Accepted Manuscript Development of a new catalase activity assay for biological samples using optical CUPRAC sensor Burcu Bekdeşer, Mustafa Özyürek, Kubilay Güçlü, Fulya Üstün Alkan, Reşat Apak PII: DOI: Reference:
S1386-1425(14)00752-5 http://dx.doi.org/10.1016/j.saa.2014.04.178 SAA 12143
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received Date: Revised Date: Accepted Date:
19 March 2014 25 April 2014 30 April 2014
Please cite this article as: B. Bekdeşer, M. Özyürek, K. Güçlü, F. Alkan, R. Apak, Development of a new catalase activity assay for biological samples using optical CUPRAC sensor, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa.2014.04.178
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Development of a new catalase activity assay for biological samples using optical CUPRAC sensor Burcu Bekdeşer1, Mustafa Özyürek1, Kubilay Güçlü1*, Fulya Üstün Alkan2, Reşat Apak1 1
Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar 34320, Istanbul,
Turkey 2
Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Istanbul University,
Avcılar 34320, Istanbul, Turkey
ABSTRACT A novel catalase activity assay was developed for biological samples (liver and kidney tissue homogenates) using a rapid and low-cost optical sensor−based ‘cupric reducing antioxidant capacity’ (CUPRAC) method. The reagent, copper(II)-neocuproine (Cu(II)-Nc) complex, was immobilized onto a cation-exchanger film of Nafion, and the absorbance changes associated with the formation of the highly-coloured Cu(I)-Nc chelate as a result of reaction with hydrogen peroxide (H2O2) was measured at 450 nm. When catalase was absent, H2O2 produced the CUPRAC chromophore, whereas catalase, being an effective H2O2 scavenger, completely annihilated the CUPRAC signal due to H2O2. Thus, the CUPRAC absorbance due to H2O2 oxidation concomitant with Cu(I)-Nc formation decreased proportionally with catalase. The developed sensor gave a linear response over a wide concentration range of H2O2 (0.68-78.6 µM). This optical sensor−based method applicable to tissue homogenates proved to be efficient for low hydrogen peroxide concentrations (physiological and nontoxic levels) to which the widely used UV method is not accurately responsive. Thus, conventional problems of the UV method arising from relatively low sensitivity and selectivity, and absorbance disturbance due to gaseous oxygen evolution were overcome. The catalase findings of the proposed method for tissue homogenates were statistically alike with those of HPLC. Keywords: Catalase activity, Optical sensor, Cupric reducing antioxidant capacity (CUPRAC) assay, Nafion membrane film. *
Corresponding authors: Tel: +90 212 473 7070, Fax: +90 212 473 7180
E-mail addresses: [email protected]
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Introduction
Hydrogen peroxide (H2O2) is one of the most frequently occurring reactive oxygen species in the biosphere. It emerges in the environment as a by-product of aerobic metabolism in respiratory and photosynthetic electron-transport chains, and of enzymatic activity mainly by oxidases. Both excessive hydrogen peroxide and its decomposition product hydroxyl radical (•OH), formed in a Fenton-type reaction, are harmful for almost all cell components [1]. Reactive oxygen species (ROS) give rise to severe damage to the cell membrane as well as to biological macromolecules like lipid, protein and DNA. Enzymatic antioxidant defense systems can eliminate the hazards of excessive ROS, e.g., superoxide dismutase eliminates superoxide anion radical by dismutation to H2O2 and O2, and the accumulation of H2O2 is prevented by the actions of catalases and peroxidases [2]. Catalase (EC 1.11.1.6, CAT) is an ubiquitous antioxidant enzyme present in most aerobic cells, and is involved in the detoxification of H2O2. Catalase catalyzes the dismutation of H2O2 by two types of reactions. Both reactions include a first step of formation of an intermediate (compound I) consisting of the enzyme and H2O2. The catalytic activity associated with 2 moles H2O2 ends up with reduction to 1 mole H2O and oxidation to 1 mole O2 in the overall reaction [3]. CAT + H2O2 → compound I + H2O compound I + H2O2 → CAT + H2O + O2 Net reaction: 2 H2O2 → 2 H2O + O2 (catalytic activity) The peroxidatic activity of catalase involves the catalytic reaction of compound I with hydrogen donors (AH2 such as methanol, ethanol, phenols) other than H2O2 [4,5]. compound I (CAT-H2O2) + AH2 → CAT + 2 H2O + A (peroxidatic activity)
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Catalase can be found in all aerobic microorganisms, plant, and animal cells and is especially concentrated in the liver, kidney and erythrocyte [6]. It is generally regarded as the primary enzymatic defense against H2O2 generation [7]. Moreover, measurement of changes in endogenous antioxidant enzyme activity can be used to indirectly quantify ROS [8]. Thus, the measurement of catalase activity in biological samples is important. A large number of assays were developed for the determination of catalase activity [9-11]. The most common method for detection of catalase activity is the UV spectrophotometric method making use of the change of 240 nm−absorbance when H2O2 is consumed by catalase. This assay is not suitable for the measurement of CAT activity of UV-absorbing substances causing interference [12]. Moreover, the UV method has low H2O2 sensitivity and cannot measure concentrations below 1.0 mM H2O2 (i.e. at physiological and nontoxic levels). The unphysiologically high substrate concentrations in the millimolar range result in rapid CAT inactivation. Additionally, molecular oxygen may be liberated in gaseous form leading to disturbance of absorbance [6,9]. Catalase activity may be measured quantitatively by the conventional method of Von Euler and Josephson (1927) via permanganate titration of the remaining excess of H2O2 [13], which may suffer from human error in end-point reading [14]. Determination of peroxidatic function of catalase is based on the reaction of the enzyme with methanol (acting as H-donor), where the produced formaldehyde is measured at 550 nm using 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (Purpald reagent) as chromogen [4]. A commercially available [15] fluorescent detection kit for catalase activity makes use of diluted horseradish peroxidase (HRP) to react with a substrate in the presence of H2O2 to convert the colorless substrate into a fluorescent product (λex=570 nm, λem=590 nm), where increasing levels of catalase in the samples causes a decrease in H2O2 concentration and a reduction in fluorescent product formation. However, HRP−linked assays have certain disadvantages, such as those mentioned by Halliwell, i.e. peroxidase can also oxidize certain substrates without
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neecssarily adding H2O2 [16]. In addition, several biological compounds including thiols and ascorbate can serve as substrate for HRP and thus compete with the probe for oxidation, leading to underestimation of H2O2 formation [17]. In the literature, there are a number of enzyme-based electrochemical sensors for H2O2, manufactured by immobilizing catalase on the modified electrode, which are naturally not directly applicable to catalase assay in complex samples. As one of the few exceptions, Cohen & Weber (1993) described the use of a gold-coated optical fiber as a reagentless photoelectrochemical sensor for the in situ photogeneration and electrochemical detection of the catalase enzyme substrate, hydrogen peroxide [18]. The H2O2 was generated from the photochemical reduction of molecular oxygen, mediated by tris(2,2’-bipyridyl)ruthenium: Ru(bpy)32+, isolated on the device with the aid of the cation-exchange membrane: Nafion, where the peroxide was detected amperometrically. The peroxide oxidation current was sensitive to catalase; about 25 nM catalase could rapidly reduce the device's photosignal by 50%. The reversible hydrogen peroxide optical sensor described by Mills et al. (2007), based on the fluorescent quenching of the dye ion-pair [Ru(bpy)32+(Ph4B−)2], by O2 produced by the catalytic breakdown of H2O2, was not so sensitive: it could detect H2O2 over a range of 0.01 to 1 M, with a calculated lower limit of detection of 0.1 mM [19]. A paper-based colorimetric detection device for H2O2 vapor sensing relies upon the cellulose microfibril network of paper towels, which provide a tunable interface for modification with Ti(IV)-oxo complexes for reacting with H2O2 and yielding maximal absorbance at 400 nm [20], but a linear concentration/response curve could not be observed. Consequently, the idea in the present study is to develop a novel catalase activity assay for biological samples (such as liver and kidney tissue) using an optical sensor−based ‘cupric reducing antioxidant capacity’ (CUPRAC) method, which has originally emerged as a widely applicable antioxidant capacity assay [21] but also given rise to many other modified (e.g.,
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antioxidant and antiradical activity) assays. One of these modifications is the optical sensor−based CUPRAC method using an immobilized chromogenic reagent, copper(II)neocuproine (Cu(II)-Nc) complex on a cation-exchanger polymer membrane (Nafion), for the assessment of antioxidant capacity of non-enzymatic antioxidants [22]. The tissue homogenates were evaluated for their catalase activity using the proposed sensing method −associated with the changes of the CUPRAC absorbance due to H2O2 oxidation concomitant with Cu(I)-Nc formation on the membrane− in comparison with the UV reference method.
