Method for the Sequential Online Analysis of Enzyme Reactions

Article pubs.acs.org/ac

Method for the Sequential Online Analysis of Enzyme Reactions Based on Capillary Electrophoresis Yuanfang Chen,† Liangliang Xu,‡ Wenwen Zhao,† Liping Guo,† and Li Yang*,† †

Faculty of Chemistry, Northeast Normal University, Changchun, Jilin, 130024, P.R.China College of Optical and Electronical Information, Changchun University of Science and Technology, Changchun, Jilin, 130012, P.R. China

‡

ABSTRACT: We have developed an easy-to-operate and effective method for performing the sequential online analysis of enzyme reactions based on capillary electrophoresis (CE). The system was constructed by passing two capillaries through a sample vial at a distance of 5 μm between the capillary ends. Direct online sample injection and sequential CE analysis were achieved by periodically switching the highvoltage power supply off and on, without any physical disturbance of the capillary inlet. The sample was injected via concentration diffusion with in-column derivatization of the amino acids occurring at the interface of the capillaries. High reproducibility of the sequential injections was demonstrated with relative standard deviation values (n = 20) of 1.01%, 1.25%, and 0.80% for peak height, peak area, and migration time, respectively. Sequential online CE enzyme assay of a glutamate pyruvate transaminase catalyzed enzyme reaction was carried out by simultaneously monitoring the substrate consumption and the product formation every 30 s from the beginning to the end of the reaction. The Michaelis constants for the reaction were obtained and were found to be in good agreement with the results of traditional off-line enzyme assays. Our method has great potential for usage in sequential online CE analysis of chemical reactions with in-column chemical derivatization of the analytes for ultraviolet or laser-induced fluorescence detection.

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running buffer for CE analysis after each sample injection, it is unlikely that EMMA or microreactor techniques could be successfully used to perform sequential online analysis. A combination of the flow injection (FI) technique and CE separation can be utilized to carry out automatic online analysis.16,17 An FI-CE technique allows one to sequentially inject the sample without interrupting the CE separation. The possibility of using an FI-CE system for sequential online analysis of enzyme inhibition has been demonstrated.18 However, special interfaces must be designed for the FI-CE coupling, making the instrument rather complicated. The potential of the combined system should be extended to the continuous monitoring of online reactions.17 Another method, Optically Gated Vacancy Capillary Electrophoresis (OGVCE)LIF, which couples optically gated sample injection19,20 with laser-induced fluorescence (LIF) detection in capillary electrophoresis, was reported for the online monitoring of an enzyme reaction with high temporal resolution from the beginning to the end of the reaction.21,22 To achieve an optically gated injection and to reduce background noise in the LIF detection, the fluorescein-labeled analytes (usually created by off-line derivatization) must have low photostability for photobleaching at low laser power.

ecause of its unique advantages, such as high efficiency and sensitivity, rapid analysis, extremely low sample volume requirements, and the ability to utilize several detection methods, capillary electrophoresis (CE) has become a powerful tool for the quantitative study of enzyme-catalyzed reactions and has been widely applied in nearly all aspects of enzyme assays,1,2 including the evaluation of enzymatic activity,3−6 enzyme kinetics,7,8 enzyme inhibition and activation,9,10 and the investigation of enzyme-mediated metabolic pathways.11,12 For enzyme assays, online monitoring of the enzymatic reaction from beginning to end is vitally important to fully understand the metabolic enzyme functions and to determine their uses for clinical diagnostics. Online monitoring of the enzymatic reaction requires that the substrate and product be sequentially injected into the capillary without any physical disturbance of the capillary inlet so that both substrate and product may be simultaneously monitored once the reaction occurs. Considering the need for reproducibility in the enzyme assay with a small amount of injected sample (usually less than 10 nL), sequential online CE analysis of enzyme reactions is challenging, especially when the enzymatic reaction requires a closed system. Electrophoretically mediated microanalysis (EMMA)1 and capillary microreactors13 may be the two most commonly used techniques for online CE enzyme assays. In either technique, the samples are usually introduced into the capillary by electrokinetic injection14 or by hydrodynamic injection.15 Because either injection method requires that the capillary inlet be physically moved from the sample container to the © 2012 American Chemical Society

