Assays for Methionine γ-Lyase and - American Chemical Society

Letter pubs.acs.org/ac

Assays for Methionine γ‑Lyase and S‑Adenosyl‑L‑homocysteine Hydrolase Based on Enzymatic Formation of CdS Quantum Dots in Situ Laura Saa,† José M. Mato,‡ and Valeri Pavlov*,† †

CIC biomaGUNE, Parque Tecnologico de San Sebastian, Paseo Miramon 182, 20009, San Sebastian, Spain CIC bioGUNE, CIBERehd, Parque Tecnologico de Bizkaia, Ed. 801 A, 48160, Derio, Spain

‡

S Supporting Information *

ABSTRACT: S-Adenosyl-L-homocysteine hydrolase (AHCY) hydrolyzes its substrate S-adenosyl-L-homocysteine (AdoHcy) to L-homocysteine (Hcy). Methionine γ-lyase (MGL) catalyzes the decomposition of Hcy to hydrogen sulfide which forms fluorescent CdS nanoparticles in the presence of Cd(NO3)2. On the basis of these enzymatic reactions, two new simple and robust fluorogenic enzymatic assays for MGL and AHCY were developed and applied to detection of AHCY inhibitors.

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performance of the analytical assays based on enzymatic signal amplification and the detection of the resulting metal NPs is limited by the sensitivity of UV−vis spectroscopy used to quantify the readout signal. An amplification method employing the enzymatic generation of fluorescent QDs can take advantage of extreme sensitivity of fluorescence spectroscopy. The majority of conventional assays employ presynthesized semiconductor QDs as labels modified with recognition molecules to detect affinity interactions or as donor/quencher fluorescence resonance energy transfer (FRET) pairs to probe enzymatic activities. These systems suffer from high background signals due to unavoidable adsorption of decorated QDs on surfaces even in the absence of analytes or insufficient quenching of a donor couple.14 On another hand, the broad application of organic fluorogenic enzymatic substrates in enzymatic assays is limited by their high costs and low stability. We report two novel sensitive fluorogenic assays for enzymatic activity of S-adenosyl-L-homocysteine hydrolase (AHCY) and methionine γ-lyase (MGL) based on generation of CdS QDs in situ using natural substrates. MGL is responsible for the α,β γ-elimination and γ,β-replacement in analogues of Lmethionione such as L-homocysteine (Hcy), through degradation to keto acids, ammonia, and H2S in anaerobic bacteria and parasitic protozoa.15 The implication of MGL in the pathogenicity of periodontal bacterium Porphyromonas gingivalis was reported. The inhibitors of MGL may be useful to prevent oral malodor and periodontal diseases.16 On another hand, MGL helps to inhibit the growth of mouse and human

he purpose of biosensing is the detection of analytes with the help of biological recognition elements such as antibodies, DNA, enzymes, and oligosaccharides. Usually, a recognition event requires amplification and transduction to yield a signal detectable by a number of physical techniques including electrochemistry, UV−vis and fluorescence spectroscopy, Raman spectroscopy, and so on. Optical and electrical sensing of biorecognition events frequently rely on metal and semiconductor nanoparticles (NPs).1 Gold and silver NPs have very significant adsorption in UV−vis spectra; they can be conveniently modified with thiol containing molecules and employed as labels in analysis of different targets. The use of metal NPs tethered to oligonucleotides in assays for quantification of DNA was pioneered by Chad Mirkin.2 NPs decorated with DNA aptamers3 were utilized for detection of thrombin.4 NPs can be formed by semiconductor materials too. Their photoexcitation results in formation of electron/hole couples which yield fluorescent emission of light upon recombination. The quantum effects govern the optical properties of semiconductor NPs; therefore, they are called quantum dots (QDs) in the literature.5 QDs demonstrate reduced photobleaching, higher quantum yield, and higher molar extinction coefficients in comparison with organic fluorophores. QDs decorated with peptide ligands, antibodies, DNA, and small molecules6 find application as biological imaging agents.7 Their application in bioanalytical assays was also demonstrated. For instance, QDs modified with antibodies were employed in affinity assays.8 Some examples of enzymatic generation of metal NPs catalyzed by alcohol dehydrogenase,9,10 glucose oxidase,11 and acetylcholine esterase12,13 were reported in the literature. The © 2012 American Chemical Society