Experimental details Reagents and apparatus The following chemicals of analytical reagent grade were purchased from the corresponding sources: Neocuproine (2,9-dimethyl-1,10-phenanthroline) (Nc), ethanol (EtOH), Catalase from bovine liver (CAT), Nafion 115 perfluorinated membran (thickness 0.005 in.): Sigma-Aldrich (St. Louise, USA); copper(II) chloride dihydrate, hydrogen peroxide (35% by wt.), Na2HPO4, NaH2PO4.2H2O, ammonium acetate: Merck (Darmstadt, Germany). The biological samples were kindly donated by the Faculty of Veterinary Medicine of Istanbul University. The visible spectra and absorption measurements were recorded using a Varian CARY Bio 100 UV-Vis spectrophotometer (Mulgrave, Victoria, Australia). Mixing was performed with a BIOSAN Programmable rotator-mixer Bulti Bio RS-24 (Riga, Latvia). The chromatograph was from A Waters Breeze TM 2 Model HPLC system (Milford, MA, USA) equipped with a 1525 binary pump, a 2465 Electrochemical Detector (ECD) (Chelmsford, MA, USA), a Hamilton 25mL-syringe (Reno, NV, USA), and reverse phase ACE C18 column (4.6 mm×250 mm, 5 μm particle size) (Milford, MA, USA) was used for 5
chromatographic measurements. Data acquisition was accomplished using Empower PRO (Waters Associates, Milford, MA).
Preparation of solutions
The original catalase solution of initial activity 3691 U mg-1 solid was diluted with 0.2 M phosphate buffer (pH 7.4) to a concentration of 738 U mL-1. Hydrogen peroxide at 1.0 mM concentration was prepared from a 0.5 M intermediary stock solution, the latter being prepared from 35% H2O2 and standardized with permanganate titration. The NaH2PO4– Na2HPO4 buffer solution (pH 7.4) at 200 mM was prepared in distilled water. Copper(II) at 1.0×10−2 M was prepared by dissolving 0.4262 g of CuCl2·2H2O, and diluting to 250 mL. Ammonium acetate (NH4Ac: 1.0 M) aqueous solution contained 19.27 g of the salt in 250 mL. Neocuproine (Nc) at 7.5×10−3 M was prepared by dissolving 0.078 g of the free base in EtOH, and diluting to 50 mL with ethanol (prepared fresh). The mobile phase for HPLC analysis with isocratic elution was prepared from 50 mM ammonium acetate. This solution was prepared by dissolving 0.9635 g of ammonium acetate in 250 mL of bidistilled water.
Preparation of tissue homogenates
CD-1 mice were obtained from the Faculty of Veterinary Medicine of Istanbul University. The mice were housed in polycarbonate cages (450 cm2 area per animal), acclimatized under laboratory conditions (23±2 °C, humidity 50-60 %, 12 light/dark cycle), and fed with water and standard mice food. Liver and kidney tissues were isolated after sacrifice by decapitation from mice. The tissue samples were washed with 0.9% (w/v) NaCl solution, weighed and homogenized by adding cold 1.15% (w/v) KCl solution in a glass
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homogenizer. Homogenates were immediately frozen in liquid nitrogen and kept at −80 °C until analysis [23]. Homogenates were filtered through a 0.45 µm membrane filter and diluted (at a ratio of 1:500) with ammonium acetate (1.0 M) solution before analysis. There was no effect of tissue homogenate on the CUPRAC absorbance of hydrogen peroxide because of the high dilution ratio.
Preparation of the CUPRAC sensor
The commercial Nafion membrane was cut into sliced 4.5×0.5 cm parts, engrossed into a tube containing 2.0 mL of 1.0×10-2 M Cu(II) + 2.0 mL of 7.5×10-3 M Nc + 2.0 mL of 1 M NH4Ac + 2.2 mL of H2O, and agitated for 30 min in a rotator [22].
Optical sensor based CUPRAC (proposed) method
To a test tube were added 0.5 mL of 1.0 mM H2O2, (2−x) mL H2O, x mL catalase solution at different concentrations (x varying between 0.1 and 2.0 mL) or tissue homogenate in this order. The reaction mixture in a total volume of 2.5 mL was incubated for 30 min in a water bath kept at 25◦C. At the end of this period, the reagent-impregnated membrane (developed CUPRAC sensor) was taken out and immersed in a tube containing 2.0 mL of the reaction mixture + 6.2 mL of EtOH. The tube was placed in a rotator and agitated for 30 min so as to achieve full color development. The colored membrane was taken out and placed in a 1 mm optical cuvette containing H2O (to prevent sticking of slices to the walls of the cuvette), and its absorbance at 450 nm was read against that of a blank membrane prepared under identical conditions excluding analyte. The absorbance values were linearly correlated to H2O2 concentrations, and the decrease in absorbance was due to catalase activity.
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UV (reference) method
The UV method was applied as the reference method of comparison for determining the catalase enzyme activity of biological samples. The reaction mixture for the UV method [24] contained in a final volume of 3.0 mL the following reagents: 1.5 mL pH 7.4 NaH2PO4– Na2HPO4 buffer solution (200 mM), 0.5 mL H2O2 (75 mM), x mL catalase solution at various concentrations (x varying between 0.1 and 2.0 mL) or tissue homogenate, (1−x) mL H2O. Reaction mixtures were incubated at 25 oC for ½ h. At the end of the incubation period, the absorbance of the resulting solution was measured at 240 nm. The absorbance values were linearly correlated to H2O2 concentrations, and the decrease in absorbance was due to catalase activity.
HPLC method
A suitable aliquot of the incubation mixture solution was injected (injection volume: 20 μL) without derivatization (i.e. without conversion into a fluorophore or a chromophore, enabling post-column detection) into the HPLC column (5 mm ODS) at ambient temperature, and isocratic elution was employed for detection of the hydrogen peroxide. The HPLC–ECD method developed by Yue et al. (2009) [25] was adapted with slight modifications, namely the use of a reverse phase ACE C18 column (4.6 mm×250 mm, 5 μm particle size) (Milford, MA, USA) in conjunction with an ECD system comprising Ag/AgCl reference and glassy carbon working electrodes. The mobile phase consisting of 50 mM ammonium acetate in water was delivered at a rate of 1 mL min-1 (as ammonium ions may cause the precipitation of proteins, statistical comparison of the proposed method with HPLC was performed only in synthetic solutions of CAT). The column effluent was pumped to an ECD detector (as
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recommended by Takahashi et al., 1999) [26]. The detector was operated in direct current (DC) mode. Each run was 5 min long. Quantification of H2O2 was performed using the data generated at 500 mV potential [25].
Statistical analysis
Statistical analyses were realized using Excel software (Microsoft Office 2002) for calculating the means and the standard error of the mean. Results were expressed as the mean ± standard deviation (SD). Using SPSS software for Windows (version 13), the data were interpreted by two-way ANalysis Of VAriance (ANOVA) [27].
