Characterization of basic drug-human serum protein interactions by capillary electrophoresis

Drug-protein interactions are determining factors in the therapeutic, pharmacodynamic and toxicological drug properties. The affinity of drugs towards plasmatic proteins is apparently well established in bibliography. Albumin (HSA) especially binds neutral and negatively charged compounds; alpha(1)-acid glycoprotein (AGP) binds many cationic drugs, lipoproteins bind to nonionic and lipophilic drugs and some anionic drugs while globulins interact inappreciably with the majority of drugs. In this paper, the characterization of the interaction between cationic drugs, beta-blockers and phenotiazines towards HSA, AGP, and both HSA + AGP mixtures of proteins under physiological conditions by CE-frontal analysis is presented. Furthermore, the binding of these drugs to all plasmatic proteins is evaluated by using ultrafiltration and CE. The results indicate that the hydrophobic character of compounds seems to be the key factor on the interaction between cationic drugs towards proteins. In fact, hydrophobic basic drugs bind in great extension to HSA, while hydrophilic basic drugs present low interactions with proteins and bind especially to AGP.     

Characterization of interactions between polyphenolic compounds and human serum proteins by capillary electrophoresis

The interaction of ten natural polyphenolic compounds (chlorogenic acid, apigenin, catechin, epicatechin, flavanone, flavone, quercetin, rutin, vicenin-2 and vitexin) with human serum albumin and mixtures of human serum albumin and α₁-acid glycoprotein under near physiological conditions is studied by capillary electrophoresis–frontal analysis. Furthermore, the binding of these polyphenolic compounds to total plasmatic proteins is evaluated using ultrafiltration and capillary electrophoresis. In spite of the relatively small differences in the chemical structures of the compounds studied, large differences were observed in their binding behaviours to plasmatic proteins. The hydrophobicity, the presence/absence of some functional groups, steric hindrance and spatial arrangement seem to be key factors in the affinity of natural polyphenols towards plasmatic proteins.     

Characterization of protein conjugates using capillary electrophoresis

With the aim of generating antibodies, a calix[4]arene-crown-6 was coupled to bovine serum albumin. For that purpose, a complete procedure to optimize and characterize the coupling of hydrophobic haptens based on capillary electrophoresis (CE) was developed. We demonstrated the existence of a polynomial relationship between the electrophoretic mobility (mu(ep)) and the hapten density. This correlation was used not only to study the coupling reaction in terms of optimization and kinetics but also to determine the average coupling molar ratio of any given conjugate. An estimation of the heterogeneity of these conjugates by simulation of experimental peaks was also proposed.     

Routine serum protein analysis by capillary zone electrophoresis: Clinical evaluation

Summary To measure the five classical protein fractions in human serum several electrophoretic techniques are available. Besides separation on cellulose acetate membrane or agarose gel, capillary zone electrophoresis (CZE) may be a useful analytical alternative in clinical routine. We have compared the Dionex CES I capillary electrophoresis system with that of the Olympus Fractoscan using specimens submitted for routine analysis. For clinical evaluation 102 samples from patients with various diseases have been analysed. Serum protein fractions were judged on separation performance, precision and the regression method ofBablok-Passing. Regression analysis revealed variable agreement between both methods with a slope ± intercept of 2.10–0.52 (α₁-fraction) and 1.0–0.20 (α₂-fraction) as worse and best, resectively; and the coefficient of variation of migration time: 5.9 %–6.8 % (between-run imprecision). Differences in the comparison of fractions are mainly caused by the improved resolution of CZE; e.g. one β-globulin peak on cellulose acetate is separated into two distinct protein fractions in CZE, including more detailed diagnostic information—as is also the case with γ-fraction. In some cases monoclonal gammopathy with low concentrations of immunglobulin clone can only be detected in CZE, whereas the cellulose acetate membrane (CAME) electropherogram is inconspicuous. The within-run precision (N=18) gave coefficients of variation of peak areas 1.3–5.9 % (CZE) and 1.0–3.8 % (cellulose acetate membrane). This is the first time that a complete clinical evaluation of CZE serum protein fraction analysis has been performed. CZE with its higher resolution and hence more detailed diagnostic information in some cases, showed good separation patterns, precision and correlation. Interchangeability of results showed that this CZE method is well suited for analysis of serum protein fractions in clinical routine. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996.     

