ARTICLE pubs.acs.org/ac
Detection and Quantification of Carboxylesterase 2 Activity by Capillary Electrophoresis Joana Lamego, Ana Sofia Coroadinha, and Ana Luísa Simplício* Instituto de Biologia Experimental e Tecnologica, Instituto de Tecnologia Química e Biologica, Universidade Nova de Lisboa, Avenida da Republica, Estac-~ao Agronomica Nacional, 2780-157 Oeiras, Portugal
bS Supporting Information ABSTRACT: The purpose of this study was to develop an analytical method to quantify the relative activities of carboxylesterases (CESs) in biological samples. Taking the advantage of loperamide, a specific carboxylesterase 2 (CES2) inhibitor, and bis-p-nitrophenyl phosphate (BNPP), an irreversible CESs inhibitor, we propose for the first time a capillary electrophoresis (CE) method that enables detecting and distinguishing CES2 activity from other CESs in complex biological samples. The capillary electrophoresis method proved to be fast, simple, repeatable, and applicable to the measurement of the specific activity of CESs. The method was successfully applied to the evaluation of human cells overexpressing human carboxylesterase 2 (hCE-2) and to several mammalian sera, using extremely small amounts of samples in comparison with traditional spectrophotometric methods. The same rationale can be applied to establish methods for determining the activity of other isoenzymes, using the appropriate specific inhibitors.
ammalian carboxylesterases (CESs) are R,β-hydrolasefolded proteins involved in the efficient hydrolysis of xenobiotics and endogenous ester- or amide-containing compounds.1-3 The classification of these enzymes is difficult due to the large number of isoenzymes, complex tissue distribution, and overlapping substrate preferences.1,4 The most recent classification of CESs categorizes these enzymes into five subfamilies, from CES1 to CES5, according to their sequence homology.3 CESs merit close attention because of their important functions in the detoxification of narcotics and other drugs, metabolism of pyrethroids, and prodrug activation.5,6 Several studies have been conducted to compare the enzymatic activities of the different CESs in mammals, due to the implications that these differences may have for (pre)clinical results.2,7,8 More recently, these enzymes have gained additional attention in the cancer treatment area. It has been discovered, for example, that the levels of some isoenzymes are different in certain tumors: human carboxylesterase 1 (hCE-1) protein levels have been found to be increased in patients suffering from hepatocellular carcinoma.9 Moreover, a correlation was established between hCE-2 expression in solid tumors and the activation of the anticancer prodrug Irinotecan (CPT-11),10 and this has led to the investigation into enzyme/prodrug strategies in which the goal is to target CESs to tumors prior to the treatment with the prodrug.11 hCE-2, the major human intestinal CES, thus attracts considerable attention. It has
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r 2011 American Chemical Society
been suggested that, prior to the treatment of human lung cancer cells with CPT-11, it is crucial to check if the cells express this enzyme.1 Considering the current trends, it becomes evident that fast, reliable, economical, and easy to perform analytical methods, enabling the evaluation of hCE-2 activity, will be needed for routine application in the near future. There are currently several ways to assess hCE-2 expression levels and activities,12 such as simple spectrophotometric assays,13 native in-gel hydrolysis assays,14 Western blot assays,15 real-time polymerase chain reaction (RT-PCR),7 and Northern blot assays.16 Simple spectrophotometric assay enables one to quantify total carboxylesterase activities, but hardly identify the relative activity of each specific carboxylesterase in a complex biological sample or enzyme mixture, since in the majority of cases overlapping absorbance occurs when inhibitors are used. Native in-gel hydrolysis assay allows one to distinguish the activities of the different carboxylesterases in complex biological samples, but no quantification assessment is possible. With Western blot assays it is possible to detect specifically the proteins, using appropriate antibodies, but only estimates concerning enzymatic activity or protein quantification are possible. RT-PCR and Northern blot assays enable one to quantify and detect, respectively, the expression Received: September 27, 2010 Accepted: December 14, 2010 Published: January 5, 2011 881