Received: September 24, 2012 Accepted: October 22, 2012 Published: October 22, 2012 8961

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Scheme 1. Reaction Process for Quantification of Enzymatic Activities of MGL and AHCY

methionine γ-lyase in citrate-phosphate buffer (12 mM, pH 6.4) for 20 min at 37 °C. After that, Cd(NO3)2 (4 μL, 0.75 M) was added to the samples (200 μL). The emission spectra of the resulting suspensions were recorded after 10 min at λexc = 330 nm. S-(5′-Adenosyl)-L-homocysteine Hydrolase Activity Assay. Varying amounts of S-(5′-adenosyl)-L-homocysteine were incubated with different amounts of S-(5′-adenosyl)-Lhomocysteine hydrolase in the presence of saturating concentration of L-methionine γ-lyase in citrate-phosphate buffer (25 mM, pH 6.4) for 20 min at 37 °C. For inhibition assays with D-eritadenine, different concentrations of inhibitor were added to the mixture. After incubation, Cd(NO3)2 (2 μL, 0.75 M) was added to the samples (100 μL). The emission spectra of the resulting suspensions were recorded after 10 min at λexc = 330 nm.

tumors in rodents and prolongs their survival.17 Two previously reported protocols of MGL assay rely on chromogenic detection of the α-keto acids, generated by this enzyme, using 3-methyl-2-benzothiazoline hydrazone to give the azine derivative.18,19 These assays are extensively employed in biomedical research.20−24 This chromogenic reagent is a toxic mutagen;25 hence, the advent of alternative less dangerous MGL assays is desirable. AHCY participates in the activated methyl cycle catalyzing the reversible hydration of S-adenosyl- L -homocysteine (AdoHcy) to Hcy and adenosine (Ado) in mammals.26 The inhibitors of AHCY are immunosuppressive and antiinflammatory agents.27 They also induce growth inhibition, apoptosis in breast cancer cells.28 Enzymatic assays for AHCY are carried out by incubation of AHCY in the presence of AdoHcy followed by detection of the reaction products with high-pressure liquid chromatography (HPLC)29 or capillary electrophoresis.30 These methods are costly and timeconsuming. The spectrophotometric assay for AHCY recommended by Sigma-Aldrich is inconvenient because it is based on complicated transduction cascade employing three additional expensive enzymes, adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase leading to formation of uric acid followed by UV−vis spectroscopy. In this study we report the first fluorogenic, affordable, robust, and simple assay for MGL and AHCY activities by enzymatic generation of fluorescent semiconductor NPs using natural enzymatic substrates. AdoHcy is enzymatically hydrolyzed to Ado and Hcy, and the latter is decomposed by MGL to yield hydrogen sulfide. In the presence of Cd2+ ions, H2S forms fluorescent nanocrystals of CdS. Our approach does not require any expensive fluorogenic enzymatic substrates and can be performed in 96-well-plates to facilitate massive screening of AHCY inhibitors.

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RESULTS AND DISCUSSION The combination of two enzymatic assays developed by us is represented in Scheme 1. The hydrolysis of the substrate AdoHcy is catalyzed by the enzyme AHCY to give Ado and Hcy. MGL promotes the hydrolysis of Hcy to 2-oxobutanoate, NH3, and hydrogen sulfide. The latter dissociates to yield S2− anions which readily interact with Cd2+ forming fluorescent CdS nanocrystals. First, the performance of two coupled reactions leading to generation of CdS NPs was studied: MGL