毛细管区带电泳法快速测定人血血清蛋白

人血血清蛋白电泳分析是临床上诊断多种疾病的常用生化指标 ,也是临床实验室检查的常规项目。目前采用的方法有醋酸纤维薄膜电泳和琼脂糖电泳 ,尤以前者为主。由于醋酸纤维薄膜电泳法操作繁琐 ,每一步骤均需手工完成 ,影响因素较多 ,初学者往往不易掌握 ,且测定的重复性较差。高效毛细管电泳是近几年来迅速发展起来的分离分析技术 [1,2 ]。用该技术分析人血清蛋白国内外虽有报道 ,但由于人血清中蛋白质组分甚为复杂 ,采用不同的分离方法和条件可出现不同数量的组分峰 ( 6、7个甚至 1 0个以上组分 ) ,与目前临床上常用的醋酸纤维薄膜电泳图谱…     

Characterization of glycerin-based polyols by capillary electrophoresis

Methods based on capillary electrophoresis (CE) have been developed to obtain the molar mass distribution (MMD) of glycerin-based polyols and details on the presence of mono- and difunctional byproducts in technical samples. Prior to the analyses the hydroxy end-groups of the trifunctional polyols were converted to chargeable and UV-active moieties with phthalic anhydride (PhAH) as the derivatization reagent. With a method of capillary zone electrophoresis (CZE) samples of glycerin-based polyols with average molar masses up to 6000 were separated according to their charge-to-size ratio. The separations were carried out with a buffer solution containing 50% (v/v) acetonitrile and 10 mM sodium tetraborate, and for detection UV absorption at 220 nm was measured. An approximately linear relation between the reciprocal of the effective mobilities and the degree of polymerisation of the glycerin-based polyols was found. Therefore, the proposed CZE system could be used to determine the degree of polymerisation and polydispersity of technical glycerin-based polyol samples. The effect of the presence of sodium dodecyl sulfate (SDS) in the buffer solution on the CE separation of linear polyethylene glycols (PEGs), polypropylene glycols (PPGs) and ethylene oxide-propylene oxide (EO-PO) copolymers with different molar masses was investigated. The interaction between the charged polymer derivatives and SDS ions in solution increased strongly with the degree of polymerisation and the amount PO in the chain of the polymeric compounds. This behaviour made it possible to invert the migration order of EO-PO containing polymers of different size. With a background electrolyte (BGE) composition of 10mM SDS and 25% (v/v) acetonitrile in borate buffer mono- and difunctional byproducts were separated from the main glycerin-based polyols based on their number of end-groups. Accurate quantities for the mono- and difunctional impurities in technical glycerin-based polyol products were determined.     

Characterization of humic substances by capillary electrophoresis

Capillary electrophoresis was used for the separation of humic acid (HA) from peat, soil, and vermicompost. The electropherograms show the presence of at least three peaks eluted between 6 and 11 min for all HA. The best analysis resolution was obtained with the use of borate buffers at pH 8.9. The HA analyzed have structural and charge similarity, which increases the difficulty of separation. Therefore, the shape of the peaks is broad and the CE profiles of all HA are similar. It is reasonable to assume that the broad band in the three regions is due to the acidic groups that have a similar structure. By comparing the results obtained for HA extracted from soil, peat, vermicompost, and the commercial sample, HA from peat had the major carbon content.     