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of the proteins, through the analysis of mRNA expression profiles; once again, no information concerning enzyme activity is obtained.12 As reported before, the protein levels of CESs do not always correlate with their activity levels.14 As CESs are promiscuous enzymes with overlapping substrate preference, it becomes difficult to attribute a specific activity to one isoenzyme in complex protein samples such as cellular extracts, serum, or other biological samples.12,17 Accordingly, the classical substrate used for CESs activity, p-nitrophenyl acetate (p-NPA), is also hydrolyzed by cholinesterases, thus constituting an example of the promiscuity of these enzymes. This overlapping substrate capability often leads to contradictions in the literature;1 in fact, only in 2005 it was clearly demonstrated that human plasma does not contain CESs activity.8 In this paper, we provide a new analytical methodology for the CESs field, which involves the use of several substrates and inhibitors enabling the identification and quantification of CES2 activity in complex biological samples. Capillary electrophoresis (CE) was chosen as the separation technique since simple spectrophotometric assessment is precluded due to overlapping absorbance between some substrates and inhibitors. CE is a powerful separation technique that has established itself as an alternative to chromatographic techniques.18 CE advantages rely on the reduced solvent and sample needs, the low cost of the capillaries, in comparison with the high-performance liquid chromatography (HPLC) columns, faster method development, and versatility.19 Nonetheless, HPLC can still be used with the proposed method. The method was successfully applied to the quantification of CES2 activity in transfected cell extracts and animal sera, and the same rationale can be quickly extended to the study of other enzymes and/or substrates in other biological samples.
minimize solution deterioration. Injections were conducted hydrodynamically, at 0.2 psi for 10 s, with cathodic migration. Separations were performed at constant current (þ70.0 μA). UV absorption was measured at 230 (thiocholines), 350 (4MUB), and 400 nm (p-nitrophenol (p-NP)). Each new capillary was pretreated with NaOH 0.1 mM, rinsed with Milli Q water, and conditioned with background electrolyte solution (BES), 30 mM borate-phosphate buffer (30 mM disodium tetraborate, 30 mM sodium dihydrogen phosphate; pH 8.0),20 at a pressure of 5.0 psi for 20 min each, in a total of 60 min. This procedure was repeated at the end of each day. The capillary was conditioned between all runs, by a short sequence (30 s each) of this procedure. Data acquisition, storage, and analysis were performed using P/ACE MDQ version 7.0. All the standard solutions used were diluted in equal proportion (50% v/v) of ethanol as the samples to be analyzed. Production of Extracts from Human Cells. Human embryonic kidney cells (HEK-293T) (kindly provided by Dr. Amos Panet, from The Hebrew University of Jerusalem, Israel) were transfected as described before,2 following some modifications. In brief, cells were transfected at 60-80% of cell density with polycation polyethylenimine (PEI) (Polysciences; Eppelheim, Germany) using pDEST26-hCE2 plasmid vector. pDEST26hCE2 plasmid vector was acquired from imaGENES (Berlin, Germany) in which CES2 (geneID 8824) was cloned in pDEST26 plasmid (Invitrogen; Carlsbad, U.S.A.). Cells were harvested 48 h after transfection and chemically lysed with M-PER mammalian extraction reagent (Pierce Biotechnology; Rockford, U.S.A.). The supernatant, obtained by centrifugation at 10 000g for 10 min at 4 °C, was stored at -20 °C without the addition of protease inhibitors. Total protein concentration was determined using BCA protein assay kit (Pierce Biotechnology), according to the manufacturer’s instructions. Serum Preparation. Human, domestic cat, bovine, domestic dog, and equine sera, from one individual each, were collected without the addition of anticoagulants.8,21 Samples were stored at 4 °C for 24 h and centrifuged afterward at 3000 rpm for 15 min. Each sample was aliquoted and stored at -20 °C until analysis. Enzymatic Assays and Inhibition Studies. The enzymatic activity reactions were performed in a final volume of 150 μL, under substrate saturation conditions. The addition of each reaction component was performed in ice, and unless otherwise stated, reactions were undertaken at 37 °C for 8 min. Purified and/or recombinant enzymes, cells extracts, and serum were diluted in 50 mM Tris-HCl buffer (pH 7.4). Stock solutions of the substrates were prepared in ethanol, and stock solutions of the inhibitors were prepared in DMSO. Organic solvent percentage in the final reaction volume never exceeded 5% (v/v). A mixture of 90 mM KH2PO4 and 40 mM KCl (pH 7.3) was used as the reaction buffer.22 The inhibition studies were performed by preincubation with loperamide (250 μM) or BNPP (500 μM) at 37 °C for 15 or 10 min, respectively. The enzymatic reactions were stopped by the addition of equal percentage (v/v) of ethanol and centrifuged at 10 000g for 10 min before analysis. All samples were injected in triplicate, and average results are presented throughout the text. Intermediate precision was determined by pooled standard deviation, based on replicate independent assays preformed in three different days in two different concentrations and in the presence and absence of inhibitor. Mean t tests were performed for evaluation of the statistical significant differences (p = 0.01) between samples, considering intermediate precision.