Results and discussion
Optimization of the optical sensor‒based CUPRAC method parameters
Catalase enzyme activity was determined by making use of the decrease in CUPRAC absorbance of H2O2 in the presence of varying CAT concentrations. Although H2O2 is essentially known to be a strong oxidant, it can act as a reductant under favorable redox potentials, e.g., toward Cu(II)-neocuproine. The molar absorption coefficient (i.e. ΔA/Δc) of the CUPRAC sensing method for hydrogen peroxide was: ε = 1.73×104 Lmol-1 using a Nafion membrane, and linear concentration range: 6.81×10-7-7.86×10-5 M (r = 0.9922). Considering the preconcentration function of the Nafion membrane (i.e. for the cuprous-neocuproine chromophore) and its average thickness (0.1 cm), the effective molar absorptivity of the method for H2O2 is raised to the level of 1.73x105 M-1cm-1. Hydrogen peroxide is CUPRAC reactive, but its degradation products (H2O and O2) are not. The CUPRAC absorbance of 9
H2O2 is attenuated as a result of its reaction with CAT (Fig. 1). The attenuated absorbance of H2O2 is dependent upon the CAT activity of the tested sample. The linear equation for the calibration graph of CAT drawn at the wavelength of 450 nm with respect to the optical sensor–based CUPRAC method was:
A450 = −1.00 c + 0.8655 (r = 0.9861)
where c is CAT concentration (U mL-1). The limit of detection (LOD) and limit of quantification (LOQ) of the optical sensor based CUPRAC method for CAT were found to be 0.044 and 0.145 U mL-1, respectively. The precision data of the proposed assay (expressed as the % RSD values) was approximately 2.29%. On the other hand, the intra-day and inter-day precisions of the commercial catalase kit [15] for 0.6-2.9 U mL-1 activities of catalase buffer were in the ranges of 3.2-5.9% and 7.3-7.8%, respectively, indicating a distinct improvement of precision in the proposed method. Recovery values with the CUPRAC sensor varied from 96.5 to 115.0%.
Figure 1 Conversion of H2O2 to reaction products (i.e. H2O and oxygen) in the presence of CAT was followed by measuring the CUPRAC absorbance of the mixture as a function of time (Fig. 2). An optimal measurement time of 30 min was chosen during which an absorbance plateau was reached due to annihilation of H2O2 by CAT.
Figure 2
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Interferences in the optical sensor–based CUPRAC method
For the developed catalase activity assay, the possible interference effects of concomitant species commonly found in biological samples on the determination of 0.30 U mL-1 are shown in Table 1. As opposed to peroxidase based assays [17], the presence of vitamin and plasma antioxidants commonly found in biological samples did not interfere with the catalase activity assay using the proposed method.
Table 1
Linearity on tissue homogenate dilution
The dilution sensitivity of kidney homogenate was evaluated using the optical sensor– based CUPRAC method, and the found catalase activities (in U mL-1) were recorded against expected catalase activity at varying dilutions of the kidney homogenate (Fig. 3). Excellent linear curves were observed passing through the origin, i.e. having a higher linearity correlation (r = 0.9994) than that of the commercial catalase kit, where the observed versus expected catalase activity curve had a correlation coefficient of r = 0.9553 [15].
Figure 3
Determination of catalase activity in tissue homogenates The catalase activity of tissue homogenates (liver and kidney) was determined by the optical sensor–based CUPRAC and UV methods. Liver homogenates were generally shown to exhibit higher CAT activity than kidney (Fig. 4), because the liver is generally regarded as 11
the major organ of detoxication [28] and catalase in liver homogenate effectively detoxifies H2O2. Casalino et al. (2002) found that the CAT activity of rat liver tissue homogenates (383±39.1 unit per mg protein) was distinctly higher than that of kidney homogenates (158.2±16.1 unit per mg protein) [29]. The CAT activity values measured with the proposed optical sensor–based CUPRAC method and reference method (UV method) were comparatively depicted in a bar diagram (Fig. 4). The order of CAT activities of tissue homogenates found with the proposed CUPRAC method were compatible with those measured by the UV method.
Figure 4 The UV method for detection of CAT activity depends on the change of the 240 nmabsorbance when H2O2 is consumed by catalase, and the relatively high activities measured by UV (Fig. 4) probably arise from the positive interference by UV-absorbing substances. The absorbance of H2O2 at 240 nm is measured directly to calculate CAT activity since the reaction products, i.e. H2O and oxygen, do not absorb at this wavelength. The molar absorptivity for H2O2 was: ε = 37 Lmol-1cm-1, and the linear concentration range: 5.08×10-43.1×10-2 M (r = 0.9998). UV spectrophotometry for H2O2 estimation is the most preferred but interference–affected method [6]. Standard calibration curves of CAT were redrawn in liver homogenate showing good parallelism of linear curves (Fig. 5), indicating that the constituents of the real matrix solution (homogenate) did not interact to produce chemical deviations from Beer’s law.
Figure 5
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Membrane stability and chemical resistance against H2O2
The properties that make the Nafion membrane indispensable are the combination of good water uptake, ion-exchange capacity, proton conductivity, low gas permeability, and excellent electrochemical stability [30]. Nafion was found to be thermally stable up to 280 °C, at which temperature the sulfonic acid groups began to decompose [31]. After protonated Nafion membrane was tested in 30% H2O2 solution at 80 °C for 12 h, small amounts of fluoride ion and sulfate ion, which were derived from the C−F single bonds and the sulfonic acid groups, respectively, of the membrane, were detected in solution [32]. Kinumoto et al. (2006) further pointed out to a more serious decomposition possibility −via radical depolymerization mechanism− of the Nafion membrane by hydroxyl and hydroperoxyl radicals generated from Fenton-type reactions of {transition metal ion+H2O2 (concentrated)} combinations [32]. The chemical stability of Nafion membrane was also shown in the H2 and O2 environments [33]. To test the possible effect of H2O2 on a Nafion membrane, one of the membranes used in the original study was treated with 0.5 mL of 1 mM H2O2 for 30 min. CUPRAC sensor was prepared by using this membrane and an unloaded membrane as described in ‘Preparation of CUPRAC sensor’. Catalase activity of (1:500 diluted) kidney homogenate was measured and found with H2O2-treated and untreated sensors as 16.01 and 15.98 U/mL, respectively. Our experimental results showed that Nafion membrane was not affected by H2O2 within the normal time period of the test protocols (and Fenton-type degradation reactions were not possible considering that the cupric-neocuproine cationic chelate but not free Cu2+ was coated on the membrane), and no significant change on catalase activity was found with the use of this sensor for tissue homogenates. Once the cupric-neocuproine adsorbed on the membrane was reduced by hydrogen peroxide to the stable cuprous-chelate, it did not enter a back
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reaction with hydrogen peroxide producing reactive species [34]. The reagent-loaded (singleuse) sensor was tested for stability, and was shown to lose only 3% signal intensity after 15 days-storage in distilled water kept in a dessicator at room temperature.
Comparison of the results of optical sensor–based CUPRAC and HPLC methods
The chromatograms of the original and remaining H2O2 after CAT reaction for validating the developed assay (Fig. 6) showed that the retention time for the H2O2 was 3.00 min. The amount of H2O2 was found with the aid of calibration curve (y = 8.10×107 c + 97864 (r = 0.9992)) drawn as peak area versus concentration. Catalase activity was manifested by the decrease in both the absorbance of the CUPRAC chromophore and in the chromatographic peak area due to H2O2 as a result of its transformation to H2O and oxygen. The degradation ratio of H2O2 depended upon the amount of CAT. Concentrations of H2O2 (the initial concentration of H2O2 was 10-2 M) remaining after CAT reaction, found with the proposed CUPRAC and reference HPLC methods, were compatible with each other (Table 2). The two-way ANalysis Of VAriance (ANOVA) comparison, via F-test of the mean-squares of “between-treatments” (i.e. remaining H2O2 concentration, with respect to the optical sensor–based CUPRAC and HPLC methods depicted in Table 2) and of residuals enabled us to conclude that there was no significant difference between treatments. The experimentally found optical sensor–based CUPRAC results and HPLC results were statistically alike at the 95% confidence level (Fexp = 7.89, Fcrit (table)
= 10.13, Fexp < Fcrit (table) at P = 0.05). The hydrogen peroxide concentration found by the
proposed method correlated well (r = 0.9976) with those of the HPLC method (Table 2). Thus, the proposed methodology was validated against the reference HPLC method. Both methods correctly reflected the relative decrease in H2O2 concentration as a result of
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conversion into H2O and oxygen. For example, the conversion efficiency of H2O2 found with the optical sensor–based CUPRAC and HPLC methods were 54.5% and 51.5%, respectively, in the presence of 1.48 U mL-1 CAT.
