Fluorometric Assay for the Determination of Glutathione Reductase

Oct 9, 2007 - In this paper, we describe a highly sensitive fluorometric assay for free sulfhydryl groups based on FRET, which we have used to develop...
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Anal. Chem. 2007, 79, 8769-8773

Fluorometric Assay for the Determination of Glutathione Reductase Activity Andrew M. Piggott and Peter Karuso*

Department of Chemistry & Biomolecular Sciences, Macquarie University, Sydney, NSW, 2109, Australia

The determination of free sulfhydryl groups is important in many aspects of biotechnology, such as measuring the coupling efficiency of thiol-reactive probes, assaying cysteine-containing haptens, and assaying reductase/thiol transferase activity, as well as in various aspects of the food industry. This is generally achieved colorimetrically using Ellman’s reagent, although the assay is relatively insensitive and Ellman’s reagent is unstable. In this paper, we describe a highly sensitive fluorometric assay for free sulfhydryl groups based on FRET, which we have used to develop a sensitive assay for glutathione reductase activity. The assay exploits the specific increase in fluorescence intensity that occurs at 520 nm when a probe containing two molecules of fluorescein linked via a disulfide group is cleaved by glutathione. The assay is 2 orders of magnitude more sensitive than the commonly used colorimetric glutathione reductase assay and has a greater dynamic range. Free thiols play a vital role in cells to maintain redox states, as powerful nucleophiles and as metabolic intermediates. There are a wide variety of fluorescent probes that react irreversibly with free thiols,1 but these are generally unsuitable for in vivo assays or for measuring reductase activity that requires recycling of oxidized and reduced thiols. The standard reagent for thiol quantification is 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), introduced by Ellman in 1959.2 DTNB reacts with thiols in a thioldisulfide exchange reaction, liberating chromogenic 5-mercapto2-nitrobenzoic acid. Glutathione reductase (GR; EC 1.8.1.7) catalyzes the reduction of oxidized glutathione (GSSG) to glutathione (GSH) by NADPH and is an essential part of the glutathione redox cycle, which maintains adequate levels of cellular GSH. GR is known to play a key role in the response to oxidative stress in both plants and animals3 and has been linked to several other diseases and conditions, including in the genesis of anxiety.4 GSH is involved in the detoxification of many xenobiotics, and overexpression of * Corresponding author. Tel: +612-9850-8290. Fax: +612-9850-8313. E-mail: [email protected]. (1) Haugland, R. P. Handbook of Fluorescent Probes and Research Products, 9th ed.; Molecular Probes Inc., 2002. (2) Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70-77. (3) Mullineaux, P. M.; Creissen, G. P. In Oxidative Stress and the Molecular Biology of Antioxidant Defenses; Scandalios, J. G., Ed.; Cold Spring Harbor Laboratory Press: New York, 1997; Vol. 34, pp 667-713. (4) Hovatta, I. T.; Richard, S.; Helton, Robert; Marr, Robert A.; Singer, Oded; Redwine, Jeffrey M.; Ellison, Julie A.; Schadt, Eric E.; Verma, Inder, M.; Lockhart, David J.; Barlow, Carrolee Nature 2005, 438, 662-666. 10.1021/ac071518p CCC: $37.00 Published on Web 10/09/2007