’ MATERIALS AND METHODS Enzymes and Reagents. CES1 enzyme (esterase from porcine liver) was from Sigma (Buchs, Switzerland); butyrylcholinesterase (BuChE) from equine serum and acetylcholinesterase (AcChE) from electric eel were also from Sigma (St. Louis, U.S.A.). Recombinant human carboxylesterase 2 (hCE-2) was from R&D Systems, Inc. (Minneapolis, U.S.A.). All reagents were analytical grade or higher. 4-Methylumbelliferyl acetate (4-MUBA) (g98%), 4-methylumbelliferone (4MUB) (g98%), p-NPA (>99%), Trizma hydrochloride (>99%), and Trizma base (>99.9%) were from Sigma (St. Louis, U.S.A.). S-Butyrylthiocholine (BuTCh) iodide (g99%) and acetylthiocholine (AcTCh) iodide (g99%) were also from Sigma (Buchs, Switzerland). Loperamide hydrochloride (analytical grade) was from Fluka (Seelze, Germany), whereas bis-p-nitrophenyl phosphate (BNPP) (>99%) and potassium chloride (g99.5%) were from Aldrich (Steinheim, Germany). Potassium dihydrogen phosphate (g99%), disodium tetraborate (g99.5%), and ethanol (99.5%) were all from Panreac (Barcelona, Spain); dimethyl sulfoxide (DMSO) (95%) was from Lab-Scan (Dublin, Ireland), and sodium dihydrogen phosphate (>99%) was from Merck (Darmstadt, Germany). Instrumentation. CE detection was performed using a Beckman Coulter (Palo Alto, U.S.A.) P/ACE MDQ capillary electrophoresis system equipped with a diode array detector. A fusedsilica capillary (Agilent; Santa Clara, U.S.A.) with an i.d. of 75 μm, and 42 cm of total length (32 cm to detector) was used. The capillary was packed in a standard cartridge and maintained at 25 °C during all experiments. The sample tray was kept at 15 °C to 882
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Figure 1. Electropherograms of substrates, products, and inhibitors detected by CE: 4-MUB (0.25 mM at 350 nm), BuTCh (0.5 mM at 230 nm), AcTCh (0.5 mM at 230 nm), and BNPP (0.25 mM at 280 nm) in a silica capillary 75 μm 42 cm, 25 °C using hydrodynamic injections (0.2 psi for 10 s). Separation was at þ70.0 μA with cathodic migration (30 mM borate-phosphate buffer, 30 mM disodium tetraborate, 30 mM sodium dihydrogen phosphate; pH 8.0).