Received: January 16, 2012 Accepted: February 24, 2012 Published: February 24, 2012 2961

dx.doi.org/10.1021/ac3001644 | Anal. Chem. 2012, 84, 2961−2967

Analytical Chemistry

Article

In this work, we describe a novel method for the sequential online analysis of enzyme reactions based on capillary electrophoresis. A simple system was constructed by coaxially aligning two capillaries through a sample vial with a distance of 5 μm between the capillary ends. Direct online sample injection and sequential CE analysis were easily achieved by periodically switching the high-voltage power supply off (for sample injection) and on (for CE separation). No physical disturbance of the capillary inlet was necessary. Highly reproducible sequential injections were demonstrated by analyzing a standard mixture of the amino acids alanine (Ala) and glutamate (Glu). Using the presented method, we have investigated the sequential online CE enzyme assay for a GPT-catalyzed enzyme reaction by simultaneously monitoring the Ala consumption and the Glu formation every 30 s from the beginning to the end of the reaction. The Michaelis constants for the reaction were obtained and were found to be in good agreement with the results of traditional off-line enzyme assays. Finally, a brief discussion was presented to reveal the features and advantages of the method described herein.

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EXPERIMENTAL SECTION Chemicals. L-Alanine, L-glutamate, α-ketoglutarate, 2mercaptoethanol (2-ME), and o-phthaldialdehyde (OPA) were purchased from Sigma Chemical Co. (St. Louis, MO). Glutamate pyruvate transaminase (GPT, EC2.6.1.2) was purchased from MP Biomedicals Inc. (Irvine, CA). All other reagents were of analytical grade and were used without further purification. All solvents and solutions were filtered using 0.2 μm membrane filters prior to use. Sequential Online CE Enzyme Assay. The construction of our system is schematically shown in Figure 1a. Two capillaries, with a distance of 5 μm between the smooth ends, were coaxially fixed on a glass slide (2 cm × 2 cm). The slide also acted as the bottom of the sample vial (0.8 cm diameter, 1 cm height) and had two 1 mm diameter holes on opposite sides for the capillaries to pass through. The joint portions of the capillaries, the sample vial, and the slide were securely sealed to avoid any leakage. The entire process was performed under a microscope to ensure a coaxial alignment and to maintain the distance between the capillaries. A high-voltage power supply was applied across the two capillaries and was used for the CE separation and online sample injection (see details below). The test enzyme reaction used in this work was a GPTcatalyzed reaction as follows:

Figure 1. (a) Construction of the sequential online injection system. (b1−b4) Scheme of the sequential online injection and CE analysis process.

the interfaces; thus, it enhances the diffusion efficiency for the amino acids to be injected into the capillary. When the highvoltage supply is switched on again, the plug of the derivatized amino acids, Ala and Glu, migrates with the EOF (Figure 1b3); the amino acids are electrophoretically separated and detected by UV absorption (Figure 1b4). Once the separation and detection are complete, the high-voltage supply is switched off again, and another cycle of injection−separation−detection is initiated. In this way, sequential online analysis can be achieved. No physical disturbance is necessary for this sequential online CE enzyme assay. A small amount of the sample in the vial may diffuse into the capillary during the separation and detection steps (Figure 1b3,b4). This diffusion may affect the quantitative measurement of the absorption peak height (or area). However, under our experimental conditions, we have achieved highly reproducible peak heights and peak areas, and we have obtained a linear calibration curve over a 0−15 mM sample concentration range (see the following section for details). Thus, the diffusion effect during the separation and detection can be ignored over the concentration range presented herein. In the enzyme assay experiments, the substrates Ala and αketoglutarate were initially put into the sample vial. When the GPT enzyme was added to initiate the enzyme reaction, the substrate, Ala, and the product, Glu, were simultaneously detected online as a function of the reaction time to achieve the CE enzyme assay. Sequential CE Separation Conditions. All experiments were carried out in a home-built CE apparatus. The high-