Characterization of carboxylated nanolatexes by capillary electrophoresis

Poly(styrene-co-acrylic acid) (St/AA) and poly(styrene-co-methacrylic acid) (St/MA) nanolatexes with different acid contents were prepared by emulsion copolymerization and were analyzed by capillary electrophoresis (CE) and by laser doppler velocimetry (LDV). Due to the intrinsic differences in the methodologies, CE (separative technique) and LDV (zetametry, nonseparative technique) lead to very different electrophoretic mobility distributions. Beyond these differences, the variation of the electrophoretic mobility is a complex and nonlinear function of the hydrodynamic radius, the ionic strength, and the zeta potential. To gain better insight on the influence of the ionic strength and the acid content on the electrophoretic behavior of the nanolatexes, the electrophoretic mobility data were changed into surface charge densities using the O'Brien, White, and Ohshima modeling. This approach leads to the conclusion that the surface charge density is mainly controlled at high ionic strength (～50 mM) by the adsorption of anionic surfactants coming from the sample. On the contrary, at low ionic strength, and/or in the presence of neutral surfactant in the electrolyte, the acid content was the main parameter controlling the surface charge density of the nanolatexes.     

Characterization of synthetic polyelectrolytes by capillary electrophoresis

Capillary electrophoresis in entangled polymer solutions was applied to determine the molecular mass and polydispersity of polyelectrolytes. The separation selectivities of different polyethylene glycols as buffer additive can be correlated to their average molecular mass. A universal curve correlating the selectivity and the molecular mass could be obtained by using the instrinsic viscosity of the polyethylene glycol. The separation of poly(2-vinylpyridine) standards was compared to the separation of poly(4-vinylpyridine) standards. An indirect detection system was developed to characterize the cationic polyelectrolyte polydiallyldimethyl ammonium chloride. Various polymers with oppositely charged groups (polycarboxybetaines) were investigated with respect to structure dependence, pH dependence and molecular mass dependence of interand intramolecular association.     

Creatinine determination in serum by capillary electrophoresis

Creatinine in human serum was separated in a fused-silica capillary with H3PO4 (75 mmol/L, pH 2.5) as BGE, followed by UV detection at 200 nm. Serum with methylimidazole added as internal standard was deproteinized with acetonitrile and the supernatant, after dilution with water was injected at pressure mode. Creatinine and methylimidazole were baseline-resolved in 6.5 min. Linearity in the 0-880 micromol/L range gave an r2 > or = 0.998, recovery was 102 +/- 2.8% (n = 6). Enzymatic breakdown with creatininase confirmed that serum does not interfere. The within-day and between-days coefficient of variation (CV) were < or = 2.16 and 2.7%, respectively. The accuracy, determined for lyophilized samples by isotope dilution gas chromatography-mass spectrometry was < or = +/- 2.0%. The results were compared with HPLC for 32 lyophilized samples and on 27 serum pools. Capillary electrophoresis, rapid and inexpensive, seems a promising alternative to high-performance liquid chromatography (HPLC) for creatinine determination in human serum.     

Accurately describing weak analyte-additive interactions by capillary electrophoresis

When modeling analyte-additive interactions in capillary electrophoresis (CE), it is necessary to correct for all changes in the apparent electrophoretic mobility of an analyte that are not due to specific binding. Current models based on dynamic complexation have corrected for bulk viscosity changes in the background electrolyte (BGE) when additives are used, while assuming negligible changes in the dielectric constant and other physicochemical properties of the solution. In this report, a study of weak interactions between deoxyribonucleotides and hydroxypropyl-beta-cyclodextrin (HP-beta-CD) revealed significant nonideality in binding isotherms. Changes in the dielectric properties of the solution due to the addition of high concentrations of HP-beta-CD to the BGE was observed to alter the electrophoretic mobility of analytes. A relative dielectric correction factor was required to normalize analyte mobilities to a reference state of zero additive concentration. The use of both a relative dielectric factor and a viscosity correction factor was found to increase the accuracy of the model, reflected by a higher degree of correlation between predicted and measured analyte mobilities. This type of correction is particularly relevant when studying weak analyte binding interactions or when using high concentrations of additive in the BGE. This work is vital for accurate determination of weak binding constants and mobility values, as well as providing a deeper understanding of the fundamental parameters influencing a separation in CE.     

