Kinetic study of angiotensin converting enzyme activity by capillary electrophoresis after in-line reaction at the capillary inlet

The in-capillary reaction of angiotensin converting enzyme (ACE) with the tripeptide substrate hippuryl-L-histidyl-L-leucine was studied. ACE activity was determined by the quantitation of the product, hippuric acid, at 230 nm. Reaction occurred at the capillary inlet during a predetermined waiting period, followed by the electrophoretic separation of the compounds. When the set-up was reversed, i.e. reaction at the opposite side after short-end injection of enzyme and substrate, separation was achieved in less than 5 min. Using the Lineweaver-Burk equation, an average Michaelis constant for ACE from rabbit lung was calculated to be 1.16 +/- 0.12 mM, a value consistent with previously reported data.     

毛细管区带电泳用于酶微反应器酶解条件的研究

将氨基酰化酶通过戊二醛固定在毛细管内壁,制备毛细管酶微反应器,用毛细管区带电泳对毛细管酶微反应器的酶解产物进行分离,以生成物的峰面积优化底物N-乙酰-DL-蛋氨酸的酶解条件。实验结果表明,在温度37℃的条件下,10μg/mL N-乙酰-DL-蛋氨酸磷酸盐缓冲溶液(pH7.5)以4μL/min的速度通过15 cm长的毛细管酶微反应器,具有良好的酶解效果。利用毛细管酶微反应器对底物N-乙酰-DL-蛋氨酸进行酶解,每天酶解5次,10天后酶活仅下降了8.66%,说明制备的毛细管酶微反应器具有良好的稳定性。     

Analysis of angiotension-converting enzyme by capillary electrophoresis.

A method is described for determination of serum angiotension-converting enzyme by capillary electrophoresis (CE) based on incubation of the substrate, a synthetic peptide, with the serum outside the capillary and cleaving hippuric acid and a dipeptide. The reaction is stopped by the addition of acetonitrile, followed by injection of the supernatant on the capillary. The acetonitrile allows injection of a large volume of sample on the capillary. Both the substrate and the reaction product (hippuric acid) can be monitored at the same time. The CE step is rapid and can be performed in about 6 min. The CE method compared well to a kinetic assay method (= 0.98).     

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.     

Antioxidant activity of peptide-based angiotensin converting enzyme inhibitors

Angiotensin converting enzyme (ACE) inhibitors are important for the treatment of hypertension as they can decrease the formation of vasopressor hormone angiotensin II (Ang II) and elevate the levels of vasodilating hormone bradykinin. It is observed that bradykinin contains a Ser-Pro-Phe motif near the site of hydrolysis. The selenium analogues of captopril represent a novel class of ACE inhibitors as they also exhibit significant antioxidant activity. In this study, several di- and tripeptides containing selenocysteine and cysteine residues at the N-terminal were synthesized. Hydrolysis of angiotensin I (Ang I) to Ang II by ACE was studied in the presence of these peptides. It is observed that the introduction of L-Phe to Sec-Pro and Cys-Pro peptides significantly increases the ACE inhibitory activity. On the other hand, the introduction of L-Val or L-Ala decreases the inhibitory potency of the parent compounds. The presence of an L-Pro moiety in captopril analogues appears to be important for ACE inhibition as the replacement of L-Pro by L-piperidine 2-carboxylic acid decreases the ACE inhibition. The synthetic peptides were also tested for their ability to scavenge peroxynitrite (PN) and to exhibit glutathione peroxidase (GPx)-like activity. All the selenium-containing peptides exhibited good PN-scavenging and GPx activities.     