GPT

Alanine + α‐ketoglutarate ⎯⎯⎯⎯⎯→ Glutamate + Pyruvate

In Figure 1b1−b4, we illustrate the process of the sequential online CE enzyme assay, which was achieved by periodically switching the high-voltage power supply off and on. The chemical derivatization of the substrate, Ala, and the product, Glu, was carried out using OPA/2-ME in the running buffer, which can achieve rapid derivatization of amino acids for UV or LIF detection.23−25 In the experiments to obtain calibration curves and optimization, the sample vial contained a standard Ala and Glu mixture, and the running buffer contained 10 mM OPA/2-ME (Figure 1b1). When the high-voltage supply is off, the samples diffuse into the capillary because of the concentration difference between the sample buffer and the running buffer, and the rapid derivatization of the amino acids occurs at the interfaces of the capillaries (Figure 1b2). The derivatization reaction changes the concentration gradient at 2962



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Figure 2. (a) Effect of the separation voltage on the injection and separation. The investigated separation voltage range was 7−17 kV. The injection time was set at 10 s. (b) Seven sequential injections with different injection times, using a pressure of 0.5 psi instead of a high-voltage electric field as the driving force, the sample injection time (10−60 s) was referred to as the period during which the pressure was removed. In both figures, the test sample was 1.0 mM Ala and Glu prepared in 50 mM phosphate buffer at pH 7.5. The running buffer was a 25 mM borate buffer (pH 9.5) containing 10 mM OPA.

voltage power supply was designed by Yangzhou Shuanghong Electronics Co., Ltd. (Yangzhou, China) and can be automatically turned on and off at controllable times. The UV signals were recorded at a wavelength of 340 nm by a 6000PVW UV− visible detector (Cometro Technology Ltd., USA). Two fusedsilica capillaries of 50 μm i.d. and 365 μm o.d. (Hebei Yongnian Optical Fiber Factory, China) were used for the sequential CE analysis. The total length of one capillary was 11 cm and that of the other was 8 cm. The 11 cm capillary was used as the CE separation channel with an effective length of 5 cm. The sample

buffer in the sample vial was a 50 mM phosphate buffer at pH 7.5. The running buffer, that is, the reagent for the derivatization of the amino acids, was prepared by dissolving 2.7 mg of OPA in a mixture of 27 μL of ethanol and 40 μL of 2ME and diluting to 400 μL with a 25 mM borate buffer (pH 9.5), resulting in a final concentration of 10 mM OPA in the running buffer. Before CE analysis, the capillaries were pressure-rinsed with 0.1 M NaOH for 2 min, distilled water for 3 min, and the CE running buffer for 5 min, successively, 2963



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Figure 3. Typical electropherogram for the twenty sequential analysis of a 1.0 mM Ala and Glu standard mixture using the sequential online injection system. The inset shows the enlarged figure of the first three sequences. The separation voltage was 17 kV (895 V/cm). Other conditions were same as those described in Figure 2a.

sensitivity, an injection time of 10 s was chosen for all subsequent CE experiments in our study. The effect of the high voltage on the injection and separation was investigated in the range of 7−17 kV (368−895 V/cm). Figure 2a shows the electropherograms of the test sample at different voltages. An efficient baseline separation was obtained at each of the five voltages studied. For instance, at 17 kV, the separation resolution for Ala and Glu was 4.42, and the theoretical plate numbers were 4.8 × 106 and 9.8 × 106 for Ala and Glu, respectively. To achieve rapid analysis, a voltage of 17 kV was selected for all sequential online CE experiments. We did not observe any significant change in the peak area of either Ala or Glu as the high voltage changed from 7 to 17 kV. Thus, the amount of sample injected is independent of the voltage, indicating that the sample injection mechanism is not an electrokinetic injection. We also employed a pressure of 0.5 psi, rather than utilizing the electroosmotic flow, to push the sample in the capillary to the detector, to demonstrate that the sample can be injected into the capillary without the electric field. The injection time in this case was referred to the time during which the pressure was removed. The results are shown in Figure 2b for seven sequential injections with different injection times. A sharp absorption peak at each injection is observed, and the peak area increases as the injection time is increased. Because no electric field was used in Figure 2b, the results support the idea that in our system the sample is injected into the capillary via diffusion with rapid online derivatization. It should be noted that no separation of Ala and Glu can be achieved in Figure 2b because pressure was used rather than high voltage. Figure 3 shows a typical electropherogram for the 20 sequential analysis of a 1.0 mM Ala and Glu standard mixture using the presented method. As shown in the figure, two positive peaks periodically appear in the electropherogram every 30 s. During each period, the peak of Ala is 6 s ahead of that of Glu, because of their different apparent mobilities. These two peaks of the standard samples were used to identify the peaks of the substrate and the product in the subsequent enzyme assay experiments. A spike followed by a sharp dip at the beginning of each sequence is the electronic noise caused by switching off the high-voltage power supply. The time