’ RESULTS AND DISCUSSION
range, 23.4 μM to 1.5 mM 4-MUB, specifically at 1.5 mM and 750, 375, 187.5, 93.8, 46.9, and 23.4 μM. If higher sensitivity is needed, 4-MUB has the advantage of being fluorescent, and thus, using the appropriate detectors, detection and quantification limits can be further improved. Evaluation of Esterase Enzymatic Activities by CE. CESs, AcChE, and BuChE are classified as B esterases.28 As already stated, these enzymes not only show overlapping substrate preference, for example, both hCE-2 and BuChE hydrolyze CPT-11, but they also colocalize in several tissues, such as plasma, intestine, and kidney of several mammals.4 We thus further developed our method for the ability to discriminate CES2 activity from these other enzymatic activities. Commercial CESs (CES1 and hCE-2 recombinant enzyme) and cholinesterases (BuChE and AcChE) were tested toward the hydrolysis of 4-MUBA (Figure 2; Supporting Information Figure S-1). For commercial cholinesterases 50 ng of each enzyme was incubated in the same conditions as used for CESs (Supporting Information Figure S-2). Only CES enzymes were able to hydrolyze the substrate as no significant difference was detected between the spontaneous hydrolysis of 4-MUBA and the hydrolytic activity of the cholinesterases (data not shown). To distinguish specifically CES2 activity from the activity of the other carboxylesterases we propose the use of the CES2 specific inhibitor loperamide.29 Although BuChE and AcChE did not hydrolyze 4-MUBA, some biological samples may contain other enzymes that could metabolize this substrate. For this reason, we evaluated BNPP, an irreversible inhibitor specific for CESs.30,31 Taking advantage of these two cheap and commercially available inhibitors, the relative activity of CES2 can be determined in any biological sample. BNPP also absorbs at 350 nm, and therefore classical spectrophotometric enzymatic assays cannot be used. As different biological samples may contain different amounts of CESs and/or hCE-2, we decided to use high amounts of each inhibitor to ensure that maximum inhibition is achieved. Lower amounts of inhibitors can be used as demonstrated below.
Capillary Electrophoresis Method Development. There are several methods for the determination of CESs activities described in the literature, ranging from spectrophotometric11 to HPLC12 and native in-gel hydrolysis assays;12 nonetheless, to our knowledge, CES activities have never been analyzed by CE. There are substrates known to be specific for each CES. As an example, temocapril is hydrolyzed by CES1 but not by CES2;7 in contrast, procaine has been shown to be hydrolyzed by CES2 but not by CES1.23 Several efforts have been made to identify specific inhibitors that enable separation of the activities of different CESs,24-26 and there is also an effort to develop new assays that enable the evaluation of inhibitors.27 We sought to develop an analytical method that would permit the selective measurement of CES2 activity in any biological sample. Our goal was to have a simple, fast, and reliable analytical tool that would enable us to evaluate different biological samples produced in the laboratory. We started the method development using 4-MUBA as substrate, because it is more stable than the classical CESs substrate p-NPA. 4-MUBA is a general CESs substrate, and it is routinely used in native in-gel hydrolysis assays,12,13,24 but we have found that cholinesterases do not hydrolyze 4-MUBA (data not shown). To our knowledge, 4-MUBA and its hydrolysis product, 4-MUB, have not been previously analyzed by CE, and therefore we proceeded to develop a method for that purpose. When borate-phosphate buffer at pH 8.0 is used, in the CE method, 4-MUB presents an absorbance maximum at 350 nm and has a migration time of about 4.0 min (Figure 1). 4-MUBA is not detected. Equipment response was linear for this product at least in the range of 23.4 μM to 1.5 mM (R2 = 0.999). The theoretical limit of detection (13.7 μM) was calculated based on the residual standard deviation of the calibration curve and a signal-to-noise ratio of 3.3. The limit of quantification (23.4 μM) was chosen from the lowest calibration point where the coefficient of variation of repeatability (CVr) was still under 10% (n = 3). CVr was obtained in the indicated 883