Figure 6 Table 2 Conclusion
The Cu(II)-Nc reagent was electrostatically immobilized onto a cation exchanger Nafion membrane, and was demonstrated to retain its reactivity towards hydrogen peroxide after incorporation into the exchanger membrane. The absorbance of Cu(I)-Nc produced from intact H2O2 decreased proportionally with CAT activity. This offers advantages over solution–based assays, including the potential of being used as a rapid, low-cost, diversely and easily applicable method, and of future refinements of the assay to online applications and kit format. Currently, there is no commercial catalase sensor (e.g., like blood glucose sensor) in the market for spectrophotometrically (or colorimetrically) detecting/quantitating CAT in complex biological samples. Naturally with the use of electrochemical techniques enabling quantitative H2O2 determination, catalase‒ or peroxidase‒immobilized electrodes cannot be used for sensing catalase. The proposed method had better linearity and higher precision than that of the commercially available catalase kit utilized in solution phase. In conclusion, the proposed optical sensor–based method can be considered as a precise and accurate alternative for measuring the CAT activity of biological samples. We believe that this simple sensor– based assay for catalase activity measurement is a useful tool for investigating the role of catalase enzyme and its regulation under physiological conditions in various biological 15
systems, and opens the way for the future development of dip-stick type point-of-care diagnostic kits.
Acknowledgements
The authors thank TUBITAK (Turkish Scientific and Technical Research Council) for the support given to the Research Project 110T725. The authors also extend their gratitude to T.R. Ministry of Development for the Advanced Research Project of Istanbul University (2011K120320).
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[29] E. Casalino, G. Calzaretti, C. Sblano, C. Landriscina, Toxicology 179 (2002) 37-50. [30] N. H. Jalani, R. Datta, J. Membr. Sci. 264 (2005) 167-175. [31] S. R. Samms, S. Wasmus, R. F. Savinell, J. Electrochem. Soc. 143 (1996) 1498-1504. [32] T. Kinumoto, M. Inaba, Y. Nakayama, K. Ogata, R. Umebayashi, A. Tasaka, Y. Iriyama, T. Abe, Z. Ogumi, J. Power Sources, 158 (2006) 1222-1228. [33] H. Tang, S. Peikang, S. P. Jiang, F. Wang, M. Pana, J. Power Sources 170 (2007) 85-92. [34] M. Özyürek, K. Güçlü, R. Apak, Trends Anal. Chem. 30 (2011) 652-664.
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Table 1 Possible interferences of some biologically important compounds toward the optical sensor‒based CUPRAC method. Interferent
Concentration
Ascorbic acid Heparin EDTA Bilirubin Glucose Uric Acid Albumin
2.0 μM 78.4 USP/10 mL 20.0 μM 0.4 μM 0.35 mg mL-1 4.0 μM 0.4 mg mL-1
Added catalase U mL-1 0.30 0.30 0.30 0.30 0.30 0.30 0.30
19
Found catalase U mL-1 0.29 0.29 0.31 0.29 0.30 0.31 0.30
Relative error (%) -3.45 -3.45 3.22 -3.45 0 3.22 0
Table 2 Determination of H2O2 in the presence of CAT by using optical sensor‒based CUPRAC and HPLC methods (N=3). CAT (U mL-1) 0 0.30 0.89 1.18 1.48
H2O2 concentration found with respect to optical sensor‒based CUPRAC method (mM) 10.0±0.42 8.65±0.35 7.01±0.28 6.25±0.24 5.45±0.22
P = 0.05, Fexp = 7.89 Fcrit (table) = 10.13 Fexp < Fcrit (table) [H2O2]CUPRAC = 1.03[H2O2]HPLC − 0.38 (r = 0.9976).
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H2O2 concentration found with respect to HPLC method (mM) 10.0±0.32 8.50±0.28 6.95±0.31 6.16±0.29 5.15±0.17
Figure Captions: Fig. 1. Visible spectra of Cu(I)-Nc chelate produced as a result of optical sensor‒based CUPRAC reaction with varying concentrations of CAT enzyme ((a) 0 U mL-1 (b) 0.15 U mL-1 (c) 0.30 U mL-1 (d) 0.44 U mL-1 (e) 0.59 U mL-1 (f) 0.74 U mL-1) in the presence of 0.2 mM H2O2. Fig. 2. CUPRAC absorbance versus incubation time curves of H2O2 alone (▲) and H2O2 subjected to the reaction in the presence of CAT (0.59 U mL-1) (●). Fig. 3. Optical sensor‒based CUPRAC activities of catalase versus expected ones for kidney homogenate at different dilutions (r = 0.9994). Fig. 4. The CAT activity of some tissue homogenates (1:500 diluted homogenate) calculated with the optical sensor‒based CUPRAC method in comparison to that with the UV method. Data are presented as (mean ± SD) (error bars), N=3. Fig. 5. Calibration curves of CAT alone and in liver homogenate with respect to the optical sensor‒based CUPRAC method. Fig. 6. HPLC chromatograms for standard and remaining H2O2 after reaction in the presence of CAT (7.28 U mL-1): (a) 5.0×10-2 M H2O2 (standard) (b) 5.0×10-2 M H2O2 + 0.1 mL CAT (c) 5.0×10-2 M H2O2 + 0.3 mL CAT (d) 5.0×10-2 M H2O2 + 0.4 mL CAT (e) 5.0×10-2 M H2O2 + 0.5 mL CAT.
21
Fig. 1. Visible spectra of Cu(I)-Nc chelate produced as a result of optical sensor‒based CUPRAC reaction with varying concentrations of CAT enzyme ((a) 0 U mL-1 (b) 0.15 U mL-1 (c) 0.30 U mL-1 (d) 0.44 U mL-1 (e) 0.59 U mL-1 (f) 0.74 U mL-1) in the presence of 0.2 mM H2O2.
22
1.0
Absorbance
0.8
0.6
0.4
0.2
0.0 0
10
20
30
40
50
Time (min)
Fig. 2. CUPRAC absorbance versus incubation time curves of H2O2 alone (▲) and H2O2 subjected to the reaction in the presence of CAT (0.59 U mL-1) (●).
23
24
Found Catalase Activity (U/mL)
22 20 18 16 14 12 10 8 6 4 2 0 0
2
4
6
8
10
12
14
16
18
20
22
24
Expected Catalase Activity (U/mL)
Fig. 3. Optical sensor‒based CUPRAC activities of catalase versus expected ones for kidney homogenate at different dilutions (r = 0.9994).
24
Fig. 4. The CAT activity of some tissue homogenates (1:500 diluted homogenate) calculated with the optical sensor‒based CUPRAC method in comparison to that with the UV method. Data are presented as (mean ± SD) (error bars), N=3.
25
0.8
Catalase Catalase+Tissue
0.7
Absorbance
0.6
0.5
0.4
0.3
0.2 0.1
0.2
0.3
0.4
0.5
Catalase (U/mL)
Fig. 5. Calibration curves of CAT alone and in liver homogenate with respect to the optical sensor‒based CUPRAC method.
26
Fig. 6. HPLC chromatograms for standard and remaining H2O2 after reaction in the presence of CAT (7.28 U mL-1): (a) 5.0×10-2 M H2O2 (standard) (b) 5.0×10-2 M H2O2 + 0.1 mL CAT (c) 5.0×10-2 M H2O2 + 0.3 mL CAT (d) 5.0×10-2 M H2O2 + 0.4 mL CAT (e) 5.0×10-2 M H2O2 + 0.5 mL CAT.