© 2007 American Chemical Society

GR has been observed in certain drug-resistant cancer cells.5 GSH-xenobiotic conjugates are formed either spontaneously or by a glutathione-S-transferase-catalyzed coupling reaction, and ATP-dependent glutathione-S-conjugate efflux pumps have been identified in both plants6 and animals.7 GSH is also involved in many other cellular functions and metabolic pathways.8,9 Therefore, there is an increasing need for sensitive and selective assays to measure GR activity in a variety of organisms and tissue types. A classic method of determining GR activity involves measuring the rate of NADPH oxidation spectrophotometrically at 340 nm.10 This method, although still widely used, is relatively insensitive and is susceptible to interference from other NADPHconsuming enzymes present in cellular homogenates, as well as from any species that absorb UV light. An improved method,11,12 based on the increase in absorbance at 412 nm when DTNB is reduced to 5-mercapto-2-nitrobenzoic acid by GSH, overcomes some of the interference problems inherent in the classical assay. This reaction forms the basis for several commercially available glutathione reductase assay kits but is still relatively insensitive, is susceptible to species that absorb violet light, and is limited by the instability of the DTNB reagent. Moreover, absorbance-based methods are unsuitable for measurements inside cells, which generally require fluorescence-based approaches. A fluorometric GR assay,13 based on the formation of a fluorescent compound when GSH reacts with N-(9-acridinyl)maleimide (NAM), was found to be 50 times more sensitive than the classical method. However, the fluorescent compound formed has an emission maximum of 435 nm, and hence, the assay is susceptible to interference from autofluorescence and from the inherent fluorescence of NADPH, which is an integral part of the assay. This problem was overcome by Maeda et al.,14 who employed a 2,4dinitrobenzenesulfonyl fluorescein probe for thiol quantification. Cleavage of this latent fluorophore by GSH releases fluorescein, (5) McLellan, L. I.; Wolf, C. R. Drug Resist. Updates 1999, 2, 153-164. (6) Edwards, R. D.; David, P. In Molecular Ecotoxicology of Plants (Ecological Studies); Sandermann, H., Ed.; Springer: Berlin, 2004; Vol. 170. (7) Hayes, J. D.; Flanagan, J. U.; Jowsey, I. R. Ann. Rev. Pharm. Toxicol. 2005, 45, 51-88. (8) Sastre, J.; Pallardo, F. V.; Vina, J. In Handbook of Environmental Chemistry; Grune, T., Ed.; Springer: Heidelberg, 2005; Vol. 2, pp 91-108. (9) Sies, H. Free Radical Biol. Med. 1999, 27, 916-921. (10) Tietze, F. Anal. Biochem. 1969, 27, 502-522. (11) Smith, I. K.; Vierheller, T. L.; Thorne, C. A. Anal. Biochem. 1988, 175, 408-413. (12) Rahman, I.; Kode, A.; Biswas, S. K. Nat. Protocols 2007, 1, 3159-3165. (13) Kamata, T.; Akasaka, K.; Ohrui, H.; Meguro, H. Anal. Sci. 1993, 9, 867870. (14) Maeda, H.; Matsuno, H.; Ushida, M.; Katayama, K.; Saeki, K.; Itoh, N. Angew. Chem., Int. Ed. 2005, 44, 2922-2925.

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Figure 1. Schematic representation of the fluorometric GR assay. GR catalyzes the reduction of GSSG to GSH by NADPH. Subsequent cleavage of the disulfide-containing linker of FSSF by GSH results in separation of the fluorophores and a specific increase in fluorescence intensity at 520 nm.

which has an emission maximum at 520 nm and hence is less susceptible to autofluorescence than NAM. However, like DTNB, the probe is quite reactive and undergoes hydrolysis in aqueous media. Both reagents irreversibly react with GSH and would affect the redox balance of cells and thus are less suitable for highthroughput cellular assays. Fluorescence resonance energy transfer (FRET) is a nonradiative quantum mechanical transfer of energy from the excited state of a donor fluorophore to a suitable acceptor chromophore that is within 100 Å of the donor. FRET-based assays allow the signal measured to be made even more specific than normal fluorescence detection as there are essentially two filtering steps involved (first, the difference between the excitation and emission frequencies and, second, the quenching or stimulation of that emission by FRET), leading to greater sensitivity and less background interference. Recently, a FRET-based assay was reported for the determination of S-adenosylmethionine-dependent methyltransferase activity.15 In this paper, the authors used a probe consisting of two different fluorophores joined by a disulfidecontaining linker to detect the formation of homocysteine from enzymatic hydrolysis of S-adenosylmethionine. This work led us to consider the possibility of using a FRET-based assay for the determination of GR activity. Herein, we describe a highly sensitive fluorometric assay for the determination of glutathione reductase activity (Figure 1). Our assay exploits a new fluorescent probe consisting of two molecules of fluorescein joined by a short disulfide-containing linker (FSSF). Cleavage of the disulfide linker by GSH allows the two fluorescein molecules to diffuse apart, resulting in a decrease in FRETmediated quenching and a specific increase in fluorescence intensity at 520 nm (green), which is less susceptible to interference from the inherent fluorescence of NADPH and from other fluorescent species present in cellular homogenates (e.g., autofluorescence). Due to the small Fo¨rster radius of fluorescein (44 Å), there is no practical reason why a heterodimer probe is required and our approach was to make a fluorescein homodimer, which would release two identical fluorophores, making subsequent kinetic analysis simpler and it is simple to synthesize. EXPERIMENTAL SECTION Glutathione reductase assay kit (GRSA), containing GR, GSSG, NADPH, DTNB, assay buffer, and dilution buffer, was obtained (15) Wang, C.; Leffler, S.; Thompson, D. H.; Hrycyna, C. A. Biochem. Biophys. Res. Commun. 2005, 331, 351-356.

