Automated evaluation of the inhibition of glutathione reductase activity: application to the prediction of ionic liquids' toxicity

Abstract

This work is based on the automated evaluation of the influence of several ionic liquids (ILs) on the activity of immobilized glutathione reductase (GR), as an indicator of their toxicity. The reactions involved are the reduction of oxidized glutathione (GSSG) by GR, followed by the oxidation of the formed product (reduced glutathione – GSH) by the sulfhydryl reagent 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) to form a yellow derivate 2-nitro-5-thiobenzoic acid (TNB), measurable at 412 nm. The bioassay was performed in a SIA system where the ILs were submitted to a simple and precise on-line dilution strategy before evaluation of their toxicity. The activity assays were implemented in strictly aqueous media and in the presence of increasing concentrations of seven commercially available ILs. EC₅₀ values between 0.24 and 2.55 M were obtained for the tested compounds, revealing distinct inhibitory effects. The least and most toxic ILs found in this study were emim[Ms] and emim[BF₄], respectively. The automated methodology is an interesting analytical tool for the evaluation of GR activity in ILs proving to be robust while presenting good repeatability, with RSD less than 5%, in all the assay conditions, leading also to a reduction of the consumption of solvents as well as of effluent production (2 mL per cycle).

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Introduction

In recent years, the interest in ILs has increased widely and their use in the areas of organic reactions and separation technologies is already a reality.¹ These materials are a class of non-molecular solvents that consist essentially of organic cations and anions.^2,3 They have been found to possess negligible volatility, non-flammability, high thermal stability, and low melting points. These physical properties depend on both the type of cation/anion and the alkyl chains of the anions. Additionally, the polarity, hydrophobicity and solvent miscibility behavior of ILs can be finely tuned through appropriate modification of the cation and anion to meet specific requirements of the applications.⁴ Concerning the range of ILs' applications, their toxicity needs to be investigated to predict their impact on human's health and environment.⁵ This evaluation shall be part of a sustainable development of chemicals that, by means of a multidisciplinary work involving several class of researchers, can access the environmental risks of ILs by combining structure-activity relationships and (eco)toxicological tests. Data about the biological toxicity of ILs is becoming available with studies of the effect of ILs in biological elements of different levels of complexity from animals to cells. Latała et al.,⁶ presented a preliminary assessment of the toxicity of selected imidazolium ILs towards marine algae. They found that the growth of Cyclotella meneghiniana was effectively inhibited throughout the experiment regardless of the IL concentration applied. Docherty and Kulpa,⁷ reported that hexyl- and octyl-imidazolium and pyridinium bromides had significant antimicrobial activity to pure cultures of Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Pseudomonas fluorescens and Saccharomyces cerevisiae. They also found that lengthening the alkyl chain led to a concomitant increase in toxicity of ILs. A similar trend of increasing toxicity with increasing alkyl chain length was observed in other organisms, including marine bacterium Vibrio fischeri, the soil nematode Caenorhabditis elegans, and the fresh snail Physa acuta.^7–9