27
RESEARCH HIGHLIGHTS 1) Catalase activity measurement is an important part of ROS detoxification process. 2) An optical sensor was designed for catalase activity detection with CUPRAC method. 3) Developed sensor response to H2O2 concentration was linear. 4) Colorimetric sensor was statistically validated against HPLC and UV methods.
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Graphical abstract
29
S1386-1425(14)00752-5 http://dx.doi.org/10.1016/j.saa.2014.04.178 SAA 12143
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received Date: Revised Date: Accepted Date:
19 March 2014 25 April 2014 30 April 2014
Please cite this article as: B. Bekdeşer, M. Özyürek, K. Güçlü, F. Alkan, R. Apak, Development of a new catalase activity assay for biological samples using optical CUPRAC sensor, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa.2014.04.178
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Development of a new catalase activity assay for biological samples using optical CUPRAC sensor Burcu Bekdeşer1, Mustafa Özyürek1, Kubilay Güçlü1*, Fulya Üstün Alkan2, Reşat Apak1 1
Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar 34320, Istanbul,
Turkey 2
Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Istanbul University,
Avcılar 34320, Istanbul, Turkey
ABSTRACT A novel catalase activity assay was developed for biological samples (liver and kidney tissue homogenates) using a rapid and low-cost optical sensor−based ‘cupric reducing antioxidant capacity’ (CUPRAC) method. The reagent, copper(II)-neocuproine (Cu(II)-Nc) complex, was immobilized onto a cation-exchanger film of Nafion, and the absorbance changes associated with the formation of the highly-coloured Cu(I)-Nc chelate as a result of reaction with hydrogen peroxide (H2O2) was measured at 450 nm. When catalase was absent, H2O2 produced the CUPRAC chromophore, whereas catalase, being an effective H2O2 scavenger, completely annihilated the CUPRAC signal due to H2O2. Thus, the CUPRAC absorbance due to H2O2 oxidation concomitant with Cu(I)-Nc formation decreased proportionally with catalase. The developed sensor gave a linear response over a wide concentration range of H2O2 (0.68-78.6 µM). This optical sensor−based method applicable to tissue homogenates proved to be efficient for low hydrogen peroxide concentrations (physiological and nontoxic levels) to which the widely used UV method is not accurately responsive. Thus, conventional problems of the UV method arising from relatively low sensitivity and selectivity, and absorbance disturbance due to gaseous oxygen evolution were overcome. The catalase findings of the proposed method for tissue homogenates were statistically alike with those of HPLC. Keywords: Catalase activity, Optical sensor, Cupric reducing antioxidant capacity (CUPRAC) assay, Nafion membrane film. *
Corresponding authors: Tel: +90 212 473 7070, Fax: +90 212 473 7180
E-mail addresses: [email protected]
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Introduction
Hydrogen peroxide (H2O2) is one of the most frequently occurring reactive oxygen species in the biosphere. It emerges in the environment as a by-product of aerobic metabolism in respiratory and photosynthetic electron-transport chains, and of enzymatic activity mainly by oxidases. Both excessive hydrogen peroxide and its decomposition product hydroxyl radical (•OH), formed in a Fenton-type reaction, are harmful for almost all cell components [1]. Reactive oxygen species (ROS) give rise to severe damage to the cell membrane as well as to biological macromolecules like lipid, protein and DNA. Enzymatic antioxidant defense systems can eliminate the hazards of excessive ROS, e.g., superoxide dismutase eliminates superoxide anion radical by dismutation to H2O2 and O2, and the accumulation of H2O2 is prevented by the actions of catalases and peroxidases [2]. Catalase (EC 1.11.1.6, CAT) is an ubiquitous antioxidant enzyme present in most aerobic cells, and is involved in the detoxification of H2O2. Catalase catalyzes the dismutation of H2O2 by two types of reactions. Both reactions include a first step of formation of an intermediate (compound I) consisting of the enzyme and H2O2. The catalytic activity associated with 2 moles H2O2 ends up with reduction to 1 mole H2O and oxidation to 1 mole O2 in the overall reaction [3]. CAT + H2O2 → compound I + H2O compound I + H2O2 → CAT + H2O + O2 Net reaction: 2 H2O2 → 2 H2O + O2 (catalytic activity) The peroxidatic activity of catalase involves the catalytic reaction of compound I with hydrogen donors (AH2 such as methanol, ethanol, phenols) other than H2O2 [4,5]. compound I (CAT-H2O2) + AH2 → CAT + 2 H2O + A (peroxidatic activity)
2
Catalase can be found in all aerobic microorganisms, plant, and animal cells and is especially concentrated in the liver, kidney and erythrocyte [6]. It is generally regarded as the primary enzymatic defense against H2O2 generation [7]. Moreover, measurement of changes in endogenous antioxidant enzyme activity can be used to indirectly quantify ROS [8]. Thus, the measurement of catalase activity in biological samples is important. A large number of assays were developed for the determination of catalase activity [9-11]. The most common method for detection of catalase activity is the UV spectrophotometric method making use of the change of 240 nm−absorbance when H2O2 is consumed by catalase. This assay is not suitable for the measurement of CAT activity of UV-absorbing substances causing interference [12]. Moreover, the UV method has low H2O2 sensitivity and cannot measure concentrations below 1.0 mM H2O2 (i.e. at physiological and nontoxic levels). The unphysiologically high substrate concentrations in the millimolar range result in rapid CAT inactivation. Additionally, molecular oxygen may be liberated in gaseous form leading to disturbance of absorbance [6,9]. Catalase activity may be measured quantitatively by the conventional method of Von Euler and Josephson (1927) via permanganate titration of the remaining excess of H2O2 [13], which may suffer from human error in end-point reading [14]. Determination of peroxidatic function of catalase is based on the reaction of the enzyme with methanol (acting as H-donor), where the produced formaldehyde is measured at 550 nm using 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (Purpald reagent) as chromogen [4]. A commercially available [15] fluorescent detection kit for catalase activity makes use of diluted horseradish peroxidase (HRP) to react with a substrate in the presence of H2O2 to convert the colorless substrate into a fluorescent product (λex=570 nm, λem=590 nm), where increasing levels of catalase in the samples causes a decrease in H2O2 concentration and a reduction in fluorescent product formation. However, HRP−linked assays have certain disadvantages, such as those mentioned by Halliwell, i.e. peroxidase can also oxidize certain substrates without
3
neecssarily adding H2O2 [16]. In addition, several biological compounds including thiols and ascorbate can serve as substrate for HRP and thus compete with the probe for oxidation, leading to underestimation of H2O2 formation [17]. In the literature, there are a number of enzyme-based electrochemical sensors for H2O2, manufactured by immobilizing catalase on the modified electrode, which are naturally not directly applicable to catalase assay in complex samples. As one of the few exceptions, Cohen & Weber (1993) described the use of a gold-coated optical fiber as a reagentless photoelectrochemical sensor for the in situ photogeneration and electrochemical detection of the catalase enzyme substrate, hydrogen peroxide [18]. The H2O2 was generated from the photochemical reduction of molecular oxygen, mediated by tris(2,2’-bipyridyl)ruthenium: Ru(bpy)32+, isolated on the device with the aid of the cation-exchange membrane: Nafion, where the peroxide was detected amperometrically. The peroxide oxidation current was sensitive to catalase; about 25 nM catalase could rapidly reduce the device's photosignal by 50%. The reversible hydrogen peroxide optical sensor described by Mills et al. (2007), based on the fluorescent quenching of the dye ion-pair [Ru(bpy)32+(Ph4B−)2], by O2 produced by the catalytic breakdown of H2O2, was not so sensitive: it could detect H2O2 over a range of 0.01 to 1 M, with a calculated lower limit of detection of 0.1 mM [19]. A paper-based colorimetric detection device for H2O2 vapor sensing relies upon the cellulose microfibril network of paper towels, which provide a tunable interface for modification with Ti(IV)-oxo complexes for reacting with H2O2 and yielding maximal absorbance at 400 nm [20], but a linear concentration/response curve could not be observed. Consequently, the idea in the present study is to develop a novel catalase activity assay for biological samples (such as liver and kidney tissue) using an optical sensor−based ‘cupric reducing antioxidant capacity’ (CUPRAC) method, which has originally emerged as a widely applicable antioxidant capacity assay [21] but also given rise to many other modified (e.g.,
4
antioxidant and antiradical activity) assays. One of these modifications is the optical sensor−based CUPRAC method using an immobilized chromogenic reagent, copper(II)neocuproine (Cu(II)-Nc) complex on a cation-exchanger polymer membrane (Nafion), for the assessment of antioxidant capacity of non-enzymatic antioxidants [22]. The tissue homogenates were evaluated for their catalase activity using the proposed sensing method −associated with the changes of the CUPRAC absorbance due to H2O2 oxidation concomitant with Cu(I)-Nc formation on the membrane− in comparison with the UV reference method.
