresponded to the observed rate constant of the reaction (kobs ), and kobs showed good linearity with the amount of SAHase enzyme added (Fig. 3B). The observed reaction rates, kobs , for the addition of the consecutive amount of SAHase (1.6, 4.3, and 10.8 ng L−1 ) were 2.3 × 10−3 , 4.8 × 10−3 , and 1.1 × 10−2 s−1 , respectively; the values were comparable with other reported values [39]. Furthermore, it was observed that the efficiency of SAHase became attenuated upon the addition of Ade (Fig. 4). As Ade analogs have been previously used as inhibitors of SAHase activity [40], inhibition kinetics were observed for the SAHase assay when an excess amount of Ade was present in the solution. The proposed approach assumed that the reaction rate for the ring formation between the probe 1 and Hcy was so fast that hydrolysis by SAHase could be considered only as an estimation for the enzyme kinetics. Although the pseudo-first-order behavior of SAHase catalysis (Fig. 2B) corroborated this assumption, the reaction kinetics of the ring formation between 1 and Hcy were investigated using a droplet-based microfluidic channel, to confirm it. The design of the proposed droplet microfluidic device is outlined in Fig. 5A. The system consists of an inlet reservoir for injecting a carrier fluid and a Y-shaped channel for Hcy (10, 20, 30, and 40 mM) and 1 (2 mM). To determine the kinetic rate constant
Fig. 5. (A) Real-time measurement of the reaction kinetics of probe 1 when forming the enzyme product (Hcy) based on the microfluidic droplet. Left: schematic design of the microfluidic droplet; right: one-shot fluorescent images of probe 1 (2 mM) with (i) 10 mM, (ii) 20 mM, (iii) 30 mM, and (iv) 40 mM Hcy. (B) Timedependent increase in emission intensity according to various concentrations of Hcy as observed from one-shot fluorescent images.
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using the droplet system, Hcy and 1 were injected through the Y-shaped channel at 0.8 L min−1 whereas the carrier fluid was introduced into the inlet reservoir at 0.3 L min−1 . After the segmentation of the droplets, the concentration gradient profile of the binding product could be preserved in the droplets with high fidelity. Increased fluorescence was obtained from the droplet array owing to the time-dependent reaction process between Hcy and 1. The fluorescence intensities from the droplet were recorded in a one-shot image, using a CCD microscope (Fig. 5B). Assumed that the reaction followed second-order rate kinetics with respect to Hcy and 1, a rate constant (k) of 0.6 mol L−1 s−1 was estimated by fitting the intensity profiles. Given that the estimated value of kobs was approximately 100-fold lower than the value for k, the ring formation of Hcy and 1 occurred almost instantaneously and could be ignored in the SAHase assay. 4. Conclusions The SAHase activity was monitored using a simple syntheticprobe-based fluorescence real-time monitoring system. The synthetic probe 1 undergoes ring formation with Hcy, which is a product of the SAHase catalysis, and strongly fluoresces such that it is clearly visible to the naked eye. The reaction kinetics of SAHase and the inhibition effect of Ade on SAHase were observed using 1. Furthermore, the droplet-based microfluidic system was used to investigate the kinetics of 1 and Hcy. The reaction kinetics between 1 and Hcy followed a fast, second-order reaction. The method showed that the activity of SAHase can be successfully monitored real-time using the simple fluorescent approach. Acknowledgements This work was supported by the Basic Science Research Program, the Pioneer Research Program, and the Bio & Medical Technology Development Program through National Research Foundation of Korea (NRF) grants funded by the Ministry of Education, Science, and Technology (MEST) of South Korea (Grant Nos. 2012-0008314, 2012-0080734, and 2012-050100).
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Biographies
Kyung-Sik Lee obtained B.S. (2003) and M.S. (2005) in chemistry from Andong National University. He received his Ph.D. (2011), and was a postdoctoral fellow at Seoul National University under the supervision of Prof. J.-I. Hong (2011–2012). Currently he is working as a senior research engineer at the Hansol Chemical Co., Ltd. His research interests include the development of precursors for semiconductor manufacturing and the development of self-assembled superstructures based on metal-ligand.
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Seung Hwan Lee received his B.S. degree in chemical engineering at Sungkyunkwan University, Korea, in 2004. He obtained his Ph.D. degree in chemical and biological engineering from Seoul National University, Korea, in 2011. He has been working with professor Tai Hyun Park at Seoul National University as a postdoctoral fellow since 2011. His research area includes microfabrication and development of microfluidic platform for nanobiotechnology.
Jinrok Oh was born in Seoul, Korea in 1988. He obtained B.S. degree in Chemistry at KAIST in 2010 and is currently a Ph.D. candidate at Department of Chemistry, Seoul National University under the supervision of Prof. Jong-In Hong. His research focuses on the design and synthesis of new receptors for biomolecules.
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Ik-Soo Shin was born in Korea in 1975, and obtained Ph.D. degree from Seoul National University (SNU) in 2007. He was a postdoctoral fellow at the University of Texas at Austin, and SNU. Currently he is an assistant professor at Soongsil University, in Korea. His research interests include the design and development of biosensors for biological targets.
Tai Hyun Park is professor at the School of Chemical and Biological Engineering, Seoul National University. He was Director of Bio-MAX Institute, Director of Institute of Bioengineering, and Founding Chair of Engineering Biotechnology Program at Seoul National University. He is currently Executive board member of AFOB (Asian Federation of Biotechnology), Member of the National Academy of Engineering of Korea, Editor and Editorial board member of several journals. His research interests include olfactory biosensor, cellular engineering, and nanobiotechnology.
Jong-In Hong obtained his Ph.D. from Columbia University, and did his postdoctoral work at MIT. He has been a professor at Seoul National University since 1993. He currently serves a vice chairman of Department of Chemistry at SNU. He has published over 70 scientific papers. His research interests include development of optical and/or electrochemical sensors for biologically or clinically important ions and molecules.