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.     

Investigation of the Interaction Between Lidocaine and the Components of Hyaluronic Acid Using Frontal Analysis Continuous Capillary Electrophoresis

The frontal analysis continuous capillary electrophoresis (FACCE) technique was used for the characterziation of the interaction between lidocaine-HCl (Lido) and components of Na-hyaluronic acid (HA). N-Acetylglucosamine (GlcNAc) and glucuronic acid (GluA) were the components of Na-hyaluronic acid. For the investigations fused silica capillaries were used. The FACCE method was compared to affinity capillary electrophoresis (ACE). The association constants between lidocaine-HCl and the components of Na-hyaluronic acid were determined using FACCE. It was observed that only an interaction between Lido and GluA exhibited. The association constant (K _lido-GluA) between Lido and GluA was 26 ± 0.3 L mol⁻¹.

     

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.     

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.     

Investigation of the Interaction Between Lidocaine and the Components of Hyaluronic Acid Using Frontal Analysis Continuous Capillary Electrophoresis

The frontal analysis continuous capillary electrophoresis (FACCE) technique was used for the characterziation of the interaction between lidocaine-HCl (Lido) and components of Na-hyaluronic acid (HA). N-Acetylglucosamine (GlcNAc) and glucuronic acid (GluA) were the components of Na-hyaluronic acid. For the investigations fused silica capillaries were used. The FACCE method was compared to affinity capillary electrophoresis (ACE). The association constants between lidocaine-HCl and the components of Na-hyaluronic acid were determined using FACCE. It was observed that only an interaction between Lido and GluA exhibited. The association constant (K _lido-GluA) between Lido and GluA was 26 ± 0.3 L mol^?1.     

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.     

B-cell-activating factor is a regulator of adipokines and a possible mediator between adipocytes and macrophages

3T3-L1 adipocytes express the B-cell-activating factor (BAFF) and three different BAFF receptors (BAFF-Rs). Furthermore, BAFF expression is regulated by inflammatory modulators, such as tumor necrosis factor-α and rosiglitazone. Here we investigated the function of BAFF in 3T3-L1 adipocytes and RAW 264.7 macrophages. We examined adipokine expression in 3T3-L1 adipocytes treated with 10 ng ml⁻¹ BAFF. We also examined inflammatory molecule expression in RAW 264.7 macrophages treated with 10 or 100 ng ml⁻¹ BAFF. We examined BAFF expression in the coculture of 3T3-L1 adipocytes and RAW 264.7 macrophages, as well as in white adipose tissue (WAT) of diet-induced obese (DIO) mice. We found that BAFF decreases leptin and adiponectin expression, but increases the expression of proinflammatory adipokines monocyte chemotactic protein-1, interleukin-6 (IL-6), cyclooxygenase-2 (COX-2) and haptoglobin. Coculturing the two cell types resulted in increased BAFF mRNA and protein expression, as well as modulation of BAFF-R mRNA expression in both cell types. These data indicate that BAFF might mediate adipocyte and macrophage interaction. When RAW 264.7 macrophages were treated with BAFF, BAFF-R expression was modulated as in coculture, and nitric oxide synthase and IL-6 expression increased. BAFF expression also increased in WAT of DIO mice. We propose that BAFF can regulate adipokine expression and possibly mediate adipocyte and macrophage interaction.     

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。     

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.     

柚皮素、柚皮苷与溶菌酶相互作用的荧光光谱法研究

应用荧光光谱法研究了50%甲醇/水体系中柚皮素、柚皮苷与溶菌酶分子间的结合反应. 以Lineweaver-Burk双倒数方程和能量传递原理分别计算了两者与溶菌酶反应的结合常数(K)和结合距离(r): K_柚皮素^{25 ℃}＝4.00×10⁴, K_柚皮苷^{25 ℃}＝3.48×10⁴; r_柚皮素＝3.21 nm, r_柚皮苷＝3.30 nm, 以及由热力学参数的计算判断了两种分子与溶菌酶之间的作用力类型. 结果表明: 柚皮素、柚皮苷均能与溶菌酶以疏水作用相结合形成非共价化合物, 从而导致溶菌酶内在荧光的静态猝灭; 相对柚皮素, 柚皮苷与溶菌酶的结合距离增大, 作用强度减弱, 表明黄酮分子上多糖的取代不利于黄酮分子与蛋白之间的亲和作用. 根据Haslam等提出的多酚-蛋白质反应模型, 从分子水平初步探讨了糖取代对黄酮分子与蛋白相互作用减弱的原因.     

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.     

Chemical constituents from Gueldenstaedtia verna and their anti-inflammatory activity

A new triterpenoid glycoside (1) and 15 known compounds (2–16) were isolated from the whole plants of Gueldenstaedtia verna. The new compound (1) was identified as complogenin 22-O-β-d-glucopyranoside by extensive spectroscopic techniques including 1D (¹H and ¹³C) and 2D NMR experiments (HSQC, HMBC and NOESY), HR-DART-MS and chemical methods. Most of the isolates were evaluated for their inhibitory activities on LPS-induced NO production in RAW 264.7 cells. The inhibitory effects of the active compounds, sulphuretin (8) and (22E,24S)-5α,8α-epidioxy-24-methyl-cholesta-6,9(11),22-trien-3β-ol (13), on the production of pro-inflammatory mediators (including IL-6, IL1β and PGE₂) were further estimated in vitro by ELISA in RAW 264.7 macrophages.     

