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。     

Measurement of monomolecular binding constants of neutral phenols into the beta-cyclodextrin by continuous frontal analysis in capillary and microchip electrophoresis via a competitive assay

Measurement of binding constant by chip electrophoresis is a very promising technique for the high throughput screening of non-covalent interactions. Among the different electrophoretic methods available that yield the binding parameters, continuous frontal analysis is the most appropriate for a transposition from capillary electrophoresis (CE) to microchip electrophoresis. Implementation of this methodology in microchip was exemplified by the measurement of inclusion constants of 2-naphtalenesulfonate and neutral phenols (phenol, 4-chlorophenol and 4-nitrophenol) into beta-cyclodextrin by competitive assays. The issue of competitor choice is discussed in relation to its appropriateness for proper monitoring of the interaction.     

Capillary electrophoretic methods applied to the investigation of peptide complexes

This article gives an overview of the applications of capillary electrophoretic methods to investigate the non‐covalent interactions of peptides (peptide complexes) with variable middle‐ and high‐molecular‐mass receptors (ligands) as well as with small ions and molecules in the period 2007–2014. Different modes of capillary electrophoretic methods, such as mobility shift (vacancy) affinity capillary electrophoresis, multiple injection affinity capillary electrophoresis, partial filling affinity capillary electrophoresis, Hummel–Dryer method, vacancy peak method and (continuous) frontal analysis capillary electrophoresis, are briefly described and their applicability to determination of binding constants of peptide complexes is discussed. In addition, the detailed experimental conditions of individual applications and the values of binding constants of the particular peptide complexes are presented.     

Determination of Binding Constants for Basic Drugs with Serum Albumin by Affinity Capillary Electrophoresis with the Partial Filling Technique

A simple, rapid, and accurate method is presented for determination of the binding constants of eight basic drugs with serum albumin by affinity capillary electrophoresis with the partial filling technique. Molecular modeling and multivariate regression analysis were used to investigate the relationship between the binding constants and the physicochemical properties of the drugs. The results show that hydrophobic attraction is responsible for interaction of most of the drugs with HAS, although interaction between sulfamethoxazole and HSA is because of electrostatic attraction.     

亲和毛细管电泳间接紫外检测法测定金属络合物的亲和常数

 采用亲和毛细管电泳间接紫外检测方法 ,根据“峰漂移”模型 ,通过迁移时间的测定 ,可以获得在水体系中有极低亲和常数的金属络合物的亲和常数。将该方法分别应用于镁离子 柠檬酸体系和锰离子 酒石酸体系 ,在 pH为 5 .0 1,运行电压为 2 0kV ,缓冲溶液组成为咪唑和醋酸的条件下 ,测定了缓冲溶液中加入不同浓度配体后金属离子迁移时间的变化 ,经过数据处理后得出它们的亲和常数对数值分别是 3.2 7和 2 .2 8,与文献值较为一致。该方法适用于结合比为 1∶1的金属络合物的亲和常数的测定。     

Affinity capillary electrophoresis is a powerful tool to identify transthyretin binding drugs for potential therapeutic use in amyloidosis

In this work we used affinity capillary electrophoresis (ACE) to investigate the extent of interaction between a pool of drugs and wild-type transthyretin. After qualitative preliminary screening, attention was focused on the most promising molecules, flufenamic acid and flurbiprofen, which underwent a further stage of investigation, the determination of the binding constants, and, when possible, the assessment of the number of binding sites by ACE, frontal analysis (FA) capillary electrophoresis (CE) and parallel ultrafiltration (UF) experiments. Furthermore, our data demonstrate that FA CE is a suitable technique for identifying fibril ligands. This represents a novel CE application of pharmaceutical interest.     

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.     

