Multiple-step ligand injection affinity capillary electrophoresis for determining binding constants of ligands to receptors

This work demonstrates the use of multiple-step ligand injection affinity capillary electrophoresis (ACE) using two model systems: vancomycin from Streptomyces orientalis and carbonic anhydrase B (CAB, EC 4.2.1.1). In this technique a sample plug of receptor and non-interacting standards is injected by pressure and electrophoresed in a buffer containing a given concentration of ligand. The sequence is repeated for all concentrations of ligand generating a single electropherogram containing a series of individual sample plugs superimposed on environments of buffer containing increasing concentrations of ligand. Analysis of the change in the relative migration time ratio, RMTR, relative to the non-interacting standards, as a function of the concentration of the ligand, yields a value for the binding constant. A competitive assay using the technique is also demonstrated using neutral ligands for CAB. These values agree well with those estimated using other binding and ACE techniques. Data demonstrating the quantitative potential of this method are presented.     

Multiple-injection affinity capillary electrophoresis to estimate binding constants of receptors to ligands

Multiple-injection affinity capillary electrophoresis (MIACE) is used to determine binding constants (K _b) between receptors and ligands using as model systems vancomycin and teicoplanin from Streptomyces orientalis and Actinoplanes teichomyceticus, respectively, and their binding to D-Ala-D-Ala peptides and carbonic anhydrase B (CAB. EC 4.2.1.1) and the binding of the latter to arylsulfonamides. A sample plug containing a non-interacting standard is first injected followed by multiple plugs of sample containing the receptor and then a final injection of sample containing a second standard. Between each injection of sample, a small plug of buffer is injected which contains an increasing concentration of ligand to effect separation between the multiple injections of sample. Electrophoresis is then carried out in an increasing concentration of ligand in the running buffer. Continued electrophoresis results in a shift in the migration time of the receptor in the sample plugs upon binding to their respective ligand. Analysis of the change in the relative migration time ratio (RMTR) or electrophoretic mobility (μ) of the resultant receptor–ligand complex relative to the non-interacting standards, as a function of the concentration of ligand yields a value for K _b. The MIACE technique is a modification in the ACE method that allows for the estimation of binding affinities between biological interactions on a timescale faster than that found for standard ACE. In addition sample volume requirements for the technique are reduced compared to traditional ACE assays. These findings demonstrate the advantage of using MIACE to estimate binding parameters between receptors and ligands.     

Partial filling multiple injection affinity capillary electrophoresis (PFMIACE) to estimate binding constants of receptors to ligands

Partial filling multiple injection affinity capillary electrophoresis (PFMIACE) is used to determine binding constants between vancomycin (Van) from Streptomyces orientalis, teicoplanin (Teic) from Actinoplanes teicomyceticus and ristocetin (Rist) from Nocardia lurida to d-Ala-d-Ala terminus peptides and carbonic anhydrase B (CAB, E.C.4.2.1.1) to arylsulfonamides. Two variations of PFMIACE are described herein. In the first technique, the capillary is partially filled with ligand at increasing concentrations, a non-interacting standard, three or four separate plugs of receptor each separated by small plugs of buffer, a plug containing a second non-interacting standard, and then electrophoresed in buffer. Upon continued electrophoresis, equilibrium is established between the ligand and receptors causing a shift in the migration time of the receptors with respect to the non-interacting standards. This change in migration time is utilized for estimating multiple binding constants (K_b) for the same interaction. In the second technique, separate plugs of sample containing non-interacting standards, peptide one, buffer, and peptide two, were injected into the capillary column. The capillary is partially filled with a series of buffers containing an antibiotic at increasing concentrations and electrophoresed. Peptides migrate through the column at similar electrophoretic mobilities since their charge-to-mass ratios are approximately the same but remain as distinct zones due to the buffer plug between peptides. Upon electrophoresis, the plug of antibiotic flows into the peptide plugs affecting a shift in the migration time of the peptides with respect to the non-interacting standards occurs due to formation of the of the antibiotic-peptide complex. The shift in the migration time of the peptides upon binding to the antibiotic is used for the Scatchard analysis and measurement of a K_b. The PFMIACE technique expands the functionality and potential of ACE as an analytical tool to examine receptor-ligand interactions. In PFMIACE, a smaller amount of sample is required in the assay compared to both conventional ACE and MIACE. Furthermore, a wide array of data is obtained from a single experiment, thus, expediting the assay of biological species.     