and the sample vial was rinsed with distilled water and the sample buffer using a syringe. Traditional Off-line CE Enzyme Assay. The reaction mixture (200 μL) contained a 50 mM phosphate buffer (pH 7.5) and the substrates of different concentrations. Reactions were initiated by the addition of 12 μL of 500 U/mL GPT into the mixture. Aliquots of 10 μL were periodically removed from the reaction mixture, and the GPT was inactivated by the addition of 2 μL of 0.1 M HCl to each aliquot. The CE running buffer was a 25 mM borate buffer (pH 9.5) containing 10 mM OPA/2-ME. The sample was injected at a height of 10 cm for 3 s. The substrate consumption and product formation were measured at a separation electric potential of 454 V/cm. The total length of the separation capillary (50 μm i.d., 365 μm o.d.) was 55 cm with an effective length of 46 cm.

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RESULTS AND DISCUSSION Sequential Online CE Analysis for a Standard Mixture of Ala and Glu. The standard sample, a 1.0 mM Ala and Glu mixture, was first studied by the presented method to demonstrate the performance of sequential online CE analysis and to obtain a calibration curve for the quantitative measurement of the enzyme reaction. Several experimental conditions were investigated to optimize the sequential online sample injection. The effect of the distance between the two capillaries on the diffusion injection was investigated at 5, 10, and 50 μm. No significant differences in the sample injection amount, separation resolution, and efficiency were observed, and a distance of 5 μm was chosen for use in our system. The dependence of the peak area and height of the UV absorption of the derivatized Ala or Glu on the sample injection time was investigated. In this work, the injection time refers to the time during which the power supply is in the off position. It was observed that the peak areas (or the peak heights) for both Ala and Glu increase as the injection time is increased in the range of 0−300 s. For CE techniques, a small sample injection amount is required for rapid analysis and high separation efficiency. A short injection time is also necessary for high temporal resolution in sequential analysis. Thus, the injection time should be as short as possible. Considering the detection 2964



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Figure 4. Evolution of Ala and Glu peaks as a function of time in the study of a GPT-catalyzed enzymatic reaction as it related to the process of the reaction. See text for details. The initial concentrations of Ala and α-ketoglutarate were 1.5 and 5 mM, respectively. The volume of the enzyme reaction buffer was 200 μL, and the amount of GPT was 1.2 μL (500 U/mL). Other conditions were the same as those described in Figure 3.

Figure 5. Dependence of the substrate (squares) concentration and the product (circles) concentration on the reaction time. The initial reaction rate was the slope of the linear fit of the substrate consumption or product formation. All other conditions were the same as those described in Figure 4.

interval between two neighboring sequences is 10 s, which is consistent with the injection time. The results in Figure 3 clearly demonstrate the capability of the presented method for sequential online CE analysis. The temporal resolution is 30 s, and the sequential analysis is achieved automatically without any tedious or time-consuming labor. Reproducibility of the sequential online CE analysis is also demonstrated in Figure 3. Each sequence in the figure corresponds to a repeating CE run because standard samples of constant concentration are injected and separated without any physical disturbance. Excellent reproducibility can be easily observed by comparing the peak heights, peak areas, migration times, and injection times of the 20 sequences as shown in Figure 3. The relative standard deviation (RSD) (n = 20) is