Estimation of binding constants of receptors and ligands by affinity capillary electrophoresis

An estimation method for determination of binding constants of receptors to ligands by affinity capillary electrophoresis was evaluated. On the basis of the theories of pseudostationary phase or so-called dynamic stationary phase, the retention factor (k) was used to represent the interaction between the receptor and ligand. k could be easily deduced from the migration times of the ligand and the receptor. Then, with the linear relationship of k versus the concentration of ligand in the running buffer, the binding constant K _b was calculated from the slope and intercept. In order to test its feasibility, the calculation method was demonstrated using three model systems: the interactions between vancomycin and N-acetyl-d-Ala-d-Ala, ristocetin and N-acetyl-d-Ala-d-Ala, and carbonic anhydrase B and an arylsulfonamide. Estimated binding constants were compared with those determined by other techniques. The results showed that this estimation method was reliable. This calculation method offers a simple and easy approach to estimating binding constants of ligands to receptors.     

Capillary zone electrophoresis investigation of the interaction between heparin and granulocyte-colony stimulating factor

The interaction between standard heparin, low-molecular-weight heparin (LMWH), and granulocyte-colony stimulating factor (G-CSF) was studied by capillary zone electrophoresis. Both qualitative and quantitative characterizations of the heparin-protein binding were determined. The binding constants of the two different groups of heparins with G-CSF, calculated from the Scatchard plot by regression, were 4.805 x 10(5) M(-1) and 4.579 x 10(5) M(-1), respectively. The two binding constants measured are of the same order of magnitude at 10(5) M(-1), indicating that LMWH contains most of the functional groups bound to G-CSF by standard heparin.     

毛细管电泳法高效筛选8-氧代鸟嘌呤DNA糖基化酶的核酸适配体

8-氧代鸟嘌呤DNA糖基化酶(OGG1)是人体中重要的功能蛋白,在修复DNA氧化性损伤过程中起关键作用。氧化应激等引起的氧化损伤易导致炎症反应的发生,对OGG1的抑制可以一定程度上起到缓解作用;对癌细胞OGG1的抑制有望作为癌症治疗的新方法。目前的研究多集中于小分子对OGG1功能的影响和调控,而OGG1的适配体筛选尚未见报道。作为功能配体,适配体具有合成简单、高亲和力及高特异性等优点。该文筛选了OGG1的核酸适配体,结合毛细管电泳高效快速的优点建立了两种基于毛细管电泳-指数富集进化(CE-SELEX)技术的筛选方法:同步竞争法和多轮筛选法。同步竞争法利用单链结合蛋白(SSB)与核酸库中单链核酸的强结合能力,与目标蛋白OGG1组成竞争体系,并通过增加SSB浓度来增加竞争筛选压力,以去除与OGG1弱结合的核酸序列,一步筛选即可获得与OGG1强结合的核酸序列。多轮筛选法在相同孵育条件和电泳条件下,经3轮筛选获得OGG1的核酸适配体。比较两种筛选方法的筛选结果,筛选结果中频次最高的3条候选核酸适配体序列一致,其解离常数(K_D)值在1.71~2.64 μmol/L之间。分子对接分析结果表明候选适配体1(Apt 1)可能与OGG1中具有修复氧化性损伤功能的活性口袋结合。通过对两种筛选方法的对比,证明同步竞争法更加快速高效,对其他蛋白核酸适配体筛选方法的选择具有一定的指导意义。得到的适配体有望用于OGG1功能调控,以抑制其修复功能。     

Single run measurements of drug-protein binding by high-performance frontal analysis capillary electrophoresis and mass spectrometry

A novel drug-protein binding measurement method based on high-performance frontal analysis and capillary electrophoresis (HPFA/CE) is presented. A single run measurement approach is proposed to circumvent utilization of a calibration curve that is often performed with HPFA. A sensitive mass spectrometer is applied as a detector enabling the measurement of in vitro protein binding at lower drug concentrations. Unbound free fraction and binding constants can be determined by a single run measurement by consecutive injections of an internal drug standard, a buffer plug and a drug-protein mixture. Effects of injection volumes on peak height and plateau profile were investigated in two different separation systems, non-volatile buffer and volatile buffer, with UV and mass spectrometry detection, respectively. A simplified one-to-one binding model is employed to evaluate the proposed method by using both single and multiple drug concentrations to measure the unbound free fraction and calculate the binding constants of some selected compounds. The method is suitable for rapid and direct screening of the binding of a drug to a specific protein or drug-plasma protein binding.     