On the subcellular level, several enzyme inhibition assays have also provided very interesting information about the toxicity of ILs.^10–14 In fact, while test systems at higher levels of complexity allow a better understanding of the effect of ILs on a complex environment, molecular and cellular tests are useful to clarify the impact of particular structural elements and to guide their modification to reduce their hazardous potential. The evaluation of toxicity by means of enzyme inhibition presents additional advantages such as simplicity of laboratory implementation and data interpretation as well as reduction of costs and duration of the assays. Moreover, there are already authors confirming the parallelism between the results of toxicity evaluation in enzymes and plants.¹⁵ Also on this topic, it is well-known the importance of evaluating the influence of xenobiotics on enzymes of the antioxidant defense system of aquatic and terrestrial biological entities for monitoring both environmental quality and the fitness of organisms inhabiting ecosystems. Among these enzymes, GR is a very important biomarker since it plays a key role in the metabolism and clearance of xenobiotics acting as a scavenger of hydroxyl radicals, singlet oxygen, and various electrophiles. GR is a crucial enzyme that reduces glutathione disulfide (GSSG) to the sulfhydryl form GSH by the NADP-dependent mechanism, an important cellular antioxidant system. This enzyme has already been applied to investigate the toxic effects of distinct groups of compounds such as of polycyclic aromatic hydrocarbons,¹⁶ cyanotoxins,¹⁷ pharmaceuticals¹⁸ as well as ILs.^12,19 V. Paskova et al.,¹⁶ have studied the toxic effects of three homocyclic aromatic hydrocarbons (PAHs-phenanthrene, anthracene, fluorine) and their seven N-heterocyclic derivates on higher terrestrial plants Sinapis alba, Triticum aestivum, and Phaseolus vulgaris, and the effects were related to the oxidative stress as determined by biomarkers, such as GR. Other studies with GR were used to evaluate the effects of combined avian exposure to cyanotoxins.¹⁷ The results indicate that the antioxidative system plays an important role in the protective response of the tissues to multiple stressors and that its greater induction could actually help to protect the birds from more serious damage. T. Roušar et al.¹⁸ investigate a new mechanism to the toxic action of acetaminophen, especially to explore the possible inhibition of glutathione reductase through an acetaminophen-glutathione conjugate (APAP-SC). M. Yu et al.¹² M. Kumar et al.¹⁹ reported the toxicity and biological effects of some ILs on the antioxidant enzyme system of mouse liver and on the Green Seaweed Ulva lactuca, respectively, and GR was selected as biomarker to determine the oxidative stress caused by the ILs.

All these assays, however, were generally included in more global studies of the effect of xenobiotics on the antioxidant defense system of several biological entities.

Our objective is to resort to a subcellular level, and evaluate directly the inhibition effect of several ILs on the GR activity molecule, proposing a methodology capable of contributing to the prediction of human toxicity. GR activity was then studied in an aqueous media and in aqueous solutions with increasing concentrations of 1-butyl-3-methylimidazolium tetrafluoroborate (bmim[BF₄]), 1-butyl-3-methylimidazolium chloride (bmim[Cl]), 1-ethyl-3-methylimidazolium tetrafluoroborate (emim[BF₄]), 1-ethyl-3-methylimidazolium trifluoromethanesulfonate (emim[TfMs]), 1-ethyl-3-methylimidazolium methanesulfonate (emim[Ms]), tetrabutylphosphonium methanesulfonate (tbph[MS]) and 1-butyl-1-methylpyrrolidinium chloride (bmpyr[Cl]) (Fig. 1). The obtained results can be discussed in combination with those obtained through the inhibition of higher organisms with the aim of contributing to a better understanding of ILs' toxicity.

Fig. 1 Chemical structure of the studied ILs. 1: 1-butyl-3-methylimidazolium tetrafluoroborate (bmim[BF₄]); 2: 1-butyl-3-methylimidazolium chloride (bmim[Cl]); 3: tetrabutylphosphonium methanesulfonate (tbph[MS]); 4: 1-butyl-1-methylpyrrolidinium chloride (bmpyr[Cl]); 5: 1-ethyl-3-methylimidazolium tetrafluoroborate (emim[BF₄]); 6: 1-ethyl-3-methylimidazolium trifluoromethanesulfonate (emim[TfMs]); 7: 1-ethyl-3-methylimidazolium methanesulfonate (emim[Ms]).

Generally, toxicity studies have been performed till recently mainly in batch mode with drawbacks associated with analysis time, human intervention and consumption of reagents. In this context, the automation of these procedures emerged as a tool to further increase their potential and significance. In the last years, sequential injection analysis (SIA) has been profitably applied in bioassays, including enzyme based and whole cell assays, for the evaluation of the toxicity of ILs, proving to be a robust and accurate solution handling approach that guarantees precisely the reaction conditions through computer control of analytical parameters.^13,14,20,21 Features such as low-cost independent operation and drastic reduction in solutions consumed justify its choice for bioassays.