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from Sigma-Aldrich. NHS-fluorescein was obtained from Pierce. Fluorescence was measured in black microtiter plates (Becton Dickinson, with a Fluostar Galaxy plate reader, BMG Labtechnologies) using a 485-nm (14-nm band-pass at half-height) excitation filter and a 520-nm (33-nm band-pass) emission filter with 10 flashes/cycle. Nonlinear least-squares regression was performed using the Dynafit16 software package (Biokin). Molecular modeling was performed using the Chem3D software package (Cambridgesoft). Synthesis of Fluorescein Disulfide (FSSF). Fluorescein disulfide (2) was prepared in two steps from readily available starting materials (Scheme 1). Initially, 2 equiv of mono-Boc protected ethylenediamine were coupled to 3,3′-dithiodipropionic acid using EDC, yielding Boc-en-S-S-en-Boc (1). Deprotection of 1 with TFA followed by coupling to (5/6)-NHS-fluorescein yielded 2 as a mixture of isomers. Full synthetic details and characterizations are given in the Supporting Information. Measurement of Free Thiols: Cleavage of FSSF by Dithiothreitol (DTT). A stock solution of FSSF in DMSO (100 µM) was serially diluted to 0.1 µM using assay buffer (100 mM K3PO4, 1 mM EDTA, pH 7.5). A stock solution of DTT in water (100 mM) was similarly serially diluted to 0.1 mM with assay buffer. Assay buffer was used in place of FSSF and DTT for controls. For each concentration of FSSF and DTT, a 10-µL aliquot of the FSSF solution was mixed with a 90-µL aliquot of the DTT solution in a well of a black microtiter plate, and the fluorescence intensity of each well was recorded at 60-s intervals for 2 h at room temperature using a fluorescence plate reader. Each experiment was performed in triplicate, with the average of the three readings being used in all subsequent analyses. Protocol for the Fluorometric GR Assay. A stock solution of GR (0.1 U/mL) was serially diluted to 0.001 U/mL with dilution buffer (100 mM K3PO4, 1 mM EDTA, 1 mg/mL BSA, pH 7.5). Each reaction mixture consisted of GSSG (2 mM; 50 µL), FSSF (100 µM; 0-25 µL), GR (0.001 or 0.01 U/mL; 0-20 µL), NADPH (2 mM; 5 µL), and assay buffer to make a total volume of 100 µL. All reagents, except NADPH, were premixed in a standard microtiter plate and then transferred to wells of a black microtiter plate containing the NADPH solution to start the reaction. The fluorescence intensity of each well was recorded at 10-min intervals for 18 h at room temperature using a fluorescence plate reader. Each experiment was performed in triplicate, with the average of the three readings being used in subsequent analyses. RESULTS AND DISCUSSION Fluorescein Disulfide. The fluorescent probe used in our assay consisted of two molecules of fluorescein joined by a disulfide-containing linker. The linker was designed to tether two fluorescein molecules within their Fo¨rster radius such that one group acts as a fluorescence quencher for the other. This is made possible by the relatively small Stokes shift of fluorescein compared to many other fluorophores, obviating the need to synthesize an asymmetric probe. The linker was also designed to impart good water solubility on the probe by inclusion of four amide groups and leaving the phenolic groups of the fluorescein molecules unmasked. (16) Kuzmic, P. Anal. Biochem. 1996, 237, 260-273.

Scheme 1. Synthesis of FSSFa

a Reagents and conditions: (a) 3,3′-dithiodipropionic acid, EDC, TEA/CHCl , 25 °C, 18 h, 85%; (b) 20% TFA/CHCl , 25 °C, 30 min, 3 3 quant. (c) 5/6-carboxyfluorescein N-hydroxysuccinimide ester, TEA/EtOH, 25 °C, 18 h, 82%.

