Immobilized capillary adenosine deaminase microreactor for inhibitor screening in natural extracts by capillary electrophoresis

A novel strategy for the preparation of in-column adenosine deaminase (ADA) microreactor and rapid screening of enzyme inhibitors in natural extracts was demonstrated. In this approach, ADA was encapsulated in anionic polyelectrolyte alginate that was immobilized on the surface of fused-silica capillary via ionic binding technique with cationic polyelectrolyte polyethylenimine (PEI). On-line enzyme inhibition study was performed by capillary electrophoresis (CE). The substrate and product were baselined separated within 75 s. The enzyme activity was determined by the quantification of peak area of the product. Enzyme inhibition can be read out directly from the reduced peak area of the product in comparison with a reference electropherogram obtained in the absence of any inhibitor. The inhibition percentage was used to evaluate relative activity of ADA microreactor. A known ADA inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) was employed as a model compound for the validation of the inhibitor screening method, and the screening of ADA inhibitor in 19 traditional Chinese herbal medicines was performed.     

Screening of tyrosinase inhibitors by capillary electrophoresis with immobilized enzyme microreactor and molecular docking

A new method for screening tyrosinase inhibitors from traditional Chinese medicines (TCMs) was successfully developed by capillary electrophoresis with reliable online immobilized enzyme microreactor (IMER). In addition, molecular docking study has been used for supporting inhibition interaction between enzyme and inhibitors. The IMER of tyrosinase was constructed at the outlet of the capillary by using glutaraldehyde as cross‐linker. The parameters including enzyme reaction, separation of the substrate and product, and the performance of immobilized tyrosinase were investigated systematically. Because of using short‐end injection procedure, the product and substrate were effectively separated within 2 min. The immobilized tyrosinase could remain 80% active for 30 days at 4°C. The Michaelis–Menten constant of tyrosinase was determined as 1.78 mM. Kojic acid, a known tyrosinase inhibitor, was used as a model compound for the validation of the inhibitors screening method. The half‐maximal inhibitory concentration of kojic acid was 5.55 μM. The method was successfully applied for screening tyrosinase inhibitors from 15 compounds of TCM. Four compounds including quercetin, kaempferol, bavachinin, and bakuchiol were found having inhibitory potentials. The results obtained in this work were supported by molecular docking study.     

Evaluation of xanthine oxidase inhibitory activity of flavonoids by an online capillary electrophoresis-based immobilized enzyme microreactor

Xanthine oxidase (XOD) is a key enzyme in the human body to produce uric acid, and its inhibitor can be used for the treatment of hyperuricemia and gout. In this study, an online CE-based XOD immobilized enzyme microreactor (IMER) was developed for the enzyme kinetics assays and inhibitor screening. After 30 consecutive runs, the XOD activity remained about 95.6% of the initial immobilized activity. The Michaelis–Menten constant (K_m) of the immobilized XOD was determined as 0.39 mM using xanthine as substrate. The half-maximal inhibitory concentration and inhibition constant of the known inhibitor 4-aminopyrazolo[3,4-d]pyrimidine on XOD were determined as 11.9 and 5.2 μM, respectively. Then, the developed method was applied to evaluate the XOD inhibitory activity of 10 flavonoids, which indicated that dihydroquercetin, quercetin, biochanin A, and epicatechin had significant inhibitory effect on XOD. In addition, molecular docking results verified that the binding energy of the flavonoids with enzyme were in line with their inhibitory activity determined by XOD–IMER. Therefore, the developed XOD–IMER is a potential tool for the primary screening of XOD inhibitors from natural products.     

Rapid on‐line system for preliminary screening of lipase inhibitors from natural products by integrating capillary electrophoresis with immobilized enzyme microreactor

An on‐line system for preliminary screening lipase inhibitors from natural products with an immobilized lipase microreactor coupled to capillary electrophoresis was established. In this research, the lipase was anchored on the amino activated capillary inner wall using glutaraldehyde as a homobifunctional linker through Schiff base reaction. The immobilized lipase activity was evaluated by measuring the peak area of the hydrolyzate of p‐nitrophenyl acetate. In order to maintain the enzymatic activity of immobilized lipase, the acetonitrile content and the pH of the reaction solution were also optimized. Under the optimized reaction conditions, the Michaelis–Menten constant of the immobilized lipase and the half maximal inhibitory concentration for orlistat were studied, which were consistent with previous literature data. Furthermore, the developed method was applied to screen lipase inhibition activity from ten natural products. As a result, we found that six natural products have inhibitory effect on the activity of lipase, among which the inhibitory effect of Rhizoma atractylodis extract has never been reported before.     

