Comparative studies of the interaction between ferulic acid and bovine serum albumin by ACE and surface plasmon resonance

Affinity capillary electrophoresis (ACE) was used to study the interaction between ferulic acid (FA) and BSA. The interaction between FA and BSA was facilitated by injecting FA into a BSA-containing running buffer. Both mobility ratio and mobility shift assays were performed to deduce the binding constant (K(b)). However, the K(b )value obtained with the mobility ratio assay was only approximately 20% of that extracted from the mobility shift assay. The former assay yielded a K(b) value (5.6 +/- 0.4 x 10(4) M(-1)), which compares well with the result obtained with surface plasmon resonance (SPR) (5.1 +/- 0.6 x 10(4) M(-1)). The discrepancy between the mobility ratio and mobility shift assays suggests that the data extrapolation from the mobility ratio should be more reliable for cases when both changes in the EOF and viscosity of the running buffer are important. The work demonstrates that ACE, a solution-based technique, and SPR, a technique addressing interfacial processes, are highly complementary to each other and the comparative studies are confirmatory and allow binding constants to be accurately determined.     

Binding studies of porphyrins to human serum albumin using affinity capillary electrophoresis

The present work demonstrates that affinity capillary electrophoresis (ACE) can be employed as a valuable and powerful tool for studying the interactions between porphyrins and proteins in biological and biomedical research, such as the development of porphyrins and related compounds as efficient and selective photosensitizers in the photodynamic therapy of cancers. Binding constants of human serum albumin (HSA) to four biological porphyrins (uroporphyrin I, heptacarboxylporphyrin, coproporphyrin I, protoporphyrin IX), which possess a wide range of hydrophobicity, were estimated by ACE. Based on 1:1 molecular association between these individual porphyrins and HSA, the change of the electrophoretic mobility of HSA as a function of porphyrin concentration in the run buffer was measured and the binding constants were calculated from the slope of the Scatchard plots. The binding constant values were found to be 8.80 +/- 0.51 x 10(4) M(-1), 2.39 +/- 0.16 x 10(5) M(-1), 1.61 +/- 0.11 x 10(6) M(-1), and 9.34 +/- 0.30 x 10(6) M(-1) for uroporphyrin I, heptacarboxylporphyrin, coproporphyrin I, and protoporphyrin IX, respectively, and most of these results are in good agreement with those reported in the literature using conventional methods for binding measurements. Additionally, experimental binding constant data obtained using ACE was found to exhibit very good correlation with theoretical hydrophobicity values calculated using the Rekker's hydrophobic fragmental constant method, thus further supporting the hypothesis that the hydrophobicity of the porphyrin side chains play an important role in governing the hydrophobic interaction of porphyrins with serum proteins such as HSA.     

亲和毛细管电泳测定孕酮与其单克隆抗体的结合常数

采用亲和毛细管电泳的配体分离模式,以激光诱导荧光作为检测手段,测定了荧光素标记的孕酮与孕酮我隆抗体之间的结合常数,并研究了温育时间、电泳条件等因素对测定的影响。     

Determination of chlorpheniramine and its binding with human serum albumin by capillary electrophoresis with tris(2,2'-bipyridyl)ruthenium(II) electrochemiluminescence detection.

Tris(2,2'-bipyridyl)ruthenium(II) electrochemiluminescence (ECL) detection in a capillary electrophoresis separation system was used for the determination of chlorpheniramine (CPM). The experimental conditions, such as the applied potential, separation voltage, injection voltage, injection time and the pH of the separation buffer were considered in detail. The ECL intensity showed two linear responses to CPM, i.e., from 15 microM to 1 mM and from 0.8 microM to 15 microM with a detection limit of 0.5 microM. The binding of CPM with human serum albumin was also monitored using this method and the binding constant was estimated to be 4.1 x 103 M(-1).     