The protected linker (1) was synthesized in 85% yield from commercially available mono-Boc protected ethylenediamine and can be easily carried out on a gram scale. Deprotection of the linker and attachment of two fluorescein groups to yield 3,3′dithiodipropionic acid (5/6-fluorescinylcarbonylaminoethyl)amide (FSSF; 2) was effected in 82% yield from commercially available NHS-fluorescein. Unlike the commonly used colorimetric reagent (DTNB), FSSF appears to be very stable and aqueous solutions kept in the dark at room temperature for several weeks showed no appreciable decomposition, as determined by fluorescence measurements. Cleavage of Fluorescein Disulfide by Thiols. An energy minimized (MM2) model of FSSF showed the fluorophores to be no greater than 30 Å apart when the disulfide-containing linker was fully extended (Figure S-1, Supporting Information). This separation is considerably less than the 44-Å Fo¨rster radius for fluorescein,1 so the molecule should exhibit strong FRET-mediated quenching. Cleavage of the disulfide bond allows separation of the fluorophores and should result in a corresponding increase in observed fluorescence intensity. To test this hypothesis and develop a fluorometric assay for free thiols, solutions of FSSF ranging from 10 nM to 10 µM were treated with increasing concentrations of DTT and the fluorescence intensity of each solution was recorded as a function of time with the same receiver gain settings on the fluorometer. The progress curves obtained for the most concentrated (10 µM) and most dilute (10 nM) solutions of FSSF used are shown in Figure 2a and Figure 2b, respectively. As expected, addition of DTT resulted in a timedependent increase in fluorescence, while no increase was observed in the absence of DTT. The detection limit of FSSF was found to be 5 nM with the receiver gain optimized for the most concentrated solution of FSSF (10 µM), but lower concentrations should be detectable by using a higher receiver gain. Therefore, it should be possible to detect thiols directly in cells using concentrations of FSSF that do not affect cellular redox homeostasis as cellular glutathione, the dominant redox mediator, is present in the millimolar range.17 The data obtained for the reaction of DTT with FSSF were fitted to a three-component reaction model (1) using nonlinear

least-squares regression with the Dynafit16 program. Although cleavage of FSSF by DTT actually occurs in two steps, it is the initial formation of the mixed-disulfide intermediate (FSSDTT)

(17) Senft, A. P.; Dalton, T. P.; Shertzer, H. G. Anal. Biochem. 2000, 280, 8086.

(18) Song, L.; Hennink, E. J.; Young, I. T.; Tanke, H. J. Biophys. J. 1995, 68, 2588-2600.

FSSF + DTT f 2FSH + ox-DTT FSH f photobleached FSH FSSH f photobleached FSSF

(k1)

(1)

(kpb1) (kpb2)

that results in separation of the two fluorescein molecules and gives rise to the corresponding increase in fluorescence. Further reaction of FSSDTT to give FSH and oxidized DTT produces no additional increase in fluorescence, and hence, the overall cleavage reaction can be approximated to a simple one-step reaction, as shown in the first line of eq 1. Careful inspection of the progress curves obtained revealed that, after reaching a maximum, the fluorescence intensity started to drop slightly toward the end of the experiment. This small (1-2%/h) can be explained by photobleaching of the fluorophore. Photobleaching occurs when a fluorophore permanently loses the ability to fluoresce due to photon-induced chemical change (e.g., isomerization or reaction of the excited state with oxygen). Fluorescein is well-known to undergo photobleaching in solution and when bound to biomolecules, and although the mechanism of bleaching is complex, it can be approximated to a single-exponential decay.18 In this study, both the probe (FSSF) and product (FSSG) are susceptible to photobleaching, and hence, the rates of photobleaching of both these species were determined and accounted for in subsequent experiments. The first-order rate constant for the photobleaching of FSSF (kpb2) was determined independently by linear regression analysis of the data obtained from the control experiments with no DTT added (Figure S-3, Supporting Information), allowing rate constants for the cleavage of FSSF by DTT (k1) and for the photobleaching of FSSG (kpb1) to be calculated by nonlinear leastsquares regression with Dynafit (Table 1). Although the rates of photobleaching of FSSF and FSSG were found to be minimal, with only a 10% drop in fluorescence after 16 h of irradiation, the inclusion of these terms in the reaction model produced a much

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Table 1. Rate Constants Obtained from Dynafit Partial Least-Squares Regression Analysis of the GR Assay Dataa rate constant k1 kcat k2 kpb1 kpb2 kden

value

UNITS 10-7

L µmol-1 s-1 L µmol-1 s-1 L2 µmol-2 s-1 s-1 s-1 s-1

(1.1109 ( 0.0008) × 11.78 ( 0.04 (4.308 ( 0.004) × 10-12 (9.07 ( 0.05) × 10-6 (1.97 ( 0.04) × 10-6 (3.06 ( 0.01) × 10-5

a The errors shown are standard errors for the rate constants, as calculated by Dynafit.