In this context, and taking in account all the advantageous mentioned it was decided to evaluate the effect of several ILs on the GR activity by applying to an automated assay. The selected methodology is based on the DTNB-GSSG reductase recycling assay in which GSSG is reduced by GR to form GSH that is subsequently oxidized by the sulfhydryl reagent DTNB to form a yellow derivate, TNB that can be measured at 412 nm (Scheme 1).²²

Scheme 1 Schematic representation of the sequence of chemical reaction employed to measure GR activity.

Our group had already automatized this assay by sequential injection analysis for the analysis of blood samples.²³ However, in order to apply it to evaluate ILs toxicity, it was necessary to adapt the previous system to effectively obtain a versatile, low-cost and robust tool for the evaluation of enzyme inhibition by ILs. To take advantageous of these changes it was necessary to proceed again with the optimization of the SIA system before using it to ILs evaluation.

Results and discussion

Automated SIA system used in the GR activity assay

The assay used in the evaluation of ILs effect on GR activity was based on work previously implemented by our group for the determination of total and oxidized glutathione in human whole blood.²³ However, it was necessary to do the upgrade of the system, and to adapt it to the analysis of ILs, by performing a new optimization (Table 1, Fig. 2).

Table 1 Range of values used in the optimization studies and selected values for the final assay

Parameter	Range	Selected value
NADPH concentration (mM)	0.2–1.4	1.2
DTNB concentration (mM)	1–4	3
GSSG volume (μL)	50–250	100
NADPH volume (μL)	25–75	50
DTNB volume (μL)	25–75	50
Sample volume (μL)		50
Stop period (s)	0–150	120

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Fig. 2 Effect of different volumes of GSSG, NADPH and DTNB (a) and different concentrations of NADPH and DTNB (b) performed with GSSG 0.8 μM.

Briefly, it will be pointed out before, the adjustments made necessary for the establishment of the final automated system used in the bioassays. So, first the enzyme was immobilized in order to minimize its consumption. The enzymatic column was placed in the way to the detector where the reaction zone was stopped for a period of 120 s increasing about 3 times the sensitivity. Second, the reagents inserted in the system were changed. The new sequence comprised the aspiration of 100 μL of GSSG, 12.5 μL of diluted IL, 50 μL of DTNB and 50 μL of NADPH sample. These studies were performed with GSSG standards with concentration in the range of 0.2–1.5 μM and with water in the place of IL. While several optimal parameters (Fig. 2) showed to be the same, the GSSG volume was raised to 100 μL as an increase of sensitivity of 49% was obtained over 50 μL, probably due to the fact that the total amount of enzyme immobilized and present in the assay doubled. The last alteration made to the system, was very important and enabled for the first time the automated on-line dilution of ILs, increasing the robustness and the precision of this step.

With the auxiliary dilution conduit (DC) (Fig. 5) and just a single aspirated aliquot of each IL a concentration gradient was obtained and an inhibition profile could be established. The implementation of the dilution strategy involved initial theoretical experiments designed to calibrate the DC, and characterize the attained dilution profile necessary for the ILs, making use of a colored solution of bromothymol blue.

Generally, this strategy involves three main steps in the analytical cycle (Table 4) to attain the dilution of an aspirated aliquot. A volume of sample or reagent (V_S) to be diluted is aspirated to the HC; then, a portion of this zone with volume V_T (transfer volume) is transferred from the HC to the DC; finally, a portion of the transferred solution, with defined volume (V_A – analysis volume), is aspirated back to the HC from the DC and is actually used for the analysis. After the utilization of the first aliquot, several others can be aspirated back to the HC and used in the assay with distinct dilution degrees.