Use of a Dual-Marker Form of Analysis to Estimate Binding Constants Between Receptors and Ligands by Affinity Capillary Electrophoresis

This work describes the use of a dual-standard analysis approach termed the time-average ratio (TAR) in affinity capillary electrophoresis (ACE) to estimate binding constants of receptors to ligands. In this form of analysis the TAR is the migration time of the receptor divided by the average of the sum of the migration times of two non-interacting standards. This change in TAR as a function of the concentration of ligand yields a value for the binding constant. This concept is demonstrated using three model systems: carbonic anhydrase B (CAB, EC 4.2.1.1) and arylsulfonamides, vancomycin (Van) and ristocetin (Rist) from Streptomyces orientalis and Nocardia lurida, respectively, and d-Ala- d-Ala terminus peptides. Three ACE techniques are used to examine the three systems: standard ACE, flow-through partial-filling ACE (FTPFACE), and on-column derivatization coupled to ACE. The findings described here demonstrate that ACE data analyzed using the TAR form of analysis yield binding constants between receptors and ligands comparable to those estimated using other ACE forms of analysis. A comparison to three other forms of analysis is described.     

Optimization of conditions for flow-through partial-filling affinity capillary electrophoresis to estimate binding constants of ligands to receptors

This work details the determination of the minimal injection time of ligand required in flow-through partial-filling affinity capillary electrophoresis (FTPFACE) to estimate binding constants of ligands to receptors. Two model systems are examined in this study: carbonic anhydrase B (CAB, EC 4.2.1.1) and arylsulfonamides, and vancomycin from Streptomyces orientalis and d-Ala-d-Ala peptides. Using CAB, a minimal injection time of 0.07 min at high pressure was determined that provided for the accurate and reproducible measurement of binding constants. In the FTPFACE technique, the capillary is first partially filled with a zone of ligand followed by a sample plug containing receptor and non-interacting standards. Upon application of a voltage the receptor and standards flow into the zone of ligand where a dynamic equilibrium is achieved between receptor and ligand. Continued electrophoresis results in the receptor and standards flowing through the domain of the ligand plug prior to detection. Analysis of the change in the relative migration time ratio (RMTR) of the receptor, relative to the non-interacting standards, as a function of the concentration of ligand, yields a value for the binding constant. In the present study, variable injection times of 4-carboxybenzenesulfonamide (CBSA) were examined to determine the minimal injection time needed to establish an equilibrium between CAB and ligand. A mathematical relationship was derived that correlated injection time and ligand concentration to the change in RMTR and comparisons made between the experimental and calculated values. Binding constants were obtained for a series of arylsulfonamide ligands and d-Ala-d-Ala terminus peptides to CAB and Van, respectively. The results support the use of FTPFACE to estimate affinity constants under variable experimental conditions.     

On-column synthesis coupled to affinity capillary electrophoresis for the determination of binding constants of peptides to glycopeptide antibiotics