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In order to demonstrate the ability of the method to distinguish between hCE-2 and CES1, we mixed the two enzymes (50 or 25 ng of hCE-2 with 12.5 ng of CES1) and performed the same set of assays, ensuring once again substrate saturation conditions (Figure 2). As expected, the incubation of BNPP with both mixtures completely inhibited enzymatic activity. The incubation of each mixture with loperamide caused a decrease in the relative percentage of activity to the same levels, in both mixtures. Although the protein levels of CESs do not always correlate with their activity levels,14 the proposed methodology enables us to reveal the relative activities of the enzymes, through the use of the different inhibitors. Other techniques, such as Western blots, estimate the amount of an enzyme in a biological sample but do not provide any information concerning their activity. On the other hand, native in-gel hydrolysis assays permit the detection of enzyme activities, but no direct quantification is possible. This methodology also offers the possibility to determine the specific enzyme activities. For this, one has to follow classical enzymatic studies rules like ensuring that the assays are performed in the linearity range of the enzymatic reaction and that the substrate is not at a limiting concentration. In fact, we have quickly determined the linearity range of enzymatic reaction for CES1 enzyme (Supporting Information Figure S-5). In this way, there is no need to appeal to other methodologies usually used, such as spectrophotometric methods, to determine these parameters as our methodology provides quick results. The intermediate precision of the method was determined (CVi = 12.8%) for two different CES1 concentrations (25 and 12.5 ng) in the presence and in the absence of one of the inhibitors (loperamide) in three different days. Using the same rationale, the different types of assays that can be performed are unlimited. It is worth to note that the inhibition of BuChE and AcChE by loperamide does not interfere with the detection and/or quantification of hCE-2 activity in biological samples as we also saw that BuChE and AcChE do not hydrolyze 4-MUBA. CES2 Activity Determination in Complex Biological Samples. To demonstrate the potential of this methodology we have applied it to several different biological samples, with different complexities. We started analyzing hCE-2 activity in extracts from human cells that were transfected with hCE-2 to evaluate the specific activity of the enzyme. Since HEK-293T human cell line has only basal expression of esterases,2 we have also analyzed the original cells as a control. We have assayed the linearity range of the reaction and determined the specific activity of hCE-2 in the overexpressing and in the original cells (Figure 3). Total protein in each sample was determined, and all the assays were performed using the same total protein amount. Our methodology enabled the use of extremely low amounts of protein, from 3 to 6 μg (Figure 3), whereas in classical spectrophotometric assays, the minimum protein amount used in similar assays is 20 μg,2 and in native in-gel hydrolysis assays, it reaches 30 μg14 of total protein amount of tissue extracts. We are thus able to use almost 7 times less protein comparing to the classical methods. By evaluating the obtained data, we concluded that we were able to increase 15 times the specific activity of hCE-2 in the transfected cells. In addition, as reported, we can detect a basal expression of esterases in this cell line. Comparing the specific activities obtained in the presence of loperamide and in the absence of the inhibitor for the transfected cell line we observe that hCE-2 activity accounts for 93% of the total specific activity of these transfected cells.
Figure 2. Commercial CESs activity toward 4-MUBA in the presence and absence of inhibitors. Amounts of 12.5 ng of CES1 or 50 ng of hCE-2 enzymes were incubated with 0.5 mM 4-MUBA in a total reaction volume of 150 μL. Mixtures of the two enzymes included mix 12.5/50 (12.5 ng of CES1 and 50 ng of hCE-2 and mix 12.5/25 ng (12.5 ng of CES1 and 25 ng of hCE-2) Preincubation with 250 μM loperamide or 500 μM BNPP was performed as described in the Materials and Methods section. The spontaneous hydrolysis of 4-MUBA is also shown. Each result represents the average of three injections, and error bars represent the standard deviation. A / represents a statistical significant difference (p = 0.01) between the enzymatic activities in the presence (gray or dashed bars) and in the absence (black bars) of the inhibitor, based on standard deviation of intermediate precision. The amount of 4-MUB (μM) formed in each sample is mentioned on the top of each bar.