Integration of capillary isoelectric focusing with monolithic immobilized pH gradient,immobilized trypsin microreactor and capillary zone electrophoresis for on‐line protein analysis

An integrated platform consisting of protein separation by CIEF with monolithic immobilized pH gradient (M‐IPG), on‐line digestion by trypsin‐based immobilized enzyme microreactor (trypsin‐IMER), and peptide separation by CZE was established. In such a platform, a tee unit was used not only to connect M‐IPG CIEF column and trypsin‐IMER, but also to supply adjustment buffer to improve the compatibility of protein separation and digestion. Another interface was made by a Teflon tube with a nick to couple IMER and CZE via a short capillary, which was immerged in a centrifuge tube filled with 20 mmol/L glutamic acid, to exchange protein digests buffer and keep electric contact for peptide separation. By such a platform, under the optimal conditions, a mixture of ribonuclease A, myoglobin and BSA was separated into 12 fractions by M‐IPG CIEF, followed by on‐line digestion by trypsin‐IMER and peptide separation by CZE. Many peaks of tryptic peptides, corresponding to different proteins, were observed with high UV signals, indicating the excellent performance of such an integrated system. We hope that the CE‐based on‐line platform developed herein would provide another powerful alternative for an integrated analysis of proteins.     

Advances in screening enzyme inhibitors by capillary electrophoresis

Capillary electrophoresis (CE) has attracted lots of attention due to its simplicity, low sample consumption, low solvent volume, high resolution, and high speed. Based on these advantages, it has been widely used in enzyme inhibitor screening. There are two main operation modes on enzyme inhibitor screening: off‐line (precapillary enzyme assays) in which process CE was used as an analytical tool; online (in‐capillary enzyme assays) which combined the sample injection, mix, reaction, separation, and detection within a single run. Additionally, diverse of new materials were introduced to immobilize enzyme, which has been coupled with CE for the study of enzyme activity and its inhibitor screening. This review gives an overview of the developments and applications for the CE‐based enzyme inhibitor screening.     

基于毛细管电泳的天然产物中酶抑制剂筛选研究进展

人类疾病的发生往往与体内各种酶的功能失调密切相关,因此酶一直是目前新药研发的重要靶标。天然产物是发现新药的宝贵资源,但是由于成分复杂,活性筛选一直受制于耗时费力的分离纯化过程。毛细管电泳(CE)技术由于具有样品和试剂消耗少、灵活多样的分离模式且不受样品基质干扰的特点,可以直接从粗提物开始筛选活性成分,在复杂样品活性筛选中显示出独特的优势。该文综述了近十年来CE在天然产物中酶抑制剂筛选的研究进展。其中重点介绍了CE应用于重要药物靶标,包括转移酶(激酶)、水解酶以及氧化还原酶等方面的应用,总结了用于酶抑制剂筛选的电泳分离模式和酶动力学研究,并展望了CE用于天然产物中活性成分筛选的应用前景。     

Determination of 4‐hydroxyproline‐2‐epimerase activity by capillary electrophoresis: A stereoselective platform for inhibitor screening of amino acid isomerases

Isomerases involved in the metabolism of D /L ‐amino acids represent promising therapeutic targets for treatment of disease. Herein, we report a tunable platform for the assessment of enzymatic kinetics involving amino acid isomerization by CE that offers improved selectivity and sensitivity over traditional methods. Enzyme activity and competition assays were evaluated for various hydroxyproline diastereoisomers, proline enantiomers and their structural analogs using 4‐hydroxyproline‐2‐epimerase as a model system. In this work, pyrrole 2‐carboxylic acid was found to be a selective inhibitor of 4‐hydroxyproline‐2‐epimerase with a half‐maximal inhibition concentration of (2.3±0.1) mM. Reliable methods for unambiguous characterization of amino acid isomerases are required for the screening of novel inhibitors with epimerase and/or racemase activity.     