Table 2 Figures of merit of the SIA methodology for the evaluation of IL toxicity

Regression equation	(A.U.) = 0.5119 (±0.0751) CGSSG (μM) + 0.0127 (±0.0084)
R^2	0.9996
Detection limit	0.03 μM
Quantification limit	0.11 μM
Linear response range	0.2–1.5 μM
Repeatability	R.S.D.% = 4.8
Reproducibility (10 days)	R.S.D.% = 5.6

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Table 3 Toxicity of the studied ILs expressed as EC₅₀

IL	EC50 (M) ± SD^a
a SD – standard deviation of three replicates.
bmim[BF4]	1.87 ± 0.08
bmim[Cl]	2.43 ± 0.71
emim[BF4]	0.09 ± 0.01
emim[TfMs]	2.20 ± 0.85
emim[Ms]	2.55 ± 0.93
bmpyr[Cl]	0.71 ± 0.06
tbph[Ms]	0.24 ± 0.03

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Table 4 Analytical cycle for the evaluation of the inhibition effect on GR activity

Step	Valve position	Volume (μL)	Flow rate (ml min^−1)	Time (s)	Action
1	2	50	0.8	3.75	Aspiration of volume VS of the IL into HC	On line dilution of ILs
2	7	350	0.8	26	Propulsion of IL (VT) from HC to DC
3	8	—	1.5	50	Flushing of the HC
4	3	100	0.8	7.5	Aspiration of L-glutathione oxidized
5	7	12.5	0.3	2.5	Transfer of volume of the IL VA from DC into HC
6	4	50	0.8	3.75	Aspiration of DTNB
7	5	50	0.8	3.75	Aspiration of NADPH
8	1	—	1.0	22	Propulsion of the sequence to reactor
9	1	—	0	120	Stop period
10	1	—	1.5	75	Propulsion to the detector and signal acquisition
11	7	—	1.5	75	Flushing of the DC

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In this work, the volume V_S was fixed at 50 μL and the other parameters were optimized according to the intended dilution profile. The V_T was studied in the range of 300–350 μL and combined with V_A of 12.5 and 25 μL. Considering the future application of the method to the evaluation of ILs' toxicity, the combination of 350 μL of V_T with 25 μL of V_A originated dilution profiles that can be both useful for the established purpose (Fig. 3).

Fig. 3 Analytical signals (●) and respective dilution factors (▲) obtained by transferring, in consecutive analytical cycles, the analysis volume (V_A) of 25 μL (a) or 12.5 μL (b) to the holding coil. Dilution factor: dilution level attained by calculating the ratio between the analytical signals obtained with and without dilution.

In detail, the sequential aspiration of aliquots of 25 μL after transference of 350 μL to the dilution coil originates dilutions from 3.7 to 58.9 times, with the profile depicted in Fig. 3a. On the other hand, the aspiration of diluted aliquots of 12.5 μL conducts to a more broad dilution profile with dilution factors comprised between 3.6 to 80.4 times (Fig. 3b). The accuracy of the proposed on-line dilution in the SIA (SIAd) system was confirmed by comparing the results obtained with those furnished by a comparison batch procedure (Bd), being established for the two methods with 95% confidence limits: SIAd = 0.002 (0.021)+ 1.027(0.043), (R² = 0.9998).

After system optimization, in strictly aqueous media, aiming also the collection of data for comparison purposes, calibration curves, for the intercept and slope, the linear working range, detection limit, repeatability and reproducibility (Table 2) were evaluated. Comparing just the figures of merit obtained with ref. 23, one may emphasize the achievement of the double of the sensitivity, important as the inhibition assay is based on a decrease of absorbance.

GR inhibition in the presence of ILs

In this work, GR activity was evaluated in the presence of seven commercially available ILs aiming the prediction of their toxicity obtained in the optimized analytical cycle and resorting to the optimized dilutions strategy. By working with may can be present in “home synthesized” ILs. The selected compounds exhibited variable chemical structure conferred by different structural elements that determine the particular behavior of each compound. The respective inhibition profiles are shown in Fig. 4 and the calculated EC₅₀ values are represented in Table 2. As can be seen, these results showed that the evaluated ILs exhibited a relatively weak inhibitory effect on GR, with EC₅₀ values between 0.09 and 2.55 M. These concentrations are thousands of times higher than those found for these and other enzymes with protective roles, but not included in the antioxidant defense system, such as acetylcholinesterase²⁴ and carboxylesterase,¹³ whose EC₅₀ are at the μM level. Not underestimating the interest of GR as biomarker, these results reveal a significant resistance of the enzyme to the tested ILs, being in good agreement with the tendency observed with the enzymes of the antioxidant defense system when exposed to ILs. Generally, these enzymes keep their activity and increase their biological expression as a response to the oxidative stress caused by ILs.