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.     

A fast high throughput method for the determination of acidity constants by capillary electrophoresis. 3. Basic internal standards

A set of 25 monoprotic bases is proposed as internal standards for pK(a) determination by capillary electrophoresis. The pK(a) of the bases is determined and compared with available literature data. The capillary electrophoresis internal standard method offers numerous advantages over other typical methods for pK(a) determination, especially of analysis time and buffer preparation. However, it requires disposing of appropriate standards with reference pK(a) value. The set of bases established in this work together with the set of acids previously established provide a reference set of compounds with well-determined acidity constants that facilitate the process of selecting appropriate internal standards for fast pK(a) determination by capillary electrophoresis in high throughput screening of pharmaceutical drugs. In addition, the performance of the method when acidic internal standards are used for the determination of acidity constants of basic internal standards has also been tested. Although higher errors may be expected in this case, good agreement is observed between determined and literature values. These results indicate that in most cases structural similarity between the analyte and the internal standard might not be an essential requirement in the internal standard method.     

Evaluation of a fluorescein-labeled estradiol derivative for use in affinity capillary electrophoresis

A fluorescein-labeled estradiol derivative was assessed for use in affinity capillary electrophoresis (ACE) in a competitive immunoassay format, in which the fluorescently labeled estradiol competed with unlabeled estradiol for a mouse anti-estradiol antibody. The preparation of the labeled estradiol produced a mixture of fluorescein-containing compounds that led to multiple peaks in the electropherogram and to which the antibody responded differently. Two of the components of the mixture, towards which the mouse antibody showed most affinity, were isolated using fraction collection via capillary electrophoresis (CE). The two fractions of the labeled estradiol products isolated by CE were characterized using mass spectrometric methods. The two active fluorescein-conjugated products differed in the carboxylate on the fluorescein moiety, one having a methyl group instead of the acidic hydrogen for the other. The estradiol antibody showed a stronger binding for the conjugate containing the methyl group, as determined from the estimated binding constants using Scatchard analysis. The isolated fractions of labeled estradiol were shown to be applicable to the ACE immunoassay method.     

Optimization of affinity capillary electrophoresis for routine investigations of protein–metal ion interactions

To facilitate the implementation of affinity capillary electrophoresis into routine binding screening studies of proteins with metal ions, method acceleration, transfer and precision improvement were investigated. Affinity capillary electrophoresis was accelerated by using shorter capillaries, employing lower sample concentrations and smaller injection volumes. Intra‐ and inter‐instrument method transfers were investigated considering the temperature setting of the capillary cooling system. For intra‐instrument method transfer, similar results were obtained when transferring a method from a long (62 cm) to a short (31 cm) capillary. The analysis time was reduced from 9 to 4 min. In case of inter‐instrument method transfer, interaction results showed small variation on the capillary electrophoresis instrument with inefficient capillary cooling system. Binding measurement precision was enhanced by slightly pushing the sample above the beginning of the capillary. Changing the buffer vials after each 30 runs and employing extra flushing after each 60 subsequent runs further enhanced the precision. The use of 0.1 molar ethylenediaminetetraacetic acid in the rinsing solution successfully desorbs the remaining metal ions from the capillary wall. Excellent precision for apparent mobility ratio measurements was achieved for different protein–metal ion interactions (relative standard deviation of 0.16–0.89%, 15 series, 12 runs for each).     