Dual‐enzyme,co‐immobilized capillary microreactor combined with substrate recycling for high‐sensitive glutamate determination based on CE

A new method for high‐sensitive determination of glutamate was developed and evaluated based on CE by using dual‐enzyme co‐immobilized capillary microreactor combined with substrate recycling. The capillary microreactor was prepared by covalently co‐immobilizing glutamate dehydrogenase (GDH) and glutamic pyruvic transaminase (GPT) on the inner surface of a capillary and was characterized by SEM, ultraviolet‐visible spectroscopy, and fluorescence spectroscopy. The GDH‐GPT co‐immobilized capillary microreactor showed great stability and reproducibility. The apparent K_m for glutamate with GDH‐GPT coupled reaction was determined to be 0.61±0.06 mM but 2.56±0.24 mM when only GDH was immobilized. Glutamate determination was based on on‐column monitoring UV absorption at 340 nm of the reaction product reduced nicotinamide adenine dinucleotide, of which peak area was directly related to the glutamate concentration. The response of the present co‐immobilized GDH‐GPT assay for glutamate is greatly enhanced over single enzyme system, and a 15.7‐fold improvement in sensitivity was obtained. The detection limit of the proposed method is 0.15 μM glutamate (S/N=3). Selectivity for glutamate is good over most of the 20 amino acids. Finally, this method was successfully applied to determine the glutamate content in rat plasma and serum samples.     

On-line immobilized acetylcholinesterase microreactor for screening of inhibitors from natural extracts by capillary electrophoresis

In this study we developed a simple capillary electrophoresis (CE) method with an on-line acetylcholinesterase (AChE) microreactor at the inlet of capillary for inhibitor screening. The fused-silica capillary surface was modified with a polycationic polyethylenimine coating. Solutions of the enzyme and chitosan were then injected to immobilize the enzyme in approximately 2.9?cm of the capillary inlet (total length of capillary 60.2?cm) by electrostatic interaction and the film overlay technique. Separation of enzyme reaction product (thiocholine, ThCh) and unreacted substrate (acetylthiocholine, AThCh) was achieved within 3.0?min. The conditions affecting the efficiency of reaction of the enzyme were optimized by measuring the peak area of ThCh. Under the optimum conditions, using Huperzine-A as model inhibitor, K (i) and IC (50) were 0.551?μmol?L(-1) and 1.52?μmol?L(-1), respectively, for immobilized AChE. Finally, screening of a small compound library containing two known AChE inhibitors and 30 natural extracts was conducted, and species with inhibition activity were directly identified. Compared with previous publications on screening for AChE inhibitors in natural products based on CE methods, the method developed in this work has the advantages of lower cost per analysis, less leakage, and better bioaffinity for the immobilized enzyme because of the unique properties of sodium alginate and chitosan.     

Trypsin inhibitor screening in traditional Chinese medicine by using an immobilized enzyme microreactor in capillary and molecular docking study

A trypsin immobilized enzyme microreactor was successfully prepared in capillary for studying enzyme kinetics of trypsin and online screening of trypsin inhibitors from traditional Chinese medicine through capillary electrophoresis. Trypsin was immobilized on the inner wall at the inlet of the capillary treated with polydopamine. The rest of the capillary was used as a separation channel. The parameters including the separation efficiency and the activity of immobilized trypsin were comprehensively evaluated. Under the optimal conditions, online screening of trypsin inhibitors each time can be carried out within 6 min. The Michaelis–Menten constant of immobilized trypsin was calculated to be 0.50 mM, which indicated high affinity of the immobilized trypsin for the substrate. The half‐maximal inhibitory concentration of known inhibitor of benzamidine hydrochloride hydrate as a model inhibitor was 13.32 mM. The proposed method was successfully applied to screen trypsin inhibitors from 15 compounds of traditional Chinese medicine. It has been found that baicalin showed inhibitory potency. Molecular docking study well supported the experimental result by exhibiting molecular interaction between enzyme and inhibitors.     

Rapid screening of aminopeptidase N inhibitors by capillary electrophoresis with electrophoretically mediated microanalysis

In this work, an electrophoretically mediated microanalysis (EMMA) method with a partial‐filling technique was setup to evaluate the inhibitory potency of novel compounds toward aminopeptidase N (APN). It was necessary to optimize the electrophoretic conditions with respect to the kinetic constraints and for attaining high sensitivity. In our setup, a part of the capillary was filled with the incubation buffer for the enzyme reaction, whereas the rest was filled with a suitable BGE for the separation of substrates and products. To monitor the performance of the newly developed method, the kinetic constants (K_m and V_max) for the catalyzed dissociation of l ‐Leucine‐p‐nitroanilide in the presence of APN as well as the inhibition constant (IC₅₀) of a known competitive inhibitor, that is bestatin, were determined and these results were compared with those obtained by a classical spectrophotometric assay. The developed EMMA method was subsequently applied to the screening of 30 APN inhibitors. Whereas the inhibition potency of these inhibitors (expressed in IC₅₀ values) were significantly underestimated by the EMMA method, the order of the inhibitory potential of these various compounds was found in agreement with the literature.     