Experimental details Reagents and apparatus The following chemicals of analytical reagent grade were purchased from the corresponding sources: Neocuproine (2,9-dimethyl-1,10-phenanthroline) (Nc), ethanol (EtOH), Catalase from bovine liver (CAT), Nafion 115 perfluorinated membran (thickness 0.005 in.): Sigma-Aldrich (St. Louise, USA); copper(II) chloride dihydrate, hydrogen peroxide (35% by wt.), Na2HPO4, NaH2PO4.2H2O, ammonium acetate: Merck (Darmstadt, Germany). The biological samples were kindly donated by the Faculty of Veterinary Medicine of Istanbul University. The visible spectra and absorption measurements were recorded using a Varian CARY Bio 100 UV-Vis spectrophotometer (Mulgrave, Victoria, Australia). Mixing was performed with a BIOSAN Programmable rotator-mixer Bulti Bio RS-24 (Riga, Latvia). The chromatograph was from A Waters Breeze TM 2 Model HPLC system (Milford, MA, USA) equipped with a 1525 binary pump, a 2465 Electrochemical Detector (ECD) (Chelmsford, MA, USA), a Hamilton 25mL-syringe (Reno, NV, USA), and reverse phase ACE C18 column (4.6 mm×250 mm, 5 μm particle size) (Milford, MA, USA) was used for 5
chromatographic measurements. Data acquisition was accomplished using Empower PRO (Waters Associates, Milford, MA).
Preparation of solutions
The original catalase solution of initial activity 3691 U mg-1 solid was diluted with 0.2 M phosphate buffer (pH 7.4) to a concentration of 738 U mL-1. Hydrogen peroxide at 1.0 mM concentration was prepared from a 0.5 M intermediary stock solution, the latter being prepared from 35% H2O2 and standardized with permanganate titration. The NaH2PO4– Na2HPO4 buffer solution (pH 7.4) at 200 mM was prepared in distilled water. Copper(II) at 1.0×10−2 M was prepared by dissolving 0.4262 g of CuCl2·2H2O, and diluting to 250 mL. Ammonium acetate (NH4Ac: 1.0 M) aqueous solution contained 19.27 g of the salt in 250 mL. Neocuproine (Nc) at 7.5×10−3 M was prepared by dissolving 0.078 g of the free base in EtOH, and diluting to 50 mL with ethanol (prepared fresh). The mobile phase for HPLC analysis with isocratic elution was prepared from 50 mM ammonium acetate. This solution was prepared by dissolving 0.9635 g of ammonium acetate in 250 mL of bidistilled water.
Preparation of tissue homogenates
CD-1 mice were obtained from the Faculty of Veterinary Medicine of Istanbul University. The mice were housed in polycarbonate cages (450 cm2 area per animal), acclimatized under laboratory conditions (23±2 °C, humidity 50-60 %, 12 light/dark cycle), and fed with water and standard mice food. Liver and kidney tissues were isolated after sacrifice by decapitation from mice. The tissue samples were washed with 0.9% (w/v) NaCl solution, weighed and homogenized by adding cold 1.15% (w/v) KCl solution in a glass
6
homogenizer. Homogenates were immediately frozen in liquid nitrogen and kept at −80 °C until analysis [23]. Homogenates were filtered through a 0.45 µm membrane filter and diluted (at a ratio of 1:500) with ammonium acetate (1.0 M) solution before analysis. There was no effect of tissue homogenate on the CUPRAC absorbance of hydrogen peroxide because of the high dilution ratio.
Preparation of the CUPRAC sensor
The commercial Nafion membrane was cut into sliced 4.5×0.5 cm parts, engrossed into a tube containing 2.0 mL of 1.0×10-2 M Cu(II) + 2.0 mL of 7.5×10-3 M Nc + 2.0 mL of 1 M NH4Ac + 2.2 mL of H2O, and agitated for 30 min in a rotator [22].
Optical sensor based CUPRAC (proposed) method
To a test tube were added 0.5 mL of 1.0 mM H2O2, (2−x) mL H2O, x mL catalase solution at different concentrations (x varying between 0.1 and 2.0 mL) or tissue homogenate in this order. The reaction mixture in a total volume of 2.5 mL was incubated for 30 min in a water bath kept at 25◦C. At the end of this period, the reagent-impregnated membrane (developed CUPRAC sensor) was taken out and immersed in a tube containing 2.0 mL of the reaction mixture + 6.2 mL of EtOH. The tube was placed in a rotator and agitated for 30 min so as to achieve full color development. The colored membrane was taken out and placed in a 1 mm optical cuvette containing H2O (to prevent sticking of slices to the walls of the cuvette), and its absorbance at 450 nm was read against that of a blank membrane prepared under identical conditions excluding analyte. The absorbance values were linearly correlated to H2O2 concentrations, and the decrease in absorbance was due to catalase activity.
7
UV (reference) method
The UV method was applied as the reference method of comparison for determining the catalase enzyme activity of biological samples. The reaction mixture for the UV method [24] contained in a final volume of 3.0 mL the following reagents: 1.5 mL pH 7.4 NaH2PO4– Na2HPO4 buffer solution (200 mM), 0.5 mL H2O2 (75 mM), x mL catalase solution at various concentrations (x varying between 0.1 and 2.0 mL) or tissue homogenate, (1−x) mL H2O. Reaction mixtures were incubated at 25 oC for ½ h. At the end of the incubation period, the absorbance of the resulting solution was measured at 240 nm. The absorbance values were linearly correlated to H2O2 concentrations, and the decrease in absorbance was due to catalase activity.
HPLC method
A suitable aliquot of the incubation mixture solution was injected (injection volume: 20 μL) without derivatization (i.e. without conversion into a fluorophore or a chromophore, enabling post-column detection) into the HPLC column (5 mm ODS) at ambient temperature, and isocratic elution was employed for detection of the hydrogen peroxide. The HPLC–ECD method developed by Yue et al. (2009) [25] was adapted with slight modifications, namely the use of a reverse phase ACE C18 column (4.6 mm×250 mm, 5 μm particle size) (Milford, MA, USA) in conjunction with an ECD system comprising Ag/AgCl reference and glassy carbon working electrodes. The mobile phase consisting of 50 mM ammonium acetate in water was delivered at a rate of 1 mL min-1 (as ammonium ions may cause the precipitation of proteins, statistical comparison of the proposed method with HPLC was performed only in synthetic solutions of CAT). The column effluent was pumped to an ECD detector (as
8
recommended by Takahashi et al., 1999) [26]. The detector was operated in direct current (DC) mode. Each run was 5 min long. Quantification of H2O2 was performed using the data generated at 500 mV potential [25].