基于毛细管电泳的苦参碱转运过程中的血清白蛋白行为成像

利用毛细管电泳（CE）技术在体外条件下建立了苦参碱（MT）与血清白蛋白（SA）相互作用的分析方法。生理条件下通过淌度移动法和前沿分析法（FA）研究苦参碱与血清白蛋白的结合状况，构建配体（MT）-受体（SA）相互作用模型。其中，淌度移动法采用非线性拟合方法获得苦参碱与人血清白蛋白（HSA）结合参数；FA运用非线性回归方程、Scatchard方程、Klotz方程3种方程获得苦参碱与人血清白蛋白结合参数，分析其相互作用状况进而分析模型适用度。利用淌度移动法可知，人血清白蛋白与苦参碱表观结合常数K_B为8.072×10³ mol/L；利用FA法可知，采用非线性回归方程、Scatchard方程、Klotz方程3种方程获得苦参碱与人血清白蛋白表观结合常数K_B分别为1.434×10³、1.781×10³和2.133×10³ mol/L，且二者结合位点数在1.0左右，说明苦参碱与人血清白蛋白作用只有单一类型的结合位点。通过计算分析得到3个方程的相关系数（r），关系为r_Klotz > r_非线性 > r_Scatchard。结果表明：淌度移动法和FA法均适用于分析MT-HSA体系的结合状况；由于FA法可以计算出表观结合常数的同时又能计算出结合位点数，因而更适合MT-HSA体系的分析研究；分析比较得出3种方程之中Klotz方程为最适理论模型。结合参数表明，MT-HSA相互作用体系之间发生的结合反应为单一类型的结合位点且结合稳定。相关工作阐明了典型生物碱与血清白蛋白的相互作用机制，可为生物碱的血液输运机制的深入研究提供有益参考。     

Spectroscopic investigations of hematoxylin–Eu(III) complex interacting with Herring-sperm DNA

The interaction of HE–Eu(III) complex (HE?=?hematoxylin) with Herring-sperm DNA (hsDNA) has been studied by absorption spectra, fluorescence, and viscosity measurements in physiological buffer (pH?=?7.40). The binding constant of HE–Eu(III) complex to hsDNA was obtained by double reciprocal method at 298 and 310?K and the corresponding thermodynamic parameters (Δ_r Hm??=?8.55?×?10⁴?J?mol^?1, Δ_r Gm??=??3.01?×?10⁴?J?mol^?1, Δ_r Sm??=?387.95?J?mol^?1?K^?1) were calculated, showing that the interaction between HE–Eu(III) complex and hsDNA was driven mainly by entropy. The value of K indicated that the binding mode of HE–Eu(III) complex with DNA was not classical intercalation. These results were further supported by viscosity method and competitive binding experiment. Scatchard analysis suggests that the interaction mode was a mixed binding, which contains partial intercalation and groove binding.     

Determination of diclofenac sodium by capillary zone electrophoresis with electrochemical detection

Capillary zone electrophoresis was employed for the determination of diclofenac sodium using an end-column amperometric detection with a carbon fiber microelectrode, at a constant potential of 0.83 V vs. saturated calomel electrode. The optimum conditions of separation and detection are 4.90 x 10(-3) mol/l Na2HPO4-3.10 x 10(-3) mol/l NaH2PO4 (pH 7.0) for the buffer solution, 10 kV for the separation voltage, 5 kV and 10 s for the injection voltage and the injection time, respectively. The limit of detection is 2.5 x 10(-6) mol/l or 5.2 fmol (S/N=2). The relative standard deviation is 0.8% for the migration time and 4.7% for the electrophoretic peak current. The method was applied to the determination of diclofenac sodium in human urine.     

A new method of quantification of pipemidic-acid by capillary zone electrophoresis with end-column amperometric detection

Capillary zone electrophoresis was employed for the determination of pipemidic acid using an end-column amperometric detection with a carbon fiber microdisk array electrode, at a constant potential of -1.10 V vs. saturated calomel electrode. The optimum conditions of separation and detection were 1.2 x 10(-4) mol/LNaOAc - 8.8 x 10(-4) mol/ LHOAc for the buffer solution, 20 kV for the separation voltage, 5 kV and 10 s for the injection voltage and the injection time. The limit of detection was 1.05 x 10(-7) mol/L or 189 amol (S/N=3). The relative standard deviation was 0.31% for the migration time and 2.0% for the electrophoretic peak current. The method was applied to determining pipemidic acid in human serum.     

Characterization of the interaction between human complement protein C4 and a single-chain variable fragment antibody by capillary electrophoresis and surface plasmon resonance

Immunoaffinity capillary electrophoresis and surface plasmon resonance have been used for the characterization of the interaction between two large-sized proteins, the human complement protein C4 and the single-chain variable fragment C43. The rather high kinetic rate constants as determined by surface plasmon resonance pointed out that a capillary electrophoresis method had to be applied, in which the labeled C4 is preincubated with C43 before injection and the same concentration of C43 is included in the running buffer. Analysis of the concentration dependence of the small mobility shift of the fluorescent C4 signal upon binding of C43 resulted in a dissociation constant that was comparable to the one obtained with surface plasmon resonance. This study is one of the few examples where capillary electrophoresis is successfully used to characterize the interaction between large proteins.     