Binding constants of the glycopeptide antibiotics teicoplanin (Teic), ristocetin (Rist), and vancomycin (Van), and their derivatives to D-Ala-D-Ala terminus peptides were determined by on-column ligand and receptor synthesis coupled to affinity capillary electrophoresis (ACE) or partial filling ACE (PFACE). In the first technique, 9-fluorenylmethoxycarbonyl (Fmoc)-amino acid-D-Ala-D-Ala species are first synthesized using on-column techniques. The initial sample plug contains a D-Ala-D-Ala terminus peptide and two non-interacting standards. Plugs two and three contain solutions of Fmoc-amino acid-N-hydroxysuccinimide (NHS) ester and buffer, respectively. Upon electrophoresis, the initial D-Ala-D-Ala peptide reacts with the Fmoc-amino acid NHS ester yielding the Fmoc-amino acid D-Ala-D-Ala peptide. Continued electrophoresis results in the overlap of the glycopeptide in the running buffer and the plug of Fmoc-amino acid-D-Ala-D-Ala peptide and non-interacting markers. Subsequent analysis of the change in the electrophoretic mobility (mu) or relative migration time ratio (RMTR) of the peptide relative to the non-interacting standards, as a function of the concentration of the antibiotic, yields a value for the binding constant. In the second technique, derivatives of the glycopeptides Teic and Rist are first synthesized on-column before analysis by ACE or PFACE. After the column has been partially filled with increasing concentrations of D-Ala-D-Ala terminus peptides, a plug of buffer followed by two separate plugs of reagents are injected. The order of the reagent plugs containing the antibiotic and two non-interacting standards and the anhydride varies with the charge of the glycopeptide. Upon electrophoresis, the antibiotic reacts with the anhydride yielding a derivative of Teic or Rist. Continued electrophoresis results in the overlap of the derivatized antibiotic and the plug of D-Ala-D-Ala peptide. Analysis of the change in RMTR of the new glycopeptide relative to the non-interacting standards, as a function of the concentration of the D-Ala-D-Ala ligand yields a value for the binding constant.     

Through-a-chip partial filling affinity capillary electrophoresis for estimating binding constants of ligands to receptors

In this paper, we describe the development of a microfluidic/capillary electrophoresis (CE) technique employing partial filling affinity capillary electrophoresis (PFACE) to estimate binding constants of ligands to receptors using as model systems carbonic anhydrase B (CAB, EC 4.2.1.1) and vancomycin from Streptomyces orientalis. Using multilayer soft lithography (MSL), a microfluidic device (MD) consisting of fluid and control channels is fabricated and fitted with an external capillary column. Multiple flow channels allows for manipulation of a zone of ligand and sample containing receptor and non-interacting standards into the MD and subsequently into the capillary column. Upon electrophoresis the sample components migrate into the zone of ligand where equilibrium is established. Changes in migration time of the receptor are used in the analysis to obtain a value for the binding interaction. The manipulation of small volumes of solution on the MD minimizes the need of time-consuming pipetting steps.     

On-column ligand synthesis coupled to partial-filling affinity capillary electrophoresis to estimate binding constants of ligands to a receptor

This paper describes a two-step procedure whereby on-column ligand synthesis and partial-filling affinity capillary electrophoresis (PFACE) are sequentially coupled to each other to determine the binding constants of 9-fluorenylmethoxy carbonyl (Fmoc)-amino acid-D-Ala-D-Ala species to vancomycin (Van) from Streptomyces orientalis. In this technique four separate plugs of sample are injected onto the capillary column and electrophoresed. The initial sample plug contains a D-Ala-D-Ala terminus peptide and two non-interacting standards. Plugs two and three contain solutions of Fmoc-amino acid-N-hydroxysuccinimide (NHS) ester and running buffer, respectively. The fourth sample plug contains an increasing concentration of Van partially-filled onto the capillary column. Upon electrophoresis the initial D-Ala-D-Ala peptide reacts with the Fmoc-amino acid NHS ester yielding the Fmoc-amino acid D-Ala-D-Ala peptide. Continued electrophoresis results in the overlap of the plugs of Van and Fmoc-amino acid-D-Ala-D-Ala peptide and non-interacting markers. Analysis of the change in the relative migration time ratio of the Fmoc-amino acid-D-Ala-D-Ala peptide relative to the non-interacting standards, as a function of the concentration of Van, yields a value for the binding constant. These values agree well with those estimated using other binding and ACE techniques.     