In the developed CE method the BNPP absorption spectrum shows a maximum at 280 nm. Despite the fact that it is also detected at 280 nm, the migration time of this inhibitor (4.8 min) does not overlap with that of 4-MUB (Figure 1); thus, 4-MUB can be quantified by CE in the presence of BNPP. Using BuChE, AcChE, and their specific substrates BuTCh and AcTCh, respectively,32 we could observe that loperamide does in fact inhibit both enzymes (Supporting Information Figure S-3). Using the described CE method, we could also detect thiocoline (the hydrolysis product of both substrates) at 230 nm as well as both substrates at the same wavelength. Substrates and thiocoline migration times do not overlap, being completely distinguishable (2.0 min for both substrates and 2.5 min for thiocoline, in average). According to available published data,22,33 hCE-2 shows a higher catalytic efficiency toward 4-MUBA than hCE-1, but our data is suggesting the opposite. This may simply be due to differences in assay conditions and/or to differences in enzymes sources. As expected, loperamide inhibited completely the activity of hCE-2 (Figure 2) but not commercial CES1. On the other hand, the activities of both carboxylesterases were completely inhibited by BNPP. We were not expecting to observe CES1 inhibition in the presence of loperamide,29 but Figure 2 disproved this expectation. The IC50 and Ki values for these enzymes were previously reported,34 showing a dramatic difference between the sensitivity of the enzymes toward loperamide inhibition. In fact, the Ki for hCE-2 is practically 1000 times in comparison to the one for CES1. Thus, this unexpected inhibition verified in CES1 enzyme can be easily overcome by reducing loperamide concentration. We further confirmed this by testing the same concentration of each enzyme with a lower concentration of loperamide, 50 μM. We observed that no significant inhibition is detected in CES1, whereas CES2 is completely inhibited (Supporting Information Figure S-4). 884
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Table 1. Specific Activity of CES2-like Enzymes in Mammalian Seraa nmoL 4-MUB 3 min-1 3 serum %-1 species
a
total serum
CES2-like
other CESs
equine
0.25 ( 0.01
0.13 ( 0.01
N.D.
dog
0.10 ( 0.01
N.D.
0.05 ( 0.01
rat cat
1.07 ( 0.10 0.62 ( 0.07
0.82 ( 0.07 0.40 ( 0.04
0.16 ( 0.03 0.06 ( 0.02
bovine
0.60 ( 0.13
N.D.
N.D.
human
0.60 ( 0.05
N.D.
N.D.
Specific activities were determined using 0.5 mM 4-MUBA as described before. Amounts of 10% (v/v) of equine, dog, human, and bovine serum, 1% (v/v) rat serum, and 2.5% (v/v) cat serum were used, to cope with the linearity range of the enzymatic reactions. Preincubation with 250 μM loperamide or 500 μM BNPP was performed as described in the Materials and Methods section. The spontaneous hydrolysis of 4-MUBA was accounted. Each result represents the average of three injections (( standard deviation). N.D.: not detected.
Figure 3. hCE-2 specific activity in HEK-293T transfected cells. HEK293T cells transfected with hCE-2 and nontransfected cells extracts were incubated with 0.5 mM 4-MUBA in a total reaction volume of 150 μL. An amount of 6 μg of total protein was used in each experiment. Preincubation with 250 μM loperamide or 500 μM BNPP was performed as described in the Materials and Methods section. The spontaneous hydrolysis of 4-MUBA is also shown. Each result represents the average of three injections, and error bars represent the standard deviation. A / represents a statistical significant difference (p = 0.01) between the enzymatic activities in the presence (gray or dashed bars) and in the absence (black bars) of the inhibitor, based on standard deviation of intermediate precision.