Direct electrochemical determination of pyruvate in human sweat by capillary zone electrophoresis.

Capillary zone electrophoresis was employed for the determination of pyruvate in human sweat using electrochemical detection with a carbon fiber microdisk bundle electrode at a constant potential of 1.60 V vs. saturated calomel electrode. The optimum separation conditions are 3.6 x 10(-3) mol/L Na2HPO4-1.4 x 10(-3) mol/L NaH2PO4 (pH 7.2) for the buffer solution, and 18 kV for the separation voltage. The limits of detection of pyruvate are 8.0 x 10(-6) mol/L or 24 fmol (S/N = 3) for the injection voltage of 6 kV and the injection time of 10 s. The relative standard deviation is 2.0% for the migration time and 5.7% for the electrophoretic peak current. The method was applied to determining pyruvate in human sweat.     

Capillary electrophoresis study of human serum albumin binding to basic drugs

Summary The applicability of capillary electrophoresis/frontal analysis (CE/FA) for determining the binding constants of the drugs propranolol (PRO) and verapamil (VER) to human serum albumin (HSA) was investigated. After direct hydrodynamic injection of a drug-HAS mixture solution into a coated capillary (32 cm × 50 μm i.d.), the basic drug was eluted as a zonal peak with a plateau region under condition of phosphate buffer (pH 7.4; ionic strength 0.17) at 12 kV positive running voltage. The unbound drug concentrations measured from the plateau peak heights had good correlation coefficients,r>0.999. Employing the Scatchard plot, the Klotz plot and nonlinear regression, the drug protein binding parameters, the binding constant and the number of binding sites on one protein molecule, were obtained. The binding constant obtained was compared to a reported equilibrium dialysis result and they are basically in good agreement.     

Monitoring human serum transferrin by capillary zone electrophoresis with end-column amperometric detection

Capillary zone electrophoresis was employed for the determination of human serum transferrin using end-column amperometric detection with a carbon fiber microelectrode at a constant potential of 1.9 V vs. saturated calomel electrode (SCE). The optimum conditions of separation and detection are 7.5 x 10(-4) mol/L Tris-3.44 x 10(-4) mol/L HCl for the buffer solution, 20 kV for the separation voltage, 5 kV and 10 s for the injection voltage and the injection time, respectively. The limit of detection is 6.7 x 10(-8) mol/L or 440 amol (S/N = 2). The relative standard deviations are 0.67% for the migration time and 1.5% for the electrophoretic peak current. The method was applied to the determination of transferrin in human serum. The recovery is between 93-104%.     

Affinity capillary electrophoresis applied to the studies of interactions of a member of heat shock protein family with an immunosuppressant

The bioaffinity of receptor-ligand interactions is investigated by determining the binding constant (association constant or dissociation constant) of the resulting complex utilizing affinity capillary electrophoresis (ACE). The ACE binding assay was established with a potent immunosuppressant, deoxyspergualin (DSG), that binds specifically to Hsc70, a constitutive or cognate member of heat shock protein 70 (Hsp70) family. Quantitative determination of binding constants under different running buffer systems provide comparative results. The association constants for the interaction between Hsc70 protein and DSG were found to be 5.7·10⁴ M⁻¹ in a buffer with pH 6.95 and 6.3·10⁴ M⁻¹ in a buffer with pH 5.30 (or corresponding dissociation constants, 18 and 16 μM, respectively) based on Scatchard analyses. Binding of DSG to a synthetic peptide, SINPDEAVAYGAAV-QAAILSGDK, one of the DSG-binding fragments found from tryptic digest of Hsc70 protein, provides further detailed information for the understanding of Hsc70 binding domain. The applicability of using coated capillaries was also evaluated for probing Hsc70-DSG interaction.     

Quantitation of caffeine by capillary zone electrophoresis with end-column amperometric detection at a carbon microdisk array electrode

Capillary zone electrophoresis is employed for the determination of caffeine using end-column amperometric detection with a carbon fiber microdisk array electrode at a constant potential of 1.45 V versus a saturated calomel electrode. The optimum conditions of separation and detection are 0.1 52mM NaH2PO4-0.648mM Na2HPO4 for the buffer solution, 20 kV for the separation voltage, 5 kV for the injection voltage, and 10s for the injection time. The limit of detection is 2.9 x 10(-4)mM or 1.2 fmol (signal-to-noise ratio = 2). The relative standard deviation is 0.68% for the migration time and 2.3% for the electrophoretic peak current. The method is applied to determining caffeine in human serum and a cola drink.     