Estimation of binding constants between ristocetin and teicoplanin and peptides using on-column ligand derivatization coupled to affinity capillary electrophoresis

This work utilizes on-column ligand synthesis and affinity capillary electrophoresis (ACE) to determine binding constants (K_b) of 9-flourenylmethyloxy carbonyl (Fmoc)-amino acid derivatives to the glycopeptide antibiotics ristocetin (Rist) and teicoplanin (Teic). In this technique, two separate plugs of sample are injected on to the capillary column and electrophoresed. The initial sample plug contains a d-Ala-d-Ala terminus peptide and either one or two non-interacting standard(s). The second plug contains a Fmoc-amino acid-N-hydroxysuccinimide (NHS) ester. The electrophoresis is then carried out with an increasing concentration of Rist or Teic in the running buffer. Upon electrophoresis the initial d-Ala-d-Ala peptide reacts with the Fmoc-amino acid yielding a new Fmoc-amino acid-d-Ala-d-Ala peptide derivative. Continued electrophoresis results in the binding of Rist or Teic to the Fmoc-amino acid-d-Ala-d-Ala peptide derivatives. Analysis of the change in the relative migration time ratio (RMTR) or electrophoretic mobility () of the Fmoc-amino acid-d-Ala-d-Ala peptide derivatives relative to the non-interacting standards, as a function of the concentration of Rist and Teic, yields a value for K_b. These findings demonstrate the advantage of coupling on-column ligand synthesis to ACE for estimating binding parameters between antibiotics and ligands.Abbreviations Rist Ristocetin - Teic Teicoplanin - ACE Affinity capillary electrophoresis - RMTR Relative migration time ratio     

Flow-through partial-filling affinity capillary electrophoresis can estimate binding constants of neutral ligands to receptors via a competitive assay technique

This work evaluates the use of a competitive binding assay using flow-through partial-filling affinity capillary electrophoresis (FTPFACE) to estimate binding constants of neutral ligands to a receptor. We demonstrate this technique using, as a model system, carbonic anhydrase B (CAB, EC 4.2.1.1) and arylsulfonamides. In this technique, the capillary is first partially filled with a negatively charged ligand, a sample containing CAB and two noninteracting standards, and a neutral ligand, then electrophoresed. Upon application of a voltage the sample plug migrates into the plug of negatively charged ligand (L(-)) resulting in the formation of a CAB-L(-) complex. Continued electrophoresis results in mixing between the neutral ligand (L(0)) and the CAB-L(-) complex. L(0) successfully competes out L(-) to form the new CAB-L(0) complex. Analysis of the change in the relative migration time ratio (RMTR) of CAB relative to the noninteracting standards, as a function of neutral ligand concentration, yields a value for the binding constant. These values are in agreement with those estimated using other binding and ACE techniques. Data demonstrating the quantitative potential of this method is presented.     

Determination of binding constants between the antibiotic ristocetin A and D-Ala-D-Ala terminus peptides by affinity capillary electrophoresis

Summary Binding constants between the antibiotic ristocetin A (Rist A) and D-Ala-D-Ala terminus peptides were determined using affinity capillary electrophoresis (ACE). In these experiments two techniques are used to obtain binding constants. In the first, a plug of Rist A and non-interacting standards are injected and electrophoresed. Analysis of the change in the relative migration time ratio (RMTR) of Rist, relative to the non-interacting standards, as a function of the concentration of peptide, yields a value for the binding constant (Kb). In the second, samples of peptide and standards are injected and electrophoresed in increasing concentrations of Rist A in the running buffer. Analysis using theRMTR yields aK _b. The findings described here demonstrate the advantage of using ACE for estimating binding parameters between antibiotics and ligands.     

Electrophoretic affinity measurements on microchip. Determination of binding affinities between diketopiperazine receptors and peptide ligands

ACE on a microchip (MC-ACE) is introduced as a fast and reliable method to determine binding affinities. It is based on monitoring the change in the ionic mobility of a receptor upon binding to a ligand, or vice versa. The method is complementary to other standard methods for binding affinity determinations, like isothermal titration calorimetry (ITC), NMR, UV-Vis spectroscopy, etc. and allows for affinity studies of weak to strong binding interactions. The method is attractive since it principally allows for the analysis of the binding affinity of multiple receptors to a given ligand and requires comparatively small quantities of the binding partners (particularly in comparison to affinity measurements on capillary). We demonstrate the applicability of MC-ACE for the determination of the binding affinities between acid-rich diketopiperazine receptors and basic tripeptides in aqueous solution.     