1.01%, 1.25%, and 0.80% for peak heights, peak areas, and migration times, respectively. The calibration curve of the peak height versus concentration for each sample was measured in the concentration range of 0−15 mM. The peak heights of Ala and Glu show a linear dependence on the concentration of the sample in the range of 0−15 mM (for Ala, y = 0.258x − 0.034, R2 = 0.999; for Glu, y = 0.35x − 0.046, R2 = 0.998). The detection limits for Ala and Glu are 30 and 20 μM (S/N = 3), respectively. Sequential Online CE Analysis for a GPT-Catalyzed Reaction. Figure 4 shows the evolution of the peaks as a function of time in the sequential online CE analysis of a GPTcatalyzed reaction. “Time zero” in the electropherogram was set to the time at which the enzyme GPT was added to the sample 2965



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vial to initiate the reaction. The substrate or product was sequentially monitored every 30 s from the beginning to the end of the reaction, and each sequence was related to one time point of the enzymatic reaction. The figure clearly shows that the presented method can achieve automatic and sequential measuring of the enzyme reaction. At the beginning of the reaction, when few products are created, the principal positive peak, appearing every 30 s, is due to the UV absorption of the substrate, Ala (Figure 4a). As the reaction progresses, a pair of positive peaks with a time difference of 6 s is observed once every 30 s. According to the results of the standard samples described above, these two peaks can be easily identified as the substrate, Ala, and the product, Glu. The intensity of the substrate peak gradually decreases while that of the corresponding product peak gradually increases, which directly reflects the progress of the enzymatic reaction (Figure 4b−f). Eventually, the intensities of the substrate peak and the product peak remain unchanged, indicating that the enzymatic reaction in the sample vial has ended (Figure 4g). It should be noted that the substrate peak does not completely disappear, indicating that the enzyme reaction studied in this work is a reversible reaction. To deduce the rate constant for the reaction, the kinetic curves of the substrate and the product, that is, the dependence of their concentrations on the reaction time, were measured and are shown in Figure 5. The concentration at each time point was obtained from the measured peak height and the calibration curve. The data were recorded every 30 s during the entire reaction time with sequential CE analysis. The results show that the concentration of the substrate decays from 1.5 to 0.3 mM, while that of the product grows from 0 to 1.2 mM during the reaction time of 0−22.5 min. Figure 5 also shows the linear fitting results that yield the initial reaction rate. Identical initial reaction rates for the substrate and the product were obtained. The Ala decay rate is determined to be 0.076 ± 0.003 mM/min (N = 3) and the Glu rise rate, 0.0807 ± 0.003 mM/ min (N = 3). According to the ping-pong mechanism for enzyme reactions with two or more substrates, the Michaelis constants can be deduced by the following equation

Figure 6. Lineweaver−Burk plots of the GPT-catalyzed reaction. The experiments were performed by measuring the rate constants at (a) 5 or 10 mM of the substrate Ala and (b) 2 or 5 mM of the substrate αketoglutarate. The concentration of the other substrate in each figure was varied from 2.5 to 10 mM. Each data point in the figure was the result of the average of three measurements. The reaction rate was presented as substrate consumption. The obtained kinetics curves showed characteristics typical of a ping-pong mechanism for enzyme reactions with two substrates. Other conditions were the same as those described in Figure 4.

KA ⎛ 1 ⎞ ⎛ K B ⎞⎛ 1 ⎞ 1 = m ⎜ ⎟ ⎟ + ⎜⎜1 + m ⎟⎟⎜ v Vmax ⎝ [A] ⎠ ⎝ [B] ⎠⎝ Vmax ⎠

other, demonstrating the feasibility and reliability of the presented sequential online CE analysis for enzyme reactions. To check the results of our sequential analysis, traditional offline enzyme assays were performed to measure the Km values for the GPT reaction. Maintaining the concentration of Ala at 5 or 10 mM and varying the concentration of α-ketoglutarate, Km values of 2.4 ± 0.2 for Ala and 2.3 ± 0.2 for α-ketoglutarate were obtained; keeping the concentration of α-ketoglutarate at 2 or 5 mM and varying the concentration of Ala, Km values of 2.7 ± 0.3 for Ala and 2.7 ± 0.3 for α-ketoglutarate were obtained. The resulting Km values obtained from our sequential analysis method were in good agreement with those obtained from the traditional off-line enzyme assay. Discussion. The primary purpose of the present study is to develop a method for the sequential online CE analysis of chemical reactions. To achieve this purpose, a direct sequential online sample injector is required for the CE technique. As shown in Figure 1, our system is quite simple, consisting of two-section capillaries passing through a sample vial. No complex instruments, such as those used in FI-CE16,17 or OGVCE-LIF19,20 techniques, are necessary. Samples are directly online injected via diffusion at the interfaces of the