L‐homocysteine ⎯⎯⎯⎯→ H 2S + NH3 + 2‐oxobutanoate

H 2S + Cd2 + → 2H+ + CdS

In Figure 1, the solid curve demonstrates the UV−vis absorption spectrum of the resulting CdS NPs. The absorption gradually increases starting from 500 nm and reaches a peak at 300 nm. The absorption spectrum also displays a small shoulder at about 250 nm originating from 1Sh−1Se excitonic transition (transition between the electron 1s state and the hole 1s state31) typical for semiconductor NPs having a diameter around 2 nm as described in the work by Peng et al.32 One can observe that the emission spectrum of CdS nanocrystals produced via the enzymatic reaction has a single well-defined peak with a maximum at 500 nm suggesting “focusing” of the size distribution which yields nearly monodisperse particles. High-resolution transmission electron microscopy (TEM) discovered the presence of spheroidal monodispersed CdS nanocrystals with the median diameter of 2.5 ± 0.4 nm in the

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EXPERIMENTAL SECTION Materials. L-Methionine γ-lyase (EC 4.4.1.11), S-(5′adenosyl)-L-homocysteine, S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1), DL-homocysteine, and cadmium nitrate were obtained from Sigma-Aldrich. The inhibitor D-eritadenine was from Santa Cruz Biotechnology. Absorbance spectra were recorded on a ND-1000 spectrophotometer (Nanodrop Technologies). Fluorescence measurements were performed at room temperature in a Varioskan Flash microplate reader (Thermo Scientific) using black microwell plates. L-Methionine γ-Lyase Activity Assay. Varying amounts of DL-homocysteine were incubated with different amounts of L8962

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Figure 3A represents the effect of varying quantities of Hcy at constant MGL concentration on the fluorescence registered in

Figure 1. UV−visible absorption (solid line) and emission (dashed line) spectra of CdS QDs produced by the enzymatic hydrolysis of Hcy by MGL. CdS QDs were formed in the presence of MGL (23 μg mL−1), Hcy (0.5 mM), and Cd(NO3)2 (7.5 mM) in citrate-phosphate buffer (12 mM, pH 6.4).

Figure 3. (A) Emission spectra in the system containing MGL (33 μg mL−1), Cd(NO3)2 (7.5 mM), and different concentrations of Hcy: (a) 0 mM, (b) 0.01 mM, (c) 0.02 mM, (d) 0.05 mM, (e) 0.1 mM, (f) 0.15 mM, (g) 0.2 mM, (h) 0.3 mM, and (i) 0.5 mM. Inset: Calibration curve of Hcy at λ = 500 nm. (B) Emission spectra in the system containing of Hcy (0.8 mM), Cd(NO3)2 (7.5 mM), and different concentrations of MGL: (a) 0 μg mL−1, (b) 5.5 μg mL−1, (c) 11 μg mL−1, (d) 22 μg mL−1, (e) 33 μg mL−1, and (f) 55 μg mL−1. Inset: Calibration curve of MGL at λ = 500 nm.

assay mixture (Figure 1S in Supporting Information). The presence of citrate was crucial for the formation of CdS NPs. Its stabilizing effect on NPs is well described in the literature.33 To confirm the proposed scheme of our assay, a number of control experiments in which one of the components was missing have been carried out. Figure 2 represents the

this system. The increase in the concentration of the enzymatic substrate up to 0.1 mM is linearly related with the increase in the intensity of fluorescence and asymptotically approaches its maximum starting from 0.5 mM of Hcy. The response reached the maximum value at 0.8 mM of Hcy; hence, the latter saturating substrate concentration was used in this study. The amount of formed CdS QDs depends on the rate of enzymatic production of H2S from the substrate Hcy in the assay mixture, as one can see from the increasing intensity of the fluorescence peaks. The emission peak position also depends on the amount of H2S produced enzymatically from Hcy. The increase in the concentration of the enzymatic substrate leads to a red shift of emission peaks (Figure 3A) and thereby to the increase in a diameter of the resulting CdS NPs. The value of the wavelength shift was less significant than the change in the fluorescence intensity caused by different concentrations of Hcy; therefore, the peak intensity was used as a readout signal in the consequent work. Nevertheless, the shift of the emission spectra also can be potentially employed to follow the response of our system to an analyte (Figure 2S in the Supporting Information). We compared the sensitivity of our assay for Hcy with that demonstrated by the conventional chromogenic assay based on interaction of the resulting 2-oxobutanoate, generated by MGL, with 3-methyl-2-benzothiazoline hydrazone to give the azine derivative.19 According to the calibration plots (Figures 3S−5S in the Supporting Information), our fluorogenic method was more sensitive by ∼300 times than the chromogenic test. The influence of different enzyme concentrations on the fluorescence signal arising from generated CdS QDs is shown in Figure 3B. The saturating 0.8 mM concentration of Hcy found from the previous experiment was used in this study. One can see that the increase in concentrations of MGL up to 20 μg mL−1 leads to the linear increase in the fluorescence intensities confirming the catalytic role of the enzyme in the set of reactions. Saturation was achieved starting from 30 μg mL−1.