Development and application of a capillary electrophoresis based method for the simultaneous screening of six antibiotics in spiked milk samples

An easy to use and low time consuming capillary electrophoresis (CE) method was developed and applied to the simultaneous determination of six antibiotics (ampicillin, amoxicillin, cloxacillin, penicillin, tetracycline and chloramphenicol) in spiked milk samples. Samples of milk were cleaned up by solid-phase extraction (with a C₁₈ cartridge) after protein precipitation. Analysis was performed by CE and results compared with the obtained via HPLC, both coupled to a UV-vis detector (210 nm). CE employed a 58.5 cm long fused-silica capillary (50 cm to detector), 75 μm i.d., a 2.7 × 10⁻² M KH₂PO₄, 4.3 × 10⁻² M Na₂B₄O₇ separation buffer, pH 8; an applied voltage of 18 kV; a hydrostatic injection of 0.5 psi during 3 s; and a run temperature of 25 °C. Under the described conditions, amoxicillin was not separated by HPLC, while CE was able to separate, and, therefore, allow detection. Regardless of amoxicillin, comparable results were obtained by HPLC and CE. The average recoveries of antibiotics, from milk fortified at 2.5 and 5 μg/mL, was over 72% with R.S.D.s within 5%. Recovery levels were essentially dictated by the used SPE cartridge.     

Evaluation inhibitory activity of catechins on trypsin by capillary electrophoresis–based immobilized enzyme microreactor with chromogenic substrate

In this study, a capillary electrophoresis‐based online immobilized enzyme microreactor was developed for evaluating the inhibitory activity of green tea catechins and tea polyphenol extracts on trypsin. The immobilized trypsin activity and other kinetic parameters were evaluated by measuring the peak area of the hydrolyzate of chromogenic substrate S‐2765. The results indicated that the activity of the immobilized trypsin remained approximately 90.0% of the initial immobilized enzyme activity after 30 runs. The value of Michaelis–Menten constant (K_m) was (0.47 ± 0.08) mM, and the half‐maximal inhibitory concentration (IC₅₀) and inhibition constant (K_i) of benzamidine were measured as 3.34 and 3.00 mM, respectively. Then, the inhibitory activity of four main catechins (epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate) and three tea polyphenol extracts (green tea, white tea, and black tea) on trypsin were investigated. The results showed that four catechins and three tea polyphenol extracts had potential trypsin inhibitory activity. In addition, molecular docking results illustrated that epigallocatechin gallate, epicatechin gallate, epicatechin, and epigallocatechin were all located not only in the catalytic cavity, but also in the substrate‐binding pocket of trypsin. These results indicated that the developed method is an effective tool for evaluating inhibitory activity of catechins on trypsin.     

Practical method for the rapid screening of xanthine oxidase inhibitors in herbal extracts by high‐performance liquid chromatography based on on‐line precolumn enzymatic reaction

A high‐performance liquid chromatography method with on‐line precolumn enzymatic reaction for the screening of xanthine oxidase inhibitors in natural extracts was developed. In this method, the enzymatic reaction occurred at the capillary inlet during a predetermined waiting period, after which the reaction product, uric acid, was separated and detected by liquid chromatography using ultraviolet absorption at 295 nm. Enzyme inhibition can be read out directly from the reduced peak area of uric acid in comparison to a reference chromatogram obtained in the absence of any inhibitor. In the present study, the availability of on‐line precolumn enzymatic reaction with ultraviolet detection was firstly evaluated by determining the inhibitory mechanism and IC₅₀ values of allopurinol, a commercially available positive drug. Then, the newly developed method was applied to screening of ten natural extracts from traditional Chinese medicine and as a result, the extract of Epimedium sagittatum (Sieb. et Zucc.) Maxim was found to be most positive for xanthine oxidase inhibition. The results obtained were compared with those obtained by offline enzyme assay and the effectiveness of the present method was confirmed. A rapid, low‐cost, and fully automated method for xanthine oxidase inhibitor screening was proposed.     