Statistical analysis
Statistical analyses were realized using Excel software (Microsoft Office 2002) for calculating the means and the standard error of the mean. Results were expressed as the mean ± standard deviation (SD). Using SPSS software for Windows (version 13), the data were interpreted by two-way ANalysis Of VAriance (ANOVA) [27].
Results and discussion
Optimization of the optical sensor‒based CUPRAC method parameters
Catalase enzyme activity was determined by making use of the decrease in CUPRAC absorbance of H2O2 in the presence of varying CAT concentrations. Although H2O2 is essentially known to be a strong oxidant, it can act as a reductant under favorable redox potentials, e.g., toward Cu(II)-neocuproine. The molar absorption coefficient (i.e. ΔA/Δc) of the CUPRAC sensing method for hydrogen peroxide was: ε = 1.73×104 Lmol-1 using a Nafion membrane, and linear concentration range: 6.81×10-7-7.86×10-5 M (r = 0.9922). Considering the preconcentration function of the Nafion membrane (i.e. for the cuprous-neocuproine chromophore) and its average thickness (0.1 cm), the effective molar absorptivity of the method for H2O2 is raised to the level of 1.73x105 M-1cm-1. Hydrogen peroxide is CUPRAC reactive, but its degradation products (H2O and O2) are not. The CUPRAC absorbance of 9
H2O2 is attenuated as a result of its reaction with CAT (Fig. 1). The attenuated absorbance of H2O2 is dependent upon the CAT activity of the tested sample. The linear equation for the calibration graph of CAT drawn at the wavelength of 450 nm with respect to the optical sensor–based CUPRAC method was:
A450 = −1.00 c + 0.8655 (r = 0.9861)
where c is CAT concentration (U mL-1). The limit of detection (LOD) and limit of quantification (LOQ) of the optical sensor based CUPRAC method for CAT were found to be 0.044 and 0.145 U mL-1, respectively. The precision data of the proposed assay (expressed as the % RSD values) was approximately 2.29%. On the other hand, the intra-day and inter-day precisions of the commercial catalase kit [15] for 0.6-2.9 U mL-1 activities of catalase buffer were in the ranges of 3.2-5.9% and 7.3-7.8%, respectively, indicating a distinct improvement of precision in the proposed method. Recovery values with the CUPRAC sensor varied from 96.5 to 115.0%.
Figure 1 Conversion of H2O2 to reaction products (i.e. H2O and oxygen) in the presence of CAT was followed by measuring the CUPRAC absorbance of the mixture as a function of time (Fig. 2). An optimal measurement time of 30 min was chosen during which an absorbance plateau was reached due to annihilation of H2O2 by CAT.
Figure 2
10
Interferences in the optical sensor–based CUPRAC method
For the developed catalase activity assay, the possible interference effects of concomitant species commonly found in biological samples on the determination of 0.30 U mL-1 are shown in Table 1. As opposed to peroxidase based assays [17], the presence of vitamin and plasma antioxidants commonly found in biological samples did not interfere with the catalase activity assay using the proposed method.
Table 1
Linearity on tissue homogenate dilution
The dilution sensitivity of kidney homogenate was evaluated using the optical sensor– based CUPRAC method, and the found catalase activities (in U mL-1) were recorded against expected catalase activity at varying dilutions of the kidney homogenate (Fig. 3). Excellent linear curves were observed passing through the origin, i.e. having a higher linearity correlation (r = 0.9994) than that of the commercial catalase kit, where the observed versus expected catalase activity curve had a correlation coefficient of r = 0.9553 [15].
Figure 3
Determination of catalase activity in tissue homogenates The catalase activity of tissue homogenates (liver and kidney) was determined by the optical sensor–based CUPRAC and UV methods. Liver homogenates were generally shown to exhibit higher CAT activity than kidney (Fig. 4), because the liver is generally regarded as 11
the major organ of detoxication [28] and catalase in liver homogenate effectively detoxifies H2O2. Casalino et al. (2002) found that the CAT activity of rat liver tissue homogenates (383±39.1 unit per mg protein) was distinctly higher than that of kidney homogenates (158.2±16.1 unit per mg protein) [29]. The CAT activity values measured with the proposed optical sensor–based CUPRAC method and reference method (UV method) were comparatively depicted in a bar diagram (Fig. 4). The order of CAT activities of tissue homogenates found with the proposed CUPRAC method were compatible with those measured by the UV method.
Figure 4 The UV method for detection of CAT activity depends on the change of the 240 nmabsorbance when H2O2 is consumed by catalase, and the relatively high activities measured by UV (Fig. 4) probably arise from the positive interference by UV-absorbing substances. The absorbance of H2O2 at 240 nm is measured directly to calculate CAT activity since the reaction products, i.e. H2O and oxygen, do not absorb at this wavelength. The molar absorptivity for H2O2 was: ε = 37 Lmol-1cm-1, and the linear concentration range: 5.08×10-43.1×10-2 M (r = 0.9998). UV spectrophotometry for H2O2 estimation is the most preferred but interference–affected method [6]. Standard calibration curves of CAT were redrawn in liver homogenate showing good parallelism of linear curves (Fig. 5), indicating that the constituents of the real matrix solution (homogenate) did not interact to produce chemical deviations from Beer’s law.
Figure 5
12
Membrane stability and chemical resistance against H2O2
The properties that make the Nafion membrane indispensable are the combination of good water uptake, ion-exchange capacity, proton conductivity, low gas permeability, and excellent electrochemical stability [30]. Nafion was found to be thermally stable up to 280 °C, at which temperature the sulfonic acid groups began to decompose [31]. After protonated Nafion membrane was tested in 30% H2O2 solution at 80 °C for 12 h, small amounts of fluoride ion and sulfate ion, which were derived from the C−F single bonds and the sulfonic acid groups, respectively, of the membrane, were detected in solution [32]. Kinumoto et al. (2006) further pointed out to a more serious decomposition possibility −via radical depolymerization mechanism− of the Nafion membrane by hydroxyl and hydroperoxyl radicals generated from Fenton-type reactions of {transition metal ion+H2O2 (concentrated)} combinations [32]. The chemical stability of Nafion membrane was also shown in the H2 and O2 environments [33]. To test the possible effect of H2O2 on a Nafion membrane, one of the membranes used in the original study was treated with 0.5 mL of 1 mM H2O2 for 30 min. CUPRAC sensor was prepared by using this membrane and an unloaded membrane as described in ‘Preparation of CUPRAC sensor’. Catalase activity of (1:500 diluted) kidney homogenate was measured and found with H2O2-treated and untreated sensors as 16.01 and 15.98 U/mL, respectively. Our experimental results showed that Nafion membrane was not affected by H2O2 within the normal time period of the test protocols (and Fenton-type degradation reactions were not possible considering that the cupric-neocuproine cationic chelate but not free Cu2+ was coated on the membrane), and no significant change on catalase activity was found with the use of this sensor for tissue homogenates. Once the cupric-neocuproine adsorbed on the membrane was reduced by hydrogen peroxide to the stable cuprous-chelate, it did not enter a back
13
reaction with hydrogen peroxide producing reactive species [34]. The reagent-loaded (singleuse) sensor was tested for stability, and was shown to lose only 3% signal intensity after 15 days-storage in distilled water kept in a dessicator at room temperature.