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     

Study on Interactions of Phenolic Acid-Like Drug Candidates with Bovine Serum Albumin by Capillary Electrophoresis and Fluorescence Spectroscopy

The interactions of the phenolic acids cinnamic acid (CNA), ferulic acid (FA), caffeic acid (CA) and chlorogenic acid (CLA) with bovine serum albumin (BSA) were investigated and compared using affinity capillary electrophoresis (ACE) and the fluorescence quenching methods. ACE gives binding constants (K _b) and thermodynamic parameters. The thermodynamic parameters show that each of four phenolic acids bind to BSA mainly by hydrogen bonds, electrostatic and hydrophobic interactions. The fluorescence quenching method provided quenching constant K _sv, binding site number n and K _b. The fluorescence results indicate that BSA fluorescence quenching is mainly a static quenching process. The binding constants (K _b) of CNA, FA, CA and CLA were from 2.52×10⁴ to 7.90×10⁴ L⋅mol⁻¹ from ACE experiments and 1.19×10⁴ to 5.21×10⁴ L⋅mol⁻¹ from fluorescence, their increase corresponded to the increase in the number of hydroxyl groups. These results imply that molecular structure and the number of hydroxyl groups of phenolic acids play act key roles in the affinity of natural phenolic acids towards BSA.     

Immunoaffinity capillary electrophoresis: determination of binding constant and stoichiometry for antibody-antigen interaction

Affinity capillary electrophoresis (ACE) can provide both qualitative and quantitative information on molecular interactions and affords the advantages of very low sample consumption, high mass sensitivity, short analysis time, and the use of automated instrumentation. It has been applied clinically and biochemically to the determination of the binding constant and to the measurement of the binding stoichiometry for interactions between antibodies (Ab's) and antigens (Ag's) in free solution. In many situations, the Ag molecule has two or multiple binding sites, each of which has a similar or different intrinsic affinity for binding independently to the combining site(s) on an Ab molecule. The multivalent binding reactions between Ab and Ag molecules often occur. The objective of this review is to describe the uses of ACE in the determination of binding constants and stoichiometry of Ab-Ag interactions (immunoaffnity capillary electrophoresis), focusing especially on multivalent Ab-Ag interaction modes. Five model binding systems developed recently using ACE techniques are described with principles and examples: (i) divalent mAb-monovalent Ag interaction, (ii) divalent mAb-(homo)polyvalent Ag interaction, (iii) cooperativity of two binding sites of mAb-monovalent Ag interaction, (iv) monovalent Fab-divalent Ag interaction, and (v) polyclonal Ab-monovalent Ag interaction. Finally, the determination of binding stoichiometry of Ab-Ag interactions by ACE is described.     

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.     

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.     

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.     

Molecular properties and immunomodulatory activities of a water-soluble heteropolysaccharide isolated from Plantago asiatica L. leaves

This study aims at investigating physical–chemical properties of a water-soluble heteropolysaccharide from Plnatago asiatica L. leaves, and its immunomodulatory effects on macrophages RAW264.7 cells. Hot water-extracted crude polysaccharide from the plant leaves (PLLCP) was fractionated into four fractions by DEAE Sepharose Fast Flow eluted with 0.1–0.5 M NaCl solutions. The most abundant fraction (0.3 M NaCl elution), designated PLLP-2, was identified as a heteropolysaccharide with an average molecular weight (M_w) of 3.54 × 10⁴ and composed mainly of Gal (34.4%), GalA (36.5%), Ara (10.1%) and Rha (8.4%). PLLP-2 was an acidic polysaccharide exhibiting inflaky curly aggregation with smooth surface. PLLCP and its main subfraction PLLP-2 displayed immunomodulatory activities by stimulating the production of pro-inflammatory cytokines, including TNF-α and IL-1β in macrophage RAW264.7 cells. These results indicate that the main polysaccharide fraction purified from P. asiatica L. leaves is probably pectin, and have potential immunomodulatory function.     

Comparison of various capillary electrophoretic approaches for the study of drug–protein interaction with emphasis on minimal consumption of protein sample and possibility of automation†

The binding ability of a drug to plasma proteins influences the pharmacokinetics of a drug. As a result, it is a very important issue in new drug development. In this study, affinity capillary electrophoresis, capillary electrophoresis with frontal analysis, and Hummel Dreyer methods with internal and external calibration were used to study the affinity between bovine serum albumin and salicylic acid. The binding constant was measured by all these approaches including the equilibrium dialysis, which is considered to be a reference method. The comparison of results and other considerations showed the best electrophoretic approach to be capillary electrophoresis‐frontal analysis, which is characterized by the high sample throughput with the possibility of automation, very small quantities of biomacromolecules, simplicity, and a short analysis time. The mechanism of complex formation was then examined by capillary electrophoresis with frontal analysis. The binding parameters were determined and the corresponding thermodynamic parameters such as Gibbs free energy ΔG⁰, enthalpy ΔH⁰, and entropy changes ΔS⁰ at various temperatures were calculated. The results showed that the binding of bovine serum albumin and salicylic acid was spontaneous, and that hydrogen bonding and van der Waals forces played a major role in the formation of the complex.