亲和毛细管电泳技术及其应用

对近几年新发展起来的亲和毛细管电泳技术（ACE）的原理、分类及方法作了简要介绍,着重介绍了亲和毛细管区带电泳、毛细管亲和凝胶电泳、胶束电动色谱中的亲和电泳、亲和毛细管等电聚焦、亲和探针毛细管电泳等过程和方法。对ACE在分子生物学、生物化学中的应用及该技术在亲和常数测定、核酸片段识别、竞争免疫分析、药物先导化合物的筛选等方面的应用也作了介绍。     

新多肽配体GE11与表皮生长因子受体的结合能力分析

应用亲和毛细管电泳（ACE）分析方法,对表皮生长因子受体（EGFR）和新多肽配体GE11之间的结合能力进行分析。结果表明,EGFR与多肽配体GE11之间存在特异性相互作用,考察EGFR在不同浓度GE11溶液中的迁移情况,采用非线性、双倒数、Y-倒数和X-倒数4个数据处理方法得到较好的数据拟合,并测得结合常数。该文为筛选多肽配体以及测定受体与多肽配体之间的结合常数提供了简便的方法,将有力推动肿瘤靶向药物输送的研究。     

Implementation of chemometric methodology in ACE: predictive investigation of protein-ligand binding

An ACE predictive investigation of protein-ligand binding using a highly effective chemometric response surface design technique is presented. Here, K(d) was estimated using one noninteracting standard which relates to changes in the electrophoretic mobility of carbonic anhydrase B (CAB, EC 4.2.1.1) on complexation with the ligand 4-carboxybenzenesulfonamide (CBSA) present in the electrophoresis buffer. Experimental factors including injection time, capillary length, and applied voltage were selected and tested at three levels in a Box-Behnken design. Statistical analysis results were used to create a mathematical model for response surface prediction via contour and surface plots at a given target response (K(d) = 1.19x10(-6) M). As expected, there were a number of predicted solutions that reached our target response based on the significance of each factor at appropriate levels. The adequacy of the model was validated by experimental runs with the predicted model solution (capillary length = 47 cm, voltage = 11 kV, injection time = 0.01 min) presented in detail as an example.     

亲和毛细管电泳法研究锌离子与人血清白蛋白的结合反应机制

建立了研究金属离子与人血清白蛋白(Human serum albumin,HSA)相互作用的亲和毛细管电泳(Affinity capillary electrophoresis,ACE)方法。生理条件下,构建配体(Zn2+)-受体(HSA)相互作用模型,以N,N-二甲基甲酰胺(N,N-Dimethylformamide,DMF)为内标物,基于Scatchard方程,依据有效淌度的变化,通过非线性模拟方程计算Zn2+-HSA结合反应的表观结合常数KB,定量表征了Zn2+-HSA相互作用的强度,并解析电泳谱图获得了Zn2+-HSA结合反应为一快平衡体系的结论。结果表明,建立的ACE方法简捷、有效,Zn2+-HSA相互作用的强度与Zn2+浓度之间存在明显的量效关系。     

Partial-filling affinity capillary electrophoresis

Partial-filling affinity capillary electrophoresis (PFACE) is used to examine the binding interactions between two model biological systems: D-Ala-D-Ala terminus peptides to the glycopeptide antibiotic vancomycin (Van) from Streptomyces orientalis, and arylsulfonamides to carbonic anhydrase B (CAB, EC 4.2.1.1, bovine erythrocytes). Using these two systems, modifications in the PFACE technique are demonstrated including flow-through PFACE (FTPFACE), competitive flow-through PFACE (CFTPFACE), on-column ligand synthesis PFACE (OCLSPFACE), and multiple-step ligand injection PFACE (MSLIPFACE). In PFACE small plugs of sample are injected into the capillary column and an equilibrium is established between receptor and ligand during electrophoresis. Binding constants are then obtained by Scatchard analysis using changes in the migration time of the receptor/ligand on changing the concentration of the ligand/receptor. Data demonstrating the quantitative potential of these methods are presented. This review focuses on the unique capabilities of the different PFACE techniques as applied to two model biological systems.     