Figure 2. Emission spectra in the system containing (a) MGL (23 μg mL−1), Hcy (0.5 mM), and Cd(NO3)2 (7.5 mM); (b) Hcy (0.5 mM) and Cd(NO3)2 (7.5 mM); (c) MGL (23 μg mL−1) and Cd(NO3)2 (7.5 mM); (d) MGL (23 μg mL−1) and Hcy (0.5 mM).

experimental results of the control experiments. Curve a corresponds to the emission spectrum of the system containing all components of the assay, i.e., Hcy, MGL, and Cd(NO3)2. Without MGL, the assay mixture containing only Hcy and Cd(NO3)2 showed no detectable fluorescence signal (curve b). In order to exclude the possibility that the fluorescence observed in the presence of MGL originates from its intrinsic fluorescence, the assay was performed in the mixture composed of MGL and Cd(NO3)2 without the enzymatic substrate Hcy. Curve c displays no significant fluorescence peak at 500 nm except the small peak at 400 nm, attributed to the interaction of MGL with Cd2+ ions. When the incubation of the mixture containing MGL and Hcy was performed without Cd(NO3)2 (curve d), no significant fluorescent peak was measured in the absence of Cd2+ cations. 8963

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The increase in MGL concentration resulted in a red shift of the emission spectra too (Figure 6S in the Supporting Information). The sensitivity of our assay for MGL was 200fold superior than that of the traditional chromogenic method.19(Figures 7S−9S in the Supporting Information). Having found the optimal saturating amount of MGL, we proceeded with the development of the assay for AHCY. In order to select the optimal concentration of AdoHcy, the effect of its varying amounts on the developed fluorescence in the full system composed of fixed quantities of AHCY, MGL, and Cd(NO3)2 was examined (Figure 4A). The response to the

which block the enzyme active site thanks to their structural similarity to natural substrates AdoHcy and adenosine. In MGL, two neighboring subunits containing pyridoxal-5′phosphate as a cofactor form the active site at their interface which binds specifically with Hcy.37 Adenine and adenosine derivatives are not able to block the active site of MGL; hence, the theoretical probability of false positive tests for AHCY inhibitors due to inhibition of MGL is quite low. Our experimental data prove the potent AHCY inhibitor Deritadenine does not affect the rate of decomposition of Hcy catalyzed by MGL (Figure 10S in the Supporting Information). The influence of varying concentrations of D-eritadenine on apparent activity of AHCY is shown in Figure 5. The nonlinear

Figure 4. (A) Emission spectra in the system containing of MGL (30 μg mL−1), AHCY (2.75 μg mL−1), Cd(NO3)2 (7.5 mM), and different concentrations of AdoHcy: (a) 0 μM, (b) 8.9 μM, (c) 22.4 μM, (d) 44.8 μM, (e) 89.6 μM, (f) 113 μM, (g) 226 μM, (h) 452 μM, and (i) 678 μM. Inset: Calibration curve of AdoHcy at λ = 445 nm. (B) Emission spectra in the system containing MGL (47 μg mL−1), AdoHcy (1 mM), Cd(NO3)2 (7.5 mM), and different concentrations of AHCY: (a) 0 μg mL−1, (b) 0.55 μg mL−1, (c) 1.1 μg mL−1, (d) 1.65 μg mL−1, (e) 2.2 μg mL−1. Inset: Calibration curve of AHCY at λ = 445 nm.