细管电泳-激光诱导荧光检测用于多个胞内蛋白激酶的抑制剂筛选和选择性评价

发展了一种基于毛细管电泳(CE)-激光诱导荧光(LIF)检测的多个细胞内源激酶的抑制剂平行筛选及选择性评价方法。CE高效的分离能力和LIF检测器的高选择性,使得同时测试多个胞内激酶的活性成为可能。共4种细胞系、3种特异性蛋白激酶底物肽、2种选择性蛋白激酶抑制剂和1种非选择性蛋白激酶抑制剂用于方法的建立。特异性底物肽与细胞裂解液混合后孵育,被其相应的激酶选择性地磷酸化,利用CE-LIF分离检测磷酸化产物和底物肽。同时测定一个抑制剂对几种蛋白激酶的抑制活性,用于评价抑制剂的选择性。与传统的单靶标筛选模式相比,这种基于细胞裂解液的多靶标筛选方法能提供更多的信息,更加高效,且细胞裂解液作为一种廉价的激酶来源大大降低了筛选成本。     

The next generation of capillary electrophoresis instruments: Performance of CE‐SDS protein analysis

Over the last decade, capillary electrophoresis gained tremendous importance, because it became an indispensible tool for the quality control of biologics, e.g. therapeutic antibodies. Consequently, there has been a continuous development within the CE market. Microchip techniques have been established in the last years. Further trends are complete solutions for specific applications by the usage of reagent kits. Step by step instructions and facilitated handling of the instruments are becoming more common. This work focuses on the sized‐based protein analysis with CE‐SDS. The instruments CE 7100 by Agilent Technologies, LabChip® GXII Touch HT by PerkinElmer, Maurice S. by Protein Simple and PrinCE NextI870 by Prince Technologies have been evaluated, mainly analyzing protein mixtures of different molecular weights in long series. Published data of the PA 800 plus by SCIEX are also included in the tabled results. Precision, reliability, flexibility, and speed have been identified as the most important performance parameters, others such as resolution, sensitivity, linearity, ease of use and sustainability have also been considered. All tested instruments have shown an excellent performance. Depending on application and necessities, each user can find the most appropriate one.     

Aldehyde oxidase assay by capillary electrophoresis: From off‐line,online up to immobilized enzyme reactor

Capillary electrophoresis is a modern separation technique characterized by many benefits, which qualify it also for enzyme assays and the study of enzyme kinetics during drug development. Homogeneous or heterogeneous approaches can be followed for the enzymatic incubation. In this study, an immobilization procedure of aldehyde oxidase on magnetic particles was developed considering their integration with capillary electrophoresis. A number of magnetic nano/microparticle types were tested for this purpose, showing that aldehyde oxidase was most active when immobilized on bare silica magnetic nanoparticles. Primarily, the reusability of the enzyme immobilized on bare silica nanoparticles was tested. Three consecutive incubations with substrate could be performed, but the activity considerably dropped after the first incubation. One reason could be an enzyme detachment from the nanoparticles, but no release was detected neither at 4°C nor at 37°C during 5 h. The drop in enzymatic activity observed in consecutive incubations, could also be due to inactivation of the enzyme over time at given temperature. For the immobilized enzyme stored at 4°C, the activity decreased to 83% after 5 h, in contrast with a steep decrease at 37°C to 37%.     

Enantiomeric separation of 1,3‐dimethylamylamine by capillary electrophoresis with indirect UV detection using a dual‐selector system

The CE method employing an indirect UV detection for the enantioseparation of 1,3‐dimethylamylamine (DMAA), widely used in various preworkout and dietary supplements labeled as a constituent of geranium extract has been developed. The dual‐selector system consisting of negatively charged sulfated α‐CD (1.1% w/v) and sulfated β‐CD (0.2% w/v) in 5 mM phosphate/Tris buffer (pH 3.0) containing the addition of 10 mM benzyltriethylammonium chloride (BTEAC) as the chromophoric additive was used for the enantiomeric separation of DMAA stereoisomers with the LODs in the range of 7.82–9.24 μg/mL. The method was partly validated and applied for the determination of the stereoisomeric composition of DMAA in commercial dietary supplements to verify the potential natural origin of DMAA.     

毛细管电泳法研究中草药有效成分对二氢叶酸还原酶的抑制作用

利用已建立的二氢叶酸还原酶抑制剂的毛细管电泳法筛选模型,研究了10种中草药有效成分对二氢叶酸还原酶的抑制作用.经实验测定发现木樨草素、表儿荼素、紫杉醇和槲皮素对二氢叶酸还原酶(DHFR)有抑制作用,且抑制效果为木樨草素>紫杉醇>表儿荼素>槲皮素.通过对产物NADP(氧化型辅酶Ⅱ)峰面积的定量测定,计算了4种抑制剂的抑制率.