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…     

亲和毛细管电泳法测定牛血清白蛋白和加替沙星的结合常数

利用亲和毛细管电泳法对牛血清白蛋白(BSA)与加替沙星(GT)间的结合反应及其相互作用做了初步探索,并应用淌度比(M)作为指标测定了两者的结合常数。以20 mmol/L pH 7.4的磷酸盐缓冲液作为运行缓冲液,分别以GT和BSA作为添加剂,另一组分为进样样品,内标为二甲基甲酰胺,于214 nm波长下检测。两种测定条件下得到的结合常数分别为4.4×104 L/mol和4.2×104 L/mol,与传统的荧光淬灭法测得的结果基本一致。该方法具有简单、高效的优点。     

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.     

Triplet-polaron quenching in conjugated polymers

We studied the triplet-polaron quenching in a platinum(II) porphyrin- (PtOEP-) doped polyspirobifluorene (PSF-TAD) copolymer. The copolymer contains a hole-transporting phenylenediamine unit (TAD) as a comonomer. Triplet-polaron quenching was probed by the change in PtOEP phosphorescence lifetime under an applied voltage in a unipolar device. The charge-induced reduction of the optically excited lifetime of PtOEP is one-third for the highest applied bias. The charge density can be obtained from current-voltage characteristics in the space-charge-limited (SCL) regime. The obtained hole mobility under SCL conditions is (7 +/- 2) x 10(-5) cm(2)/(V s). This result is in accord with recent mobility measurements of the time-of-flight mobility in our polymer. The triplet-polaron recombination constant was evaluated to be (4 +/- 1) x 10(-13) cm(3)/s, implying a triplet-polaron interaction radius of 2 x 10(-10) m. The results show that triplet-polaron annihilation cannot be neglected in device models for phosphorescent light-emitting diodes.     

高效毛细管电泳分析羊栖菜多糖对肿瘤机体红细胞合淌度的影响

为了分析羊栖菜多糖（SFPS）对肿瘤机体红细胞合淌度的影响,建立了肿瘤动物模型,分高、中、低剂量腹腔给予羊栖菜多糖7 d,采集并制备红细胞悬液,应用高效毛细管电泳法检测红细胞的合淌度。实验条件:毛细管为75 μm×50 cm,电泳缓冲液为含2 g/L羟丙基甲基纤维素的磷酸盐溶液(0.1 mol/L,pH 7.4),压力进样为3.448 kPa×10 s,分离电压为20 kV,柱温为25 ℃,电渗淌度为2.16×10-4 cm2·V-1·s-1。实验结果表明：羊栖菜多糖能降低肿瘤机体红细胞的电泳迁移时间(阴性对照组为15.76 min,SFPS低剂量组为13.96 min,SFPS中剂量组为12.90 min,SFPS高剂量组为13.51 min,正常对照组为11.51 min),增加红细胞的合淌度(阴性对照组为1.06×10-4 cm2·V-1·s-1,SFPS低剂量组为1.19×10-4 cm2·V-1·s-1,SFPS中剂量组为1.29×10-4 cm2·V-1·s-1,SFPS高剂量组为1.23×10-4 cm2·V-1·s-1,正常对照组为1.45×10-4 cm2·V-1·s-1),SFPS 3个剂量组红细胞的合淌度与阴性对照组比较均有非常显著的差异(P＜0.01)。羊栖菜多糖能够改变肿瘤机体红细胞的合淌度,并使之趋向于正常机体水平,这可能与其改变红细胞表面的电荷密度有关。高效毛细管电泳法可以作为检测红细胞生理状态和功能的一种辅助工具。     

Monitoring myoglobin by capillary zone electrophoresis with end-column amperometric detection

Capillary zone electrophoresis was employed for the determination of myoglobin in human urine using end-column amperometric detection with a carbon fiber microelectrode at a constant potential of 1.80 V vs. saturated calomel electrode (SCF). The optimum conditions of separation and detection are: 3.73 x 10-4 mol/L sodium diethyl malonyl urea (barbitone sodium), 1.34 x 10-4 mol/L HCl for the buffer solution, 20 kV for separation voltage, 5 kV and 5 s for injection voltage and injection time, respectively. The limit of detection is 4.4 x 10-8 mol/L or 84 amole signal to noise (S/N = 2). The relative standard deviation is 2.9% for the migration time and 2.5% for the electrophoretic peak current. The method can be used for the determination of myoglobin in human urine. The samples can be directly injected and need no pretreatment. The method is also rapid, less than 2 min, and has a recovery rate of 94-106%.