Targeted catalytic inactivation of angiotensin converting enzyme by lisinopril-coupled transition-metal chelates

A series of compounds that target reactive transition-metal chelates to somatic angiotensin converting enzyme (sACE-1) have been synthesized. Half-maximal inhibitory concentrations (IC(50)) and rate constants for both inactivation and cleavage of full-length sACE-1 have been determined and evaluated in terms of metal chelate size, charge, reduction potential, coordination unsaturation, and coreactant selectivity. Ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and tripeptide GGH were linked to the lysine side chain of lisinopril by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride/N-hydroxysuccinimide coupling. The resulting amide-linked chelate-lisinopril (EDTA-lisinopril, NTA-lisinopril, DOTA-lisinopril, and GGH-lisinopril) conjugates were used to form coordination complexes with iron, cobalt, nickel, and copper, such that lisinopril could mediate localization of the reactive metal chelates to sACE-1. ACE activity was assayed by monitoring cleavage of the fluorogenic substrate Mca-RPPGFSAFK(Dnp)-OH, a derivative of bradykinin, following preincubation with metal chelate-lisinopril compounds. Concentration-dependent inhibition of sACE-1 by metal chelate-lisinopril complexes revealed IC(50) values ranging from 44 to 4500 nM for Ni-NTA-lisinopril and Ni-DOTA-lisinopril, respectively, versus 1.9 nM for lisinopril. Stronger inhibition was correlated with smaller size and lower negative charge of the attached metal chelates. Time-dependent inactivation of sACE-1 by metal chelate-lisinopril complexes revealed a remarkable range of catalytic activities, with second-order rate constants as high as 150,000 M(-1) min(-1) (Cu-GGH-lisinopril), while catalyst-mediated cleavage of sACE-1 typically occurred at much lower rates, indicating that inactivation arose primarily from side chain modification. Optimal inactivation of sACE-1 was observed when the reduction potential for the metal center was poised near 1000 mV, reflecting the difficulty of protein oxidation. This class of metal chelate-lisinopril complexes possesses a range of high-affinity binding to ACE, introduces the advantage of irreversible catalytic turnover, and marks an important step toward the development of multiple-turnover drugs for selective inactivation of sACE-1.     

Oxidation of thiocyanate by hydrogen peroxide - a reaction kinetic study by capillary electrophoresis

The oxidation reaction kinetics of thiocyanate by excess hydrogen peroxide has been studied by using capillary electrophoresis. The paper illustrates for the first time the use of capillary electrophoresis in studying reaction kinetics and provides a non-laborious way to determine the rate law and the rate constant for the above reaction in the pH range 6–8. Standard solutions of thiocyanate were mixed with buffer solutions of different pHs (6–8) and the reactions were initiated by adding appropriate volumes of hydrogen peroxide in capillary electrophoresis vials. Each reaction mixture was sampled at regular time intervals using an automatic injection programme to follow the progress of the reaction and identify the reaction products. The peak areas, representing the products, were integrated and their concentrations were quantified using calibration plots. The concentration profiles obtained from a series of experiments at a particular pH were then used to determine the rate law and the rate constant for the reaction. Furthermore, the rate of decomposition of hypothiocyanite formed during the reaction is determined for the first time. The rate law is zero order with respect to hypothiocyanite and first order with respect to hydrogen peroxide. The results indicate that the rate law for the oxidation reaction is zero order with respect to thiocyanate and first order with respect to hydrogen peroxide. The rate constant for the reaction at 25°C and at zero ionic strength is 3.6(±0.2)×10(⁻⁴) min⁻¹.     

Online enzymatic sequencing of glycans from Trastuzumab by phospholipid-assisted capillary electrophoresis

CE separations of glycans taken from the cancer drug, Trastuzumab (Herceptin(?)), were accomplished using phospholipid additives. Glycans were labeled with 1-aminopyrene-3,6,8-trisulfonic acid and were separated with efficiencies as high as 510000 theoretical plates in a 60.2 cm 25 μm id fused-silica capillary. The thermally tunable phospholipid was loaded into the capillary when it possessed a viscosity similar to that of water. The temperature was increased, and the separations were performed when the material exhibited higher viscosity. Enzymes were integrated into the separation with the phospholipid additive. Neuraminidase, β1-4 galactosidase, and β-N-acetylglucosaminidase were injected into the capillary without covalent modification and used for enzyme hydrolysis. Exoglycosidase enzymes cleaved the terminal glycan residues. The glycan sequence could be verified based on enzyme specificity. Neuraminidase was used to determine total glycan content of the low-abundance glycans containing sialic acid. β1-4 Galactosidase and β-N-acetylglucosaminidase were used sequentially in-capillary, to determine the structure of the high-abundance glycans.     