Characterization of 10 species ofMahonia by capillary electrophoresis

Summary A simple, rapid and effective capillary electrophoresis method was developed for the characterization of 10 different species ofMahonia. A fingerprint of the extract of each species was constructed using a mixed buffer of borate and phosphate, containing methanol, pH 8.0. The effective electrophoretic mobility and % normalized area of each peak in electrophoregrams were evaluated to characterize various species. Three alkaloids: berberine, palmatine and jatrorrhizine were found in all 10 species. The CE technique appears to provide a powerful tool for the identification and quality control of plant drugs.     

Characterization of plasma apolipoproteins by capillary electrophoresis.

The main apolipoproteins of plasma high-density lipoproteins (HDL) and low-density lipoproteins (LDL) were analyzed by capillary electrophoresis. Where possible the results were compared with slab sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Addition of the detergent SDS to the running buffer was essential for separation. Separations were carried out in bare silica and polyacrylamide-coated capillaries. The main apolipoproteins of HDL could be separated in an uncoated capillary filled with borax buffer containing 0.1% SDS. Using the coated capillary, a mixture of HDL and LDL apolipoproteins was resolved in less than 12 min. These preliminary studies indicate that capillary electrophoresis is a promising technique for screening plasma apolipoproteins.     

Characterization of interactions between human serum albumin and tumor-inhibiting amino alcohol platinum(II) complexes using capillary electrophoresis

Platinum(II) complexes with amino alcohol ligands are of growing interest as anticancer agents capable of changing their reactivity toward biomolecules at different pH values. The binding of such compounds to the transport protein, human serum albumin (HSA), under simulated physiological conditions (pH 7.4, 100mM chloride, 37 degrees C) has been studied by capillary electrophoresis (CE), with the objective to acquire and compare their binding parameters. The association constants and stoichiometric ratios of the platinum-HSA adducts were determined by measuring the concentration changes of the peak area response of the Pt complex (after a 48 h incubation of the reaction mixture to attain equilibrium), constructing the binding isotherms, and their mathematical analysis. The investigated Pt(II) compounds were found to show moderate affinity toward HSA, with association constants ranging from 1.0 x 10(3) to 2.4 x 10(4)M(-1). Such binding behavior was attributed to a distinctive structural feature of bis(amino alcohol)platinum(II) complexes, that is, existence of an equilibrium between ring-opened and ring-closed forms in solution.     

Determination of antibacterial flomoxef in serum by capillary electrophoresis

A determination method of flomoxef (FMOX) concentration in serum by capillary electrophoresis is developed. Serum samples are extracted with acetonitrile. After pretreatment, they are separated in a fused-silica capillary tube with a 25 mM borate buffer (pH 10.0) as a running buffer that contains 50mM sodium dodecyl sulfate. The FMOX and acetaminophen (internal standard) are detected by UV absorbance at 200 nm. Linearity (0-200 mg/L) is good, and the minimum limit of detection is 1.0 mg/L (S/N = 3). The relative standard deviations of intra- and interassay variability are 1.60-4.78% and 2.10-3.31%, respectively, and the recovery rate is 84-98%. This method can be used for determination of FMOX concentration in serum.     

Analysis of micafungin in serum by capillary zone electrophoresis

A method is developed for measuring the concentration of micafungin, an echinocandin antifungal agent, in serum, using capillary zone electrophoresis. With a 0.1M borate buffer (pH 10.0) as the run buffer, detection is carried out at 200 nm. Pretreatment is performed by adding acetonitrile to serum (1:2) for deproteinization, allowing the supernatant fluid to be taken for measurement. The detection limit of the assay is 0.5 mg/L at a signal-to-noise ratio of 3.0. Coefficients of variation for intra- and inter-assay precision are 3.45-4.47% and 2.61-7.15%, respectively, at a nominal concentration of 5.0-25.0 mg/L. Their recovery rates are 92-110%. It is convenient for the monitoring of micafungin therapy in patients contracting clinically important deep mycoses.     