Fig. 4 Experimental toxicity data of the enzymatic assay. Inhibition profiles obtained with analytical cycle presented in Table 3 with GSSG 1.2 μM and 12.5 μL of diluted IL from DC for each point obtained.

Additionally, in this work, GR inhibition by ILs followed the tendencies described in literature regarding the impact of cation head groups and anions. Even though the studies regarding the size of the alkyl side chain of the cation were not extensive in this work, the results contradict the majority the published studies.^24,25 This particular case will be discussed considering the properties of the ILs involved in the study of GR structure.

Considering the well-known influence of the cationic head group of ILs on toxicity, three main structures were selected: imidazolium, phosphonium and pyrrolidinium. It was observed that ILs comprising the non-aromatic pyrrolidinium and phosphonium groups exhibited lower EC₅₀ values (0.24 and 0.71 M, respectively) than the ones embracing the aromatic butyl-imidazolium and ethyl-imidazolium cations with the same anion (bmim[Cl] = 2.43 M and emim[Ms] = 2.55 M). The nature of the tetrabutylphosphonium head group which exhibits a quaternary “surfactant like” structure justifies partially the observed tendency. This peculiar structure results in chemical and biological behaviors similar to those of cationic surfactants, one of the most toxic class of quaternary compounds.²⁶ Regarding the pyrrolidinium head group, the results are not surprising as it has been referred as potentially more toxic than the imidazolium one incorporating the same anion.²⁴

Still regarding the cation head group, the increase of the alkyl side chain from 2 to 4 carbons, in the ILs emim[BF₄] and bmim[BF₄], resulted in a decrease of enzyme inhibition evidenced by the increase of EC₅₀ from 0.09 M to 1.87 M. This tendency does not agree with data obtained in studies on higher organisms in which the increase of the alkylchain length of imidazolium ILs lead to an increase of lipophilicity, resulting in higher inhibition or toxicity.^24,25 However, similar observations were already reported by other authors²⁷ and by our group during the evaluation of the influence of these two ILs on the enzymes catalase and trypsin.^14,28 As before, we hypothesize that the increase of lipophilicity caused by the insertion of two carbons in the alkyl side chain to get bmim[BF₄] does not induce a sufficiently high increase of lipophilicity able to create a tendency similar to that observed with ILs with alkyl chains with 6 or more carbons. As stated before, to discuss other eventual scenarios it is necessary to take in consideration the peculiar structure of GR. This is a dimeric disulfide reductase whose catalytic center is formed from residues coming from five domains (four from one subunit and one from the other subunit) that are organized to form two deep pockets which are the binding areas of GSSG and NADPH.²⁹ It is known that these pockets are filled with solvent in the absence of substrate and cofactor. In this context, it is highly probable that the reduced size of the emim cation facilitates the access of this IL to the active site of the enzyme creating a competition with the molecules of GSSG and NADPH that difficults their entrance on the pockets and the binding to the active site causing reduction of enzyme activity when compared with bmim ILs incorporating the same anion. We postulate that this competition might not be so intense in the presence of bmim based ILs due to their longer alkyl side chain. The intriguing behavior of the enzyme in the presence of emim[BF₄], revealead by the smallest EC₅₀ of the whole study is a combination of this effect with that of the anion BF₄⁻, that will be discussed afterwards.