Comparison of the results of optical sensor–based CUPRAC and HPLC methods
The chromatograms of the original and remaining H2O2 after CAT reaction for validating the developed assay (Fig. 6) showed that the retention time for the H2O2 was 3.00 min. The amount of H2O2 was found with the aid of calibration curve (y = 8.10×107 c + 97864 (r = 0.9992)) drawn as peak area versus concentration. Catalase activity was manifested by the decrease in both the absorbance of the CUPRAC chromophore and in the chromatographic peak area due to H2O2 as a result of its transformation to H2O and oxygen. The degradation ratio of H2O2 depended upon the amount of CAT. Concentrations of H2O2 (the initial concentration of H2O2 was 10-2 M) remaining after CAT reaction, found with the proposed CUPRAC and reference HPLC methods, were compatible with each other (Table 2). The two-way ANalysis Of VAriance (ANOVA) comparison, via F-test of the mean-squares of “between-treatments” (i.e. remaining H2O2 concentration, with respect to the optical sensor–based CUPRAC and HPLC methods depicted in Table 2) and of residuals enabled us to conclude that there was no significant difference between treatments. The experimentally found optical sensor–based CUPRAC results and HPLC results were statistically alike at the 95% confidence level (Fexp = 7.89, Fcrit (table)
= 10.13, Fexp < Fcrit (table) at P = 0.05). The hydrogen peroxide concentration found by the
proposed method correlated well (r = 0.9976) with those of the HPLC method (Table 2). Thus, the proposed methodology was validated against the reference HPLC method. Both methods correctly reflected the relative decrease in H2O2 concentration as a result of
14
conversion into H2O and oxygen. For example, the conversion efficiency of H2O2 found with the optical sensor–based CUPRAC and HPLC methods were 54.5% and 51.5%, respectively, in the presence of 1.48 U mL-1 CAT.
Figure 6 Table 2 Conclusion
The Cu(II)-Nc reagent was electrostatically immobilized onto a cation exchanger Nafion membrane, and was demonstrated to retain its reactivity towards hydrogen peroxide after incorporation into the exchanger membrane. The absorbance of Cu(I)-Nc produced from intact H2O2 decreased proportionally with CAT activity. This offers advantages over solution–based assays, including the potential of being used as a rapid, low-cost, diversely and easily applicable method, and of future refinements of the assay to online applications and kit format. Currently, there is no commercial catalase sensor (e.g., like blood glucose sensor) in the market for spectrophotometrically (or colorimetrically) detecting/quantitating CAT in complex biological samples. Naturally with the use of electrochemical techniques enabling quantitative H2O2 determination, catalase‒ or peroxidase‒immobilized electrodes cannot be used for sensing catalase. The proposed method had better linearity and higher precision than that of the commercially available catalase kit utilized in solution phase. In conclusion, the proposed optical sensor–based method can be considered as a precise and accurate alternative for measuring the CAT activity of biological samples. We believe that this simple sensor– based assay for catalase activity measurement is a useful tool for investigating the role of catalase enzyme and its regulation under physiological conditions in various biological 15
systems, and opens the way for the future development of dip-stick type point-of-care diagnostic kits.
Acknowledgements
The authors thank TUBITAK (Turkish Scientific and Technical Research Council) for the support given to the Research Project 110T725. The authors also extend their gratitude to T.R. Ministry of Development for the Advanced Research Project of Istanbul University (2011K120320).
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Table 1 Possible interferences of some biologically important compounds toward the optical sensor‒based CUPRAC method. Interferent
Concentration
Ascorbic acid Heparin EDTA Bilirubin Glucose Uric Acid Albumin
2.0 μM 78.4 USP/10 mL 20.0 μM 0.4 μM 0.35 mg mL-1 4.0 μM 0.4 mg mL-1
Added catalase U mL-1 0.30 0.30 0.30 0.30 0.30 0.30 0.30
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Found catalase U mL-1 0.29 0.29 0.31 0.29 0.30 0.31 0.30
Relative error (%) -3.45 -3.45 3.22 -3.45 0 3.22 0
Table 2 Determination of H2O2 in the presence of CAT by using optical sensor‒based CUPRAC and HPLC methods (N=3). CAT (U mL-1) 0 0.30 0.89 1.18 1.48
H2O2 concentration found with respect to optical sensor‒based CUPRAC method (mM) 10.0±0.42 8.65±0.35 7.01±0.28 6.25±0.24 5.45±0.22
P = 0.05, Fexp = 7.89 Fcrit (table) = 10.13 Fexp < Fcrit (table) [H2O2]CUPRAC = 1.03[H2O2]HPLC − 0.38 (r = 0.9976).
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H2O2 concentration found with respect to HPLC method (mM) 10.0±0.32 8.50±0.28 6.95±0.31 6.16±0.29 5.15±0.17
Figure Captions: Fig. 1. Visible spectra of Cu(I)-Nc chelate produced as a result of optical sensor‒based CUPRAC reaction with varying concentrations of CAT enzyme ((a) 0 U mL-1 (b) 0.15 U mL-1 (c) 0.30 U mL-1 (d) 0.44 U mL-1 (e) 0.59 U mL-1 (f) 0.74 U mL-1) in the presence of 0.2 mM H2O2. Fig. 2. CUPRAC absorbance versus incubation time curves of H2O2 alone (▲) and H2O2 subjected to the reaction in the presence of CAT (0.59 U mL-1) (●). Fig. 3. Optical sensor‒based CUPRAC activities of catalase versus expected ones for kidney homogenate at different dilutions (r = 0.9994). Fig. 4. The CAT activity of some tissue homogenates (1:500 diluted homogenate) calculated with the optical sensor‒based CUPRAC method in comparison to that with the UV method. Data are presented as (mean ± SD) (error bars), N=3. Fig. 5. Calibration curves of CAT alone and in liver homogenate with respect to the optical sensor‒based CUPRAC method. Fig. 6. HPLC chromatograms for standard and remaining H2O2 after reaction in the presence of CAT (7.28 U mL-1): (a) 5.0×10-2 M H2O2 (standard) (b) 5.0×10-2 M H2O2 + 0.1 mL CAT (c) 5.0×10-2 M H2O2 + 0.3 mL CAT (d) 5.0×10-2 M H2O2 + 0.4 mL CAT (e) 5.0×10-2 M H2O2 + 0.5 mL CAT.
21
Fig. 1. Visible spectra of Cu(I)-Nc chelate produced as a result of optical sensor‒based CUPRAC reaction with varying concentrations of CAT enzyme ((a) 0 U mL-1 (b) 0.15 U mL-1 (c) 0.30 U mL-1 (d) 0.44 U mL-1 (e) 0.59 U mL-1 (f) 0.74 U mL-1) in the presence of 0.2 mM H2O2.
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1.0
Absorbance
0.8
0.6
0.4
0.2
0.0 0
10
20
30
40
50
Time (min)
Fig. 2. CUPRAC absorbance versus incubation time curves of H2O2 alone (▲) and H2O2 subjected to the reaction in the presence of CAT (0.59 U mL-1) (●).
23
24
Found Catalase Activity (U/mL)
22 20 18 16 14 12 10 8 6 4 2 0 0
2
4
6
8
10
12
14
16
18
20
22
24
Expected Catalase Activity (U/mL)
Fig. 3. Optical sensor‒based CUPRAC activities of catalase versus expected ones for kidney homogenate at different dilutions (r = 0.9994).
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Fig. 4. The CAT activity of some tissue homogenates (1:500 diluted homogenate) calculated with the optical sensor‒based CUPRAC method in comparison to that with the UV method. Data are presented as (mean ± SD) (error bars), N=3.
25
0.8
Catalase Catalase+Tissue
0.7
Absorbance
0.6
0.5
0.4
0.3
0.2 0.1
0.2
0.3
0.4
0.5
Catalase (U/mL)
Fig. 5. Calibration curves of CAT alone and in liver homogenate with respect to the optical sensor‒based CUPRAC method.
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Fig. 6. HPLC chromatograms for standard and remaining H2O2 after reaction in the presence of CAT (7.28 U mL-1): (a) 5.0×10-2 M H2O2 (standard) (b) 5.0×10-2 M H2O2 + 0.1 mL CAT (c) 5.0×10-2 M H2O2 + 0.3 mL CAT (d) 5.0×10-2 M H2O2 + 0.4 mL CAT (e) 5.0×10-2 M H2O2 + 0.5 mL CAT.
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RESEARCH HIGHLIGHTS 1) Catalase activity measurement is an important part of ROS detoxification process. 2) An optical sensor was designed for catalase activity detection with CUPRAC method. 3) Developed sensor response to H2O2 concentration was linear. 4) Colorimetric sensor was statistically validated against HPLC and UV methods.
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Graphical abstract
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