Characterization of polyethylene glycol-modified proteins by semi-aqueous capillary electrophoresis

A new program to characterize polyethylene glycol-modified (PEGylated) proteins is outlined using capillary zone electrophoresis (CZE). PEGylated ribonuclease A and lysozyme were selected as examples. Five separation procedures were compared to select out the mixed buffer of acetonitrile-water (1:1, v/v) at pH 2.5 as the best to characterize the PEGylated proteins without sample pretreatment. Polyethylene oxide (PEO) with a high molecular mass of 8 x 10(6) was applied to rinse the capillary to form a dynamic coating which would decrease the undesirable proteins adsorbed to the inner wall of the silica. The electroosmotic flow (EOF) mobility of the five procedures was determined, respectively. It is found that acetonitrile is mainly responsible for the good resolution of PEGylated proteins with the help of PEO coating in the semi-aqueous system. The low EOF mobility and current in the semi-aqueous system might also have some responsibility for the high resolution. The semi-aqueous procedure described in this paper also demonstrates higher resolution of natural proteins than aqueous ones.     

A preliminary evaluation of serum proteins by capillary electrophoresis

We evaluated the 5-band Serum Proteins by Capillary Electrophoresis kit (Bio-Rad Laboratories, Hercules, CA, U.S.A.) on the BioFocus 2000 CE (Bio-Rad) against conventional agarose gel electrophoresis (AGE) (Helena Laboratories, Beaumont, TX, U.S.A.). Serum from 60 patients was initially screened by AGE and divided into three groups: 1) normal electrophoretic pattern (n = 36, mean total protein 67.7 g/L), 2) monoclonal/oligoclonal gammopathy (n = 14, mean total protein 78.8 g/L), and 3) polyclonal gammopathy (n = 10, mean total protein 77.4 g/L). These samples were concurrently analyzed on the BioFocus 2000. Intraassay and interassay CVs for the five fractions for a normal sample were 0.17-1.44% and 0.42-9.11%, respectively, and 0.21-3.37% and 0.29-3.61%, respectively, for a sample with monoclonal gammopathy. Correlation coefficients for albumin and the albumin/globulin (A/G) ratio were 0.8891 (albumin range 17-93 g/L) and 0.8276 (A/G ratio range 0.39-7.81), respectively. The A/G ratio alone could not discriminate between the three groups. Capillary zone electrophoresis (CZE) correctly identified 33 of 36 samples in the normal group, and 22 of 24 samples in the other two groups, giving a clinical sensitivity and specificity of 91.7%. Our preliminary evaluation shows that protein separation by CZE is a simple, rapid, and automated alternative to conventional AGE.     

Characterization of single-stranded DNA separation by capillary gel electrophoresis

We have investigated the effect of polymer gel reconditioning, the shape of the capillary, the applied electric field, and the capillary length for single-stranded DNA. The polyethylene oxide gel had deformed under the high electric field causing the degradation of the separation power. By the reintroduction of the fresh polyethylene oxide gel for the next run, one-base resolution was recovered. It turned out that the tip of the capillary at the injection side needed to be clean and symmetric for much improved resolution. Changing DNA motion by the pulsed electric field resulted in the separation of DNA far more than 500 bases.     

Applications of affinity interactions in capillary electrophoresis

Capillary electrophoresis (CE) has proven useful for the study of reversible molecular interactions. This is because highly efficient and reproducible separations take place in an environment where molecular interactions may contribute to selectivity without being inhibited by adverse buffer conditions. Affinity CE may be used to estimate quantitative binding data (binding constants and in some cases binding stoichiometries and rate constants) for various molecular interactions. Specific binding interactions (e.g., based on antibodies or aptamers) may also be utilized to quantitatively measure specific analytes using CE. Applications within these areas are here reviewed with focus on the last three years and with emphasis on novel concepts as well as innovative methodology and technology. It is concluded that the affinity CE approach is of growing versatility and will continue to play an integral role in discovering, characterizing, and exploiting biomolecular interactions.