To evaluate the intrinsic toxicity of the anion, four commonly used anions in this kind of compounds were selected: tetrafluoroborate (BF₄⁻), chloride (Cl⁻), trifluoromethanesulfonate (TfMs⁻) and methanesulfonate (Ms⁻). In this study, as in others, the tested anions exhibited smaller effects on enzyme activity than the studied cations. In particular, the comparison of GR behavior in emim[TfMs] and emim[Ms] evidenced that these anions affect similarly enzyme activity, since they exhibited very similar EC₅₀ (2.20 and 2.55 M, respectively). Regarding BF₄⁻, even though this species has been described as not having influence on ILs toxicity, in this study its deleterious effect is well evident from the comparison of the inhibitory concentrations of emim[TfMs], emim[Ms] and emim[BF₄] with EC₅₀ of 2.2, 2.55 and 0.09 M, respectively. The same effect is also evidenced from the comparison of the EC₅₀ values obtained with bmim[BF₄] and bmim[Cl] (1.87 and 2.43 M, respectively). As it is known, this effect is related with the described partial hydrolysis of BF₄⁻ under the experiment conditions, with production of toxic hydrofluoridric acid.³⁰

Conclusions

Regarding the impact of the tested ILs on GR, it was observed that the inhibitory concentrations are high considering the possible human and environmental threshold values. However, the studied combinations evidenced a preferential inhibition by the BF₄⁻ anion specially when combined with an emim based cation head group. Even though, the comparative analysis of bmim based ILs with non-aromatic cations highlighted the toxic potential of pyrrolidinium and tetrabutylphosphonium groups.

The automated method used for the evaluation of the activity and inhibition of GR, evidence once again the adequacy of SIA for this kind of study mainly due to the precise control of the reaction conditions, namely the on-line dilutions of the inhibitors, associated to a significant reduction of costs and diminished operator intervention. Enzyme immobilization further enhanced the applicability of the methodology with benefits in terms of enzyme consumption with good repeatability and with an increment of sensitivity. The application of the developed methodology to the evaluation of ILs' toxicity confirmed its interest while providing important toxicity data. Due to its versatility and simplicity, it is expected that the presented automated assay can be integrated in the process of development of new ILs as rapid screening method that in combination with similar and higher level assays can contribute to a more sustainable expansion of these solvents.

Experimental

Materials

All chemicals were of analytical reagent grade with no further purification. Milli-Q plus water with a specific conductance less than 0.1 μS cm⁻¹ was used throughout.

L-Glutathione oxidized disodium salt (GSSG), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), ethylenediaminetetraacetic acid (EDTA) dipotassium salt dehydrate were purchased from Fluka. Glutathione redutase (GR; EC 1.6.4.2, from baker's yeast, 160 U per mg protein) and β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt (NADPH) were obtained from Sigma-Aldrich. Potassium dihydrogen phosphate (KH₂PO₄) was supplied by Riedel-de-Haen and potassium hydroxide from Merck.

Buffer solution was prepared with 100 mM KH₂PO₄, 1 mM dipotassium EDTA and pH was adjusted to 7.0 by addition of potassium hydroxide solution. This solution was used as carrier in the SIA system. Solutions of 3 mM DTNB and 1.2 mM NADPH were prepared daily in the buffer solution. A stock solution of 2 mM GSSG was prepared daily and working standard solutions were obtained from this stock solution by serial dilution. All these solutions were shielded from light throughout use.

In the inhibition studies, aqueous solutions of 1-butyl-3-methylimidazolium tetrafluoroborate (bmim[BF₄]), 1-butyl-3-methylimidazolium chloride (bmim[Cl]), 1-ethyl-3-methylimidazolium tetrafluoroborate (emim[BF₄]), 1-ethyl-3-methylimidazolium trifluoromethanesulfonate (emim[TfMs]), 1-ethyl-3-methylimidazolium methanesulfonate (emim[Ms]), tetrabutylphosphonium methanesulfonate (tbph[MS]) and 1-butyl-1-methylpyrrolidinium chloride (bmpyr[Cl]) were prepared daily. All the ILs were purchased from Sigma-Aldrich Cooperation (Fig. 1).