In human serum, for example, we can detect esterase activity that is not inhibited by loperamide or BNPP as no significant statistical difference (p = 0.01) is observed between the enzymatic activities obtained in each assay. It is known that human plasma contains four types of esterases, namely, BuChE, AcChE (present in negligible amounts only), paraoxonase, and albumin.8 We thus hypothesize that the esterase activity detected in human serum is due to one or several of the enzymes that can possibly hydrolyze 4-MUBA but that are not CESs. In fact, albumin is known to hydrolyze this substrate.13 Nonetheless, the method proves unaffected by basal activity due to esterases other than CESs, with inhibition by BNPP and by loperamide being valuable indicators of CESs and CES2 activity, respectively. Assuming that loperamide inhibits CES2-like enzymes from mammalian species as well as it inhibits hCE-2, we determined the specific activities of CES2-like and total CESs in the different mammals in the linearity range of the enzymatic reaction of each serum (Table 1). CES2-like activity was detected and quantified in equine, rat, and cat sera. In fact, in equine serum it is apparently the only CES present. The highest CES2-like activity is observed in rat serum; on the contrary, dog serum shows no significant CES2-like activity. Once again, our results are consistent with the trends reported in the literature.8,35 One should note, however, that our purpose is to demonstrate that we achieve quantifiable and consistent values of specific CES2-like activities with our proposed methodology. A deeper evaluation of the CESs present in each mammalian serum would require a higher number of individuals of each species. To quantify esterase activity and to distinguish CES2 activity from other carboxylesterases, we propose the following steps depicted in Figure 5. Starting with an unknown sample, an appropriate dilution has to be determined in order to detect or to quantify the specific activity of CES2. This can be achieved with 4-MUBA or other substrate hydrolyzed by CES2. The ability of an unknown sample to hydrolyze 4-MUBA indicates that it contains esterases able to hydrolyze this substrate; if no activity is detected, the presence of CES2 activity can be excluded and the analysis will be ended. Confirmation of activity exclusively due to carboxylesterases is performed in the presence of BNPP, which inhibits these enzymes. If no activity is detected in this step, no CESs shall be present, and thus the analysis will be
Figure 4. Mammalian sera activities toward 4-MUBA in the presence and absence of inhibitors. An amount of 10% (v/v) of serum from one individual of each mammalian species was incubated with 0.5 mM 4-MUBA in a total reaction volume of 150 μL. Preincubation with 250 μM loperamide or 500 μM BNPP was performed as described in the Materials and Methods section. The spontaneous hydrolysis of 4-MUBA is also shown. Each result represents the average of three injections, and error bars represent the standard deviation. An asterisk (/) represents a statistical significant difference (p = 0.01) between the enzymatic activities in the presence (gray or dashed bars) and in the absence (black bars) of the inhibitor, based on standard deviation of intermediate precision.
We further applied our methodology in the evaluation of a more complex matrix in terms of enzyme activity. We have chosen different mammalian sera, which are known to contain different types and different amounts of esterases.3,4,32 We tested the serum from one individual of human, bovine, equine, domestic dog, domestic cat, and rat species. Using 10% (v/v) of serum of each mammal we obtained consistent results for the CESs relative activities to the ones described in the literature8,35 (Figure 4). In fact, we did not observe any CESs activity in human or bovine sera and the highest levels of CESs activities were found in rat, equine, and cat sera. 885
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Figure 5. Flow diagram for CES2 activity identification and determination.
ended. The analysis of the enzymatic activity in the presence of loperamide enables one to discriminate and quantify the activity of CES2. Loperamide inhibits CES2 but not other CESs, and therefore CESs activities other than CES2 can be determined by the difference between the inhibitions observed with BNPP and loperamide.
e a Tecnologia (SFRH/BD/44025/2008, PTDC/EBB-BIO/ 111530/2009).
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’ CONCLUSIONS Capillary electrophoresis was used for quantification of CESs activities. The methodology proved fast, repeatable, and requires up to 7 times less sample, than classical spectrophotometric methods. Moreover, this approach proved to be applicable even when substrates and products absorb at the same wavelength. The proposed methodology should be applicable to a wide variety of samples with esterase activity. For example, the analysis and determination of hCE-1 exclusive activity would only imply the replacement of loperamide by a specific inhibitor of this enzyme such as the partially noncompetitive inhibitor 27hydroxycholesterol.30 For the analysis of the activities of other esterases, appropriate inhibitors should be used. ’ ASSOCIATED CONTENT
bS
Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
’ ACKNOWLEDGMENT The authors thank Faculdade de Medicina Veterinaria, Universidade Tecnica de Lisboa, particularly Dr. Paula Tilley, Dr. Rui Caldeira, and Dr. George Stilwell, for donating the serum samples. The authors also wish to thank Dr. J. F. Gilmer, from Trinity College Dublin, for fruitful discussions. This work was funded by the Portuguese Fundac-~ao para a Ci^encia 886
dx.doi.org/10.1021/ac102547c |Anal. Chem. 2011, 83, 881–887
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ARTICLE
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