in which ν is the enzymatic reaction rate, Vmax is the maximum reaction rate, KmA and KmB are the Michaelis−Menten constants for substrates A and B, respectively, and [A] and [B] are the concentrations of substrates A and B, respectively. Figure 6 shows the Lineweaver−Burk plots of the GPTcatalyzed reaction used in this study. The experiments were performed by measuring the rate constants at different concentrations of the substrate α-ketoglutarate (Figure 6a) or the substrate Ala (Figure 6b). The concentration of the other substrate was held constant at two different values in each figure. Each point on the diagram was the result of an average of three measurements. Linear fitting of the experimental data in each figure results in two parallel lines, demonstrating a pingpong mechanism for the GPT-catalyzed reaction. From Figure 6a, the Km values are determined to be 1.8 ± 0.1 for Ala and 2.6 ± 0.2 for α-ketoglutarate, while from Figure 6 b, the Km values are found to be 2.3 ± 0.1 for Ala and 2.5 ± 0.2 for αketoglutarate. The Km values obtained from the two different experimental configurations were in good agreement with each 2966



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Notes

capillaries. Compared with the widely used electrokinetic or hydrodynamic injection methods, diffusion injection, which utilizes the concentration difference between two solutions, generally requires a relatively long time to achieve efficient CE separation. 26 However, in the present work, a rapid derivatization reaction of amino acids occurring at the interface of the capillaries changes the concentration gradient of the samples, thus greatly enhancing the diffusion efficiency. In this work, an injection time of 10 s is sufficient for the sensitive detection of derivatized Ala and Glu. Several essential benefits of the method presented for the sequential online CE analysis of chemical reactions have been demonstrated. Each sequence for analysis is performed simply by switching a high-voltage power supply off (for sample injection) and on (for CE separation). No physical disturbance of the capillary inlet is necessary, making a CE enzyme assay possible in a closed system configuration. Great reproducibility of the sequential online sample injection [1.01% RSD (peak height, n = 20), see Figure 3] ensures the accuracy of the enzyme assay. As demonstrated by analysis of an example GPTcatalyzed reaction, the presented method can monitor the enzyme reaction online by simultaneously measuring the substrate consumption and the product formation at different time points from the beginning to the end of the reaction (Figure 4). These measurements can provide more insight into the enzyme activity and enzyme reaction kinetics. The presented method also provides a new setup for automatic in-column chemical derivatization technique in CE, as illustrated in Figure 1b and described in the Experimental Section. The in-column derivatization has important advantages over precolumn or postcolumn derivatization methods.23,24 For example, derivatization and separation can be integrated into one column, and the entire process can be performed automatically with a minimal amount of sample and reduced consumption of the reagents. Traditionally, in-capillary derivatization is accomplished via a sandwich procedure in which the sample is inserted between two reagent segments. This procedure requires at least three injections for one CE analysis, and optimization of the injection time for each injection is necessary to obtain high derivatization efficiency and reproducibility. In the presented method, the sample is sandwiched by the reagent automatically and reproducibly because the running buffer contains the reagent (OPA/2-ME). Thus, only one injection is needed for each CE analysis. The sample injection and optimization procedure is simplified and high reproducibility can be obtained by automatic control of the injection time. In conclusion, we have described a novel method for the sequential online CE analysis of enzyme reactions using twosection capillaries and power cycling of a high-voltage power supply. The feasibility and reliability of the method for sequential online CE enzyme assays was demonstrated. Although an enzyme reaction was investigated in this work, the presented method can also be used for sequential online analysis of other chemical reactions that are based on capillary electrophoresis in which the analytes are required to be chemically derivatized for UV detection or fluorescein-labeled for LIF detection.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China under Grants 21175018 and 20805005. REFERENCES

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Method for the Sequential Online Analysis of Enzyme Reactions

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