Figure 5. Emission spectra in the system containing of MGL (50 μg mL−1), AdoHcy (1 mM), AHCY (1.1 μg mL−1), Cd(NO3)2 (7.5 mM), and different concentrations of D-eritadenine: (a)100 nM, (b) 50 nM, (c) 25 nM, (d) 16 nM, (e) 12 nM, (f) 8 nM, (g) 4 nM, and (h) 0 nM. Inset: Calibration curve of D-eritadenine at λ = 445 nm.

regression applied to fitting the calibration curve displayed in the inset of Figure 5 gave the IC50 value equal to 15 ± 1.7 nM. This number corroborates well with that found in the literature.35,38

increasing amounts of AdoHcy was linear in the range from 9 to 200 μM and asymptotically reached the maximum fluorescence intensity starting from 300 μM. The shape of the calibration plot indicates the reaction proceeds through reversible formation of the enzyme−substrate complex, which further dissociates to give reaction products. This experimental data provides additional evidence to support the proposed Scheme 1 of assay operation. According to the calibration plot in Figure 4A, the saturating concentration of AdoHcy for further assays was chosen equal to 1 mM. Figure 4B shows the dependence of the readout fluorescent signal developed by our system on the quantity of AHCY injected into the assay mixture. The response to the enzyme was linear throughout the whole range of tested affordable AHCY concentrations up to 2.5 μg mL−1. The assay demonstrated the detection limit of 63 ± 10 ng mL−1 (S/N = 3) and the sensitivity of 1.76 ± 0.1 AU mL μg−1. The designed assay was applied to detection of Deritadenine, which is a previously described potent inhibitor of AHCY.34,35 The aliphatic adenine analogues including Deritadenine do not induce reduction of enzyme-bound NAD+ and inhibit the enzymatic transformation of AdoHcy to Hcy and Ado in a dose dependent manner.34 These adenine analogues cannot completely inactivate AHCY, and a residual enzyme activity is usually observed. The majority of known AHCY inhibitors are derivatives of adenine or adenosine36

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CONCLUSIONS In conclusion, the new fluorogenic assay for MGL activity based on formation of CdS QDs demonstrated 200-fold better sensitivity than the conventional chromogenic assay. The present simple fluorogenic test can find broad application in development of therapies relying on the use MGL. In addition, it was demonstrated that the fluorogenic MGL test can be employed in screening of AHCY inhibitors. Two described systems can serve as models for the design of sensitive and simple fluorogenic enzymatic assays using natural enzymatic substrates.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Fax: +34 943 005 314. Phone: +34 943 005 308. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 8964

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Letter

(31) Shi, J.-J. Chin. Phys. 2002, 11, 1286. (32) Yu, W. W.; Qu, L.; Guo, W.; Peng, X. Chem. Mater. 2003, 15, 2854−2860. (33) Serrano, T.; Gomez, I.; Colas, R.; Cavazos, J. Colloids Surf., A 2009, 338, 20−24. (34) Schanche, J. S.; Schanche, T.; Ueland, P. M.; Holy, A.; Votruba, I. Mol. Pharmacol. 1984, 26, 553−558. (35) Ctrnacta, V.; Fritzler, J. M.; Surinova, M.; Hrdy, I.; Zhu, G.; Stejskal, F. Exp. Parasitol. 2010, 126, 113−116. (36) Chiang, P. K. Pharmacol. Ther. 1998, 77, 115−134. (37) Alexander, F. W.; Sandmeier, E.; Mehta, P. K.; Christen, P. Eur. J. Biochem. 1994, 219, 953−960. (38) Huang, Y.; Komoto, J.; Takata, Y.; Powell, D. R.; Gomi, T.; Ogawa, H.; Fujioka, M.; Takusagawa, F. J. Biol. Chem. 2002, 277, 7477−7482.

ACKNOWLEDGMENTS This work was supported by the Spanish Ministry of Science and Innovation (Project BIO2011-26356), Plan Nacional (Grant SAF 2011-29851), and the Departamento de Educación del Gobierno Vasco 2011. V.P. acknowledges the contract Ramon y Cajal from the Spanish Ministry of Science and Innovation. The authors acknowledge the efforts by Marco Möller of the CIC biomaGUNE Electron Microscopy Platform.

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