GR immobilization

The immobilization procedure was similar to that described by Peña et al.³¹ In each immobilization procedure, 0.125 g of aminoalkylated glass beads were incubated in 2.5 ml of glutaraldehyde 2.5% for 1 h, at room temperature, with brief nitrogen deoxygenation every 10 minutes, for the first half hour. The activated glass beads (AGB) were washed with water and 0.1 M phosphate buffer, pH 7.5. Immobilization was then performed by adding 10 U ml⁻¹ of GR to the AGB. Incubation in an ice bath was performed during 4 h, encompassing nitrogen deoxygenation every 10 minutes during the first half hour. After incubation, the glass beads were filtered off and washed with water and Tris-HCl/CaCl₂ (50/20 mM) buffer, pH 8, to eliminate any non-immobilized enzyme. Thereafter, immobilized GR was packed in a home-made polimethylmethacrylate (PMMA) column (3 cm length; i.d. 4 mm). Two filters of 35 μm pore size (MobiTec M2235) were placed at both ends of the reactor to entrap the glass beads. The GR reactor was stored at 4 °C, in KH₂PO₄ 1 mM buffer, pH 7 in a tightly closed tube.

Flow manifold and instrumentation

The SIA flow system (Fig. 5) consisted of a Gilson Minipuls peristaltic pump, equipped with a PVC pumping tube (1.2 mm) and a Crison module that incorporates an 8-port selection valve. All connections, including the holding coil (HC) were made with 0.8 mm i.d. PTFE tubing. The HC was 2 m in length and eight-shaped in configuration. The dilution coil (DC) was 2 m.

Fig. 5 Schematic representation of the SIA system. C, carrier (phosphate buffer, pH 7); PP, peristaltic pump; HC, holding coil; DC, dilution coil; W, waste; R, GR reactor; D, detector.

The flow system was controlled by means of a microcomputer equipped with an interface card (Advantech Corp., PCL 711B, San Jose, CA). Software was developed in QuickBasic 4.5 (Microsoft) and permitted operation of the peristaltic pump and multi-position selection valve, enabling the run-time definition of all analytical parameters such as flow rate, flow direction, sample volume, reagents volume and valve positioning as well as data acquisition and processing.

As detection system, an Ocean Optics USB4000 miniature fiber optic spectrophotometer equipped with a LS-1-LL tungsten halogen light source was used. Data acquisition was performed by Auto-analysis computer software.

Analytical cycle for the evaluation of GR activity

The analytical cycle established for evaluation of GR activity, in the SIA system, is summarized in Table 3. Initially, 50 μL of IL was aspirated to the HC (step 1). The flow was reversed and the IL was propelled to the dilution coil (DC, step 2) and the holding coil was flushed for the remaining reagents (step 3). Then, 100 μL of GSSG, 12.5 μL of diluted IL (from the DC), 50 μL of DTNB and 50 μL of NADPH were aspirated sequentially to the HC (steps 4–7). The reaction zone was propelled to the enzyme reactor, where it remained for 120 s (steps 8–9). Finally, the reaction zone was directed to the detector and a signal proportional to the enzyme activity was monitored (step 10). Then, and until a complete inhibition profile for the IL in analysis was obtained, only steps 4–11 were repeated as much times as needed. Finally, the DC was flushed with carrier to eliminate the remaining IL (step 11).

GR activity and inhibition assays

The range of concentrations of IL used was stipulated specifically for each compound in order to get inhibition profiles amenable to mathematical treatment. The maximum value 100% was defined as the activity of GR immobilized in aqueous media, in the absence of inhibitor and with a GSSG standard concentration of 1.2 μM. The results of inhibition of GR by the selected ILs were expressed as EC₅₀ calculated as the effective concentration of inhibitor causing a decrease of 50% on enzyme activity, comparing with its activity in buffer media. The calculations were performed by means of polynomial correlations established in the inhibitions assays for each tested compound (Fig. 4).

Acknowledgements

This work received financial support National Funds (FCT, Fundação para a Ciência e Tecnologia) through project Pest-C/EQB/LA0006/2013 and from the European Union (FEDER funds) under the framework of QREN through Project NORTE-07-0162-FEDER-000124. Marieta L. C. Passos thanks FCT for her pos-doc grant (SFRH/BPD/72 378/2010) in the ambit of “POPH - QREN - Tipologia 4.1 - Formação Avançada” co-sponsored by FSE and national funds of MCTES.

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