Pharmacophore-based structure optimization of angiotensin converting enzyme inhibitory peptide

Chemical feature based pharmacophore models were generated for an angiotensin converting enzyme(ACE) inhibitory peptide using the Discovery Studio 2.0 pharmacophore modeling approach. The pharmacophore hypothesis selected has five features(one negative ionizable region,one hydrogen bond donor,one hydrogen bond acceptor and two hydrophobic functional groups). Additionally,ACE inhibitory hexapeptide previously obtained from silkworm pupae protein was optimized to target the ACE based on the selected pharmacophore. The results suggest that tri-peptide(thr-val-phe) may be structural determinant of ACE activity. Docking studies further provided confidence for the validity of the selected pharmacophore model to perform structure optimization of the ACE inhibitory peptide.     

Determination of rhodanese enzyme activity by capillary zone electrophoresis.

A new sensitive method has been developed for the determination of rhodanese activity. The enzymatic reactions were carried out directly in thermostatted autosampler vials and the formation of SCN- was monitored by sequential capillary zone electrophoretic runs. The determinations were performed in a 75-micron fused-silica capillary using 0.1 M beta-alanine-HCl (pH 3.50) as a background electrolyte, a separation voltage of 18 kV (negative polarity), a capillary temperature of 25 degrees C and direct detection at 200 nm. Short-end injection or long-end injection procedures were used for sample application. The method is rapid, able to be automated and requires only small amounts of sample and substrates, which is especially important in the case of highly toxic cyanide. The developed capillary electrophoretic method also has great potential for thiocyanate determinations in other applications.     

毛细管电泳序列分析技术用于在线酶分析研究进展

毛细管电泳技术具有操作简单、样品消耗量少、分离效率高和分析速度快等优势,不仅是一种高效的分离分析技术,而且已经发展成为在线酶分析和酶抑制研究的强有力工具。酶反应全程的实时在线监测,可以实现酶反应动力学过程的高时间分辨精确检测,以更准确地获得反应机制和反应速率常数,有助于更好地了解酶反应机制,从而更全面深入地认识酶在生物代谢中的功能。此外,准确、快速的在线酶抑制剂高通量筛选方法的发展,对加快酶抑制类药物的研发以及疾病的临床诊断亦具有重要意义。电泳媒介微分析法(EMMA)和固定化酶微反应器(IMER)是毛细管电泳酶分析技术中常用的在线分析方法。这两种在线酶分析法的进样方式通常为流体动力学进样和电动进样,无法实现酶反应过程中的无干扰序列进样分析。近年来,基于快速序列进样的毛细管电泳序列分析技术已经发展成为在线酶分析的另一种强有力手段,以实现高时间分辨和高通量的酶分析在线检测。该文从快速序列进样的角度,综述了近年来毛细管电泳序列分析技术在线酶分析的研究进展,并着重介绍了各种序列进样方法及其在酶反应和酶抑制反应中的应用,包括光快门进样、流动门进样、毛细管对接的二维扩散进样、流动注射进样、液滴微流...     

Miniaturization of sampling for chemical reaction monitoring by capillary electrophoresis

Purpose-made capillary electrophoresis set-ups for reaction kinetics monitoring featuring two automated injectors allowed the easy reduction of the needed reactant amount down to 500 microL. The first set-up is similar to the cross injector used frequently in lab-on-chip designs while the other uses falling droplets for sample/buffer delivery. The versatility of the system was demonstrated by the analysis of oxidation of C-vitamin by hydrogen peroxide. Pseudo first order reaction rates about 10(-3) s(-1) were measured with RSD = 1-3% in one experiment and RSD = 20% in interday/person experiment. Plate numbers were typically around 5000-20,000.     

Inactivation of human angiotensin converting enzyme by copper peptide complexes containing ATCUN motifs

The copper complex [KGHK-Cu]+ demonstrates catalytic inactivation of human angiotensin converting enzyme at sub-saturating concentrations, under oxidative conditions, with an observed rate constant k approximately 2.9 +/- 0.5 x 10(-2) min(-1).     