Application of cyclodextrin-mediated capillary electrophoresis to simultaneously determine the apparent binding constants and thermodynamic parameters of naphthalenesulfonate derivatives

Application of capillary electrophoresis (CE) to simultaneously determine the apparent binding constants and thermodynamic parameters for six positional and structural naphthalenesulfonate derivatives with β-cyclodextrin (β-CD) is presented. The change in electrophoresis mobilities was used to assess the binding constants by non-linear regression and three different linear plots methods (named double reciprocal, x-reciprocal and y-reciprocal). The substituent group(s) attached to the naphthalene ring considerably affected the inclusion behaviors of these naphthalenesulfonate derivatives. The binding constant varies over almost one order of magnitude and a highly selective sequence is obtained between these guest model compounds. Naphthalenesulfonates with the substituent(s) at the 2-position(s) displayed stronger interaction with β-CD, and gave well compatible results by these four plot methods. While at least one substituent was substituted into the 1-position of naphthalene showed the weak interaction or no interaction with β-CD. Comparison to three linear regression methods, the non-linear regression method proves to be the most suitable for these determinations. Additionally, apparent binding constants for each structural isomer with β-CD at several temperature, and thermodynamic parameters for binding were also calculated and discussed.     

Assessment of the binding performance of histamine‐imprinted microspheres by frontal analysis capillary electrophoresis

Frontal analysis capillary electrophoresis was used to evaluate the binding performance of molecularly imprinted microspheres (MIM) toward its template histamine and analogs at pH 7, and compared to the high performance liquid chromatographic method. In both methods, batch binding was employed and the binding parameters were calculated from the measured concentration of unbound amine analytes and afforded comparable histamine equilibrium dissociation constants (K _d ∼ 0.4 mM). FACE was easily carried out at shorter binding equilibration time (i.e. 30 min) and without the need to separate the microspheres, circumventing laborious and, in the case of the system under study, inefficient sample filtration. It also allowed for competitive binding studies by virtue of its ability to distinctly separate intact microspheres and all tested amines which could not be resolved in HPLC. K _d’s for nonimprinted (control) microspheres (NIM) from FACE and HPLC were also comparable (∼ 0.6 mM) but at higher histamine concentrations, HPLC gave lower histamine binding. This discrepancy was attributed to inefficient filtration of the batch binding samples prior to HPLC analysis resulting in an over‐estimation of the concentration of free histamine brought about by the presence of unfiltered histamine‐bound microspheres.     

On-column derivatization of the antibiotics teicoplanin and ristocetin coupled to affinity capillary electrophoresis

Binding constants between the glycopeptides teicoplanin (Teic) and ristocetin (Rist) and their derivatives to D-Ala-D-Ala terminus peptides were determined by on-column receptor synthesis coupled to partial-filling affinity capillary electrophoresis (PFACE) or affinity capillary electrophoresis (ACE). In these techniques, the column is first partially filled with increasing concentrations of D-Ala-D-Ala terminus peptides. This is followed by plugs of buffer, antibiotic and two noninteracting standards, and acetic and/or succinic anhydride (and buffer in the case of ACE). The order of the reagent plugs containing the antibiotic and 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 the relative migration time ratio (RMTR) of the new glycopeptide relative to the standards, as a function of the concentration of the D-Ala-D-Ala ligand yields a value for the binding constant K(b). The techniques described here can be used to assess how the derivatization of drugs alters their affinities for target molecules.     

Protonated diamines as anion-binding agents and their utility in capillary electrophoresis separations

Capillary zone electrophoresis is a proven method for separating small ions because of the inherent charge and differences in mobility of these analytes. Despite its resolving power, CZE can be insufficient for separating ions with similar mobilities. One remedy is to modify mobilities via the addition of background electrolyte complexation agents. However, this approach is not straightforward for inorganic anions, which lack complexation options. To address this shortfall, the diprotonated diamine moiety was investigated for complexation of dianions. Dicationic diamines significantly complexed dianions, and this interaction was not purely electrostatic in nature because affinities varied with dianion identity. Aqueous association constants were measured with affinity capillary electrophoresis (ACE) and found to be similar in magnitude but different in selectivity to those of dianions with magnesium ion. Binding was also investigated for zwitterionic buffers containing the protonated diamine moiety. Zwitterions exhibited binding constants as high as 18 M^?1 (30‐mM ionic strength). This work discusses the observed binding constants and their potential usefulness in CZE separations of inorganic anions. Also covered are improvements to ACE methodology and an evaluation of some of the assumptions employed.