Solutions containing GSSG, FSSF, and NADPH were treated with varying amounts of GR from 0.05 to 2 mU mL-1, and the fluorescence intensity of each solution was recorded as a function of time (Figure 3). The sigmoidal shape of the progress curves results from the enzymatic production of GSH being considerably faster than the subsequent cleavage of FSSF. However, the data obtained were easily fitted to a five-component reaction model (2) using nonlinear least-squares regression with the Dynafit program. GSSG and NADPH were present in excess throughout all the experiments, and hence, the enzymatic cleavage of GSSG by GR to give GSH was approximated to a one-step reaction to simplify the analysis. The cleavage of FSSF by GSH

GSSG + GR f 2 GSH + GR 2GSH + FSSF f 2 FSSG

better fit of the experimental data and allowed the rate of cleavage of the probe by DTT to be determined more accurately (lower standard error). A calibration plot of the initial rate of increase in fluorescence intensity versus time was constructed and was found to be directly proportional to the amount of DTT added over at least 4 orders of magnitude of FSSF concentration (Figure 2C). This large dynamic range should make FSSF useful for the determination of free thiol concentration in a variety of different applications, such as the quantification of cysteine-containing peptides (e.g., haptens) in solution or the determination of thiols inside cells. Fluorometric GR Assay. The fluorometric GR assay was performed in essentially the same manner as the standard colorimetric assay by simply exchanging DTNB with FSSF. 8772

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(2)

(k2)

FSSG f photobleached FSSG

(kpb1)

FSSF f photobleached FSSF

(kpb2)

GR f denatured GR

Figure 2. Fluorescence intensity of (A) 10 µM and (B) 10 nM FSSF in 100 mM K3PO4, 1 mM EDTA, pH 7.5, after addition of DTT at room temperature. Calibration plot (C) showing the initial rate of increase in fluorescence intensity of FSSF solutions in 100 mM K3PO4, 1 mM EDTA, pH 7.5, after addition of DTT (0.1-10 mM) at room temperature.

(kcat)

(kden)

was also approximated to a one-step reaction in a manner analogous to the DTT cleavage reaction. The rate constants for photobleaching of FSSG (kpb1) and FSSF (kpb2) were fixed to the values obtained in the DTT cleavage experiments, thereby allowing the values of kcat and k2 to be calculated by nonlinear least-squares regression (Table 1). The overall fit of the model was then further improved by including a reaction to account for the slow denaturation of the enzyme during the course of the experiment (kden). Due to the lag phase at the beginning of the assay, the initial rates of fluorescence increase after GR is added are unsuitable for constructing a calibration plot to determine GR concentration. However, as the concentration of GSH builds up following enzymatic hydrolysis of GSSG, the maximum rate of increase in fluorescence intensity is proportional to the concentration of GR added. Double-reciprocal plots of the maximum rates of increase in fluorescence intensity vs time (Figure 3B) were found to be linear between 0.05 and 2 mU mL-1 of GR for all concentrations of FSSF tested and hence are suitable calibration plots for determining unknown GR concentrations within this range and possibly well outside this range. The commercially available kit

Figure 3. Fluorescence intensity (A) of 25 µM FSSF, 1 mM GSSG, and 100 µM NADPH in 100 mM K3PO4, 1 mM EDTA, pH 7.5, after addition of GR at room temperature. Double-reciprocal calibration plot (B) of the maximum rate of increase in fluorescence intensity vs time when solutions of FSSF containing 1 mM GSSG, 100 µM NADPH in 100 mM K3PO4, 1 mM EDTA, pH 7.5, were treated with GR (0.05-2 mU) at room temperature.

for the DTNB colorimetric assay claims the concentrationdependent enzymatic reaction is linear between 3 and 30 mU mL-1, making our fluorometric assay at least 60 times more sensitive and having a dynamic range at least 1 order of magnitude more than the colorimetric assay.

the detection of glutathione, GR or glutathione transferase activity in live cells may also be feasible and this is currently under investigation.

CONCLUSIONS We have developed an accurate and sensitive fluorometric GR assay based on the specific increase in fluorescence at 520 nm that occurs when a disulfide-containing fluorescein homodimer is cleaved by GSH. The substrate is very stable, unlike the commonly used colorimetric reagent, and 2 orders of magnitude more sensitive. The probe can be used to determine free thiol concentrations over several orders of magnitude, as demonstrated with DTT, and quantification of free sulfhydryl groups in other formats may be possible. The assay is sufficiently sensitive that

Complete synthetic procedures, characterization of new compounds, molecular modeling results, and nonlinear least-squares fitting methods. This material is available free of charge via the Internet at http://pubs.acs.org.

SUPPORTING INFORMATION AVAILABLE

Received for review July 18, 2007. Accepted August 28, 2007. AC071518P

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