亲和色谱法筛选中药中血管紧张素转化酶抑制剂

以壳聚糖微球为载体、戊二醛（glutaraldehyde,GA）为交联剂对血管紧张素转化酶（Angiotensin converting enzyme,ACE）进行固定化．用固化的ACE作为亲和介质,利用血管紧张素转化酶抑制剂（Angiotensin convertingenzym einhibitor,ACEI）与ACE之间的亲和作用,结合高效液相色谱对亲和前后的体系进行检测,比较两者各组分色谱峰的差异,以此实现快速筛选复杂体系中的ACE抑制剂．应用赖诺普利（Lisinopril）、九肽抑制剂、依那普利（Enalapril）、培哚普利（Perindopril）、卡托普利（Captopril）等已上市的ACEI对方法进行验证,反映方法具有高度选择性．将方法应用于中药地龙及山楂筛选,发现共有5个组分与ACE有亲和作用,并且都能抑制ACE酶活性,它们对酶活性抑制的IC50值在0．45～4．62μg／mL范围．通过对亲和方法重现性考察,6次测定的相对标准偏差小于1％,说明方法可靠．提出的亲和色谱．色谱指纹差异法非常适合于从中药及天然产物等复杂混合物库中快速筛选靶点活性物质．     

High temporal resolution monitoring of enzyme reaction and inhibition using optically gated vacancy capillary electrophoresis and immobilized enzyme

A novel method for monitoring of enzyme reaction and inhibition with high temporal resolution was developed by using optically gated vacancy capillary electrophoresis (OGVCE) with laser-induced fluorescence (LIF) detection and immobilized enzyme. Trypsin cleavage reaction and inhibition were investigated by the presented OGVCE-LIF assay, using carboxyfluorescein (FAM) end-labeled Angiotensin as the substrate and commercially available immobilized trypsin. The substrate and the product were continuously loaded into the capillary by the electroosmotic flow while the immobilized enzyme remained in the sample vial. Substrate consumption and product formation were monitored simultaneously at 5 s interval during the whole reaction time. The enzymatic reaction rates obtained from the substrate and the product were highly consistent. The enzyme activity and the Michaelis constants of trypsin cleavage reaction, as well as the inhibition constant (for reversible competitive inhibitor) and the inhibition fraction (for irreversible inhibitor), were obtained. It was showed that the reported OGVCE-LIF method can perform fast, accurate, sensitive and reproducible CE enzyme assay with high temporal resolution, thus has great potential in application of the enzyme-substrate systems with fast reaction rate and the fluorescent substrate and products.     

Stability study of somatropin by capillary zone electrophoresis

Human Growth Hormone (hGH) is a monomeric 22 kilo Dalton (kDa), 191 amino acid protein with an isoelectric point (pI) close to pH 5, produced in the anterior pituitary gland. High level production of somatropin (recombinant hGH) is done in Escherichia coli (E. coli) to meet the demand and to avoid possible Creutzfeldt-Jacob disease (CJD).The present study was initiated on the basis of results from post-marketing control of all somatropin preparations on the Norwegian market. The samples consisted of preparations presented as somatropin in solution, and some freeze-dried preparations were also included for comparison.The present study showed a significant degree of degradation of somatropin in solution. Deamidation increased over time for preparations in solution, as well as for freeze-dried preparations after dissolution. Preparations in solution showed high content of deamidated and cleaved forms. Freeze-dried preparations after dissolution and storage showed high content of deamidated forms, but low content of cleaved forms. Also, in one preparation, an unknown peak was detected in the electropherogram from capillary zone electrophoresis (CZE), eluting after the principal peak, in front of the Gln-18 somatropin peak.     

Separation of polymerase chain reaction amplified bird genes by capillary electrophoresis

Female birds possess one Z and one W chromosome, whereas male birds possess two identical Z chromosomes. Thus, the presence of a W genetic marker is diagnostic of the female sex. Capillary electrophoresis with buffer containing an entangled solution of hydroxyethylcellulose was used to separate the polymerase chain reaction (PCR) amplified bird sexing genes CHD-Z and CHD-W. The relative standard deviations (RSD) were less than 0.6% for the male genes and less than 0.4% for the female genes for six runs and detection limits of 0.1 ng/microL were obtained with laser-induced fluorescence (LIF) detection. Using a DNA ladder and theoretical models for DNA separation in sieving media, the sizes of the two bird genes were determined.