Multiple-injection affinity capillary electrophoresis to examine binding constants between glycopeptide antibiotics and peptides

Multiple-injection affinity capillary electrophoresis (MIACE) was used to determine binding constants (K(b)) between vancomycin, ristocetin, and teicoplanin from Streptomyces orientalis, Nocardia lurida, and Actinoplanes teichomyceticus, respectively, and fluorenylmethoxycarbonyl (Fmoc)-(Gly, Ala, Val, and Phe)-D-Ala-D-Ala peptides. In this technique, separate plugs of sample containing non-interacting standards, peptide one, buffer, and peptide two, were injected into the capillary column and electrophoresed. Peptides migrate through the column at similar electrophoretic mobilities but remain as distinct zones due to the buffer plug between peptides. The electrophoresis is then carried out in an increasing concentration of antibiotic in the running buffer. Continued electrophoresis results in a shift in the migration time of the peptides upon binding to the antibiotic. Analysis of the change in the relative migration time ratio (RMTR) of the resultant complexes relative to the non-interacting standards, as a function of the concentration of antibiotic yields a value for K(b). MIACE is a versatile technique that can be used to measure affinity constants between ligands of similar relative molecular mass and charge without the need of separate binding experiments. The findings described, herein, demonstrate the advantages of using MIACE to estimate binding parameters between ligands and receptors.     

亲和毛细管电泳激光诱导荧光法测定牛血清白蛋白与其单克隆抗体的结合常数

生物分子之间的特异性相互作用是生物界普遍存在的现象．研究这些现象,对揭示生物化学作用机理、药物研究等具有重要意义．结合常数Ｋｂ是描述生物分子之间特异性相互作用最主要的参数,测定结合常数的传统方法包括平衡透析、凝胶过滤色谱和分光光度法等［１］．亲和毛细管电泳（Ａｆｆｉｎｉｔｙｃａｐｉｌｌａｒｙｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ,简称ＡＣＥ）是近几年发展起来的毛细管电泳的一个分支,在研究生物分子之间特异性相互作用等方面有很好的应用前景［２～５］．与上述传统方法相比,ＡＣＥ具有测定速度快,样品用量少,有多…     

Peak shape modeling by Haarhoff-Van der Linde function for the determination of correct migration times: a new insight into affinity capillary electrophoresis

Among the different experimental strategies available in capillary electrophoresis (CE) to determine binding parameters, affinity capillary electrophoresis (ACE) has been the most widely embraced due to its easiness of implementation and of data handling. Ligand-substrate binding constants are thus directly derived from the substrate migration time shifts resulting from the variation of ligand concentration introduced in a background electrolyte. Classically, the substrate migration time is measured on top of the electrophoretic peak, assuming symmetrical peak shape. Depending on both substrate and ligand concentrations that may be required to meet detection sensitivity or complexation conditions, zonal migrations in ACE may, however, produce triangular peak shape, most often due to pronounced electromigration dispersion (EMD), and this may result in positively or negatively erroneous migration time assessments. In this work, EMD distorted triangular peak shapes obtained in the course of host-guest complexation studies were fitted with the Haarhoff-Van der Linde function, allowing better estimation of migration time. The model systems studied were those of beta-cyclodextrin and naproxen, 2-naphthalenesulfonate, or 1-adamantanecarboxylate. The impact of this correction on binding isotherms and binding constant evaluation was exemplified. Furthermore, in situations where the substrate concentration injected by far overtakes that of the ligand in the electrolyte, the interest in this peak shape correction was discussed in connection with the question of whether the free ligand concentration can be still considered equal to the ligand concentration introduced, a question that still remains under debate nowadays.