摘要:

AbstractAffinity capillary electrophoresis (ACE) is a technique that is used to measure the binding affinity of receptors to neutral and charged ligands. ACE experiments are based on differences in the values of electrophoretic mobility of free and bound receptor. Scatchard analysis of the fraction of bound receptor, at equilibrium, as a function of the concentration of free ligand yields the dissociation constant of the receptor‐ligand complex. ACE experiments are most conveniently performed on fused silica capillaries using a negatively charged receptor (molecular mass<50 kDa) and increasing concentrations of a low molecular weight, charged ligand in the running buffer. ACE experiments that involve high molecular weight receptors may require the use of running buffers containing zwitterionic additives to prevent the receptors from adsorbing appreciably to the wall of the capillary. This review emphasizes ACE experiments performed with two model systems: bovine carbonic anhydrase II (BCA II) with arylsulfonamide ligands and vancomycin (Van), a glycopeptide antibiotic, withD‐Ala‐D‐Ala (DADA)‐based peptidyl ligands. Dissociation constants determined from ACE experiments performed with charged receptors and ligands can often be rationalized using electrostatic arguments. The combination of differently charged derivatives of proteins ‐ protein charge ladders ‐ and ACE is a physical‐organic tool that is used to investigate electrostatic effects. Variations of ACE experiments have been used to estimate the charge of Van and of proteins in solution, and to determine the effect of the association of Van to Ac2KDADA on the value of pKaof itsN‐ter

ISSN:0173-0835    

DOI:10.1002/elps.1150190303

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractThe analyte migration behavior in any chemical separation system can be described using a single equation that unifies all areas of separation science. This equation can be used in capillary electrophoresis (CE) to design separation systems, and to study interactions between analytes and additives. By using individual capacity factors for each analyte species present in the system, and with the knowledge of the characteristics of each interaction, one can predict the analyte migration behavior in complicated CE systems, including systems with multiple 1:1 interactions and/or higher order interactions.

ISSN:0173-0835    

DOI:10.1002/elps.1150190304

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractMulticomponent additives, such as derivatized cyclodextrins with various degrees of substitution, can be considered single‐component additives as long as the fraction of each component remains constant. In this paper, equations are derived describing the effect of such additives on the migration behavior of analytes. These equations are used in the study of capillary electrophoresis (CE) systems with differentially charged cyclodextrins as additives. For weakly acidic analytes, the binding with highly negatively charged sulfobutyl ether β‐cyclodextrin (SBE‐β‐CD) increases their negative electrophoretic mobility, while the binding with neutral hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) decreases their negative mobility. By obtaining the equilibrium constants and mobilities for each additive with each analyte (in this case, phenol, 2‐naphthol and 1‐naphthol), the migration behavior of these analytes in CE systems is quantitatively predicted at various concentrations of mixtures of the two additives. The properties of the contour lines in the binding isotherm surfaces of such C

ISSN:0173-0835    

DOI:10.1002/elps.1150190305

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractAffinophoresis is a type of affinity electrophoresis using an affinophore, a soluble ionic carrier bearing affinity ligand(s). It was reported previously that an affinophore, prepared by coupling multiplep‐aminophenyl α‐D‐mannoside ligands to a part of the carboxyl groups of succinylated polylysine, specifically changed the mobility of pea lectin in agarose gel. The affinophoresis of this divalent lectin with the polyliganded affinophore was investigated by using capillary electrophoresis. Analysis of the mobility change of the lectin in the presence of differently modified affinophores showed that the affinity was larger for affinophores having higher ligand density. Analysis of the inhibition of the mobility change by a neutral ligand, with a known affinity constant for the lectin, allowed estimation of the contributions of monovalent and divalent interactions to the binding in the lectin‐affinophore complex. The proportion of divalent complexes was greater for affinophores having higher ligand density. This approach to estimate the contribution of divalency in complex formation should be generally applicable to the analysis of divalent interactions with different techniques other than electro

ISSN:0173-0835    

DOI:10.1002/elps.1150190306

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractAn automated on‐line competitive immunoassay based on capillary electrophoresis (CE) was utilized to monitor secretion of insulin from single islets of Langerhans stimulated by glucose and tolbutamide. In the instrument, fluorescein isothiocyanate‐labeled insulin (FITC‐insulin), monoclonal anti‐insulin and perifusate of single islets were mixed on‐line while islets were exposed to different levels of glucose and tolbutamide. Insulin released from single islets competed with FITC‐insulin for antibody binding sites. Therefore, the amounts of bound and free FITC‐insulin were modulated by insulin released from islets. The bound and the free FITC‐insulin were separated by CE every 3 s and the bound over free ratio (B/F) was measured. Insulin levels were obtained by comparing B/F with calibration curves obtained under the same conditions except that the islet perfusate was replaced with various concentrations of insulin. Patterns of insulin secretion stimulated by glucose and tolbutamide observed were comparable to what has been seen previously using radioimmunoassay or enzyme‐linked immunoassay. This on‐line competitive immunoassay system provided a fast and direct way to measure insulin release from single islets. The effects of temperature on antibody‐antigen reaction rate and binding equilibr

ISSN:0173-0835    

DOI:10.1002/elps.1150190307

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractScrapie in sheep and goats is the prototype of transmissible spongiform encephalopathies found in humans and animals. A feature of these diseases is the accumulation of rod‐shaped fibrils in the brain that form from an aggregated protein. This protein (PrPSC) is a protease‐resistant form of a normal host cell protein. When the aggregated protein is denatured in sodium dodecyl sulfate (SDS) and β‐mercaptoethanol, a monomer form of ∼27 kDa molecular mass is observed. A competition immunoassay to detect PrPSCfrom scrapie‐infected sheep was developed using free zone capillary electrophoresis with laser‐induced fluorescence (LIF) for detection and flourecein‐labeled sysnthetic peptides from PrPSC. Antibodies were made to each respective peptide and used in the competition assay. The fluorescent‐labeled peptides bound to the antibody were separated from the unbound peptides using 200 mMTricine, pH 8.0, containing 0.1%n‐octylglucoside and 0.1% bovine serum albumin (BSA). The amount of antibody that would bind ∼50% of the fluorescent‐labeled peptide was determined for each peptide. When unlabeled peptide was added to the assay, ∼2 fmoles of the peptide could be measured. When PrPSCextracted from infected sheep brain was added to the assay, approximately 135 pg of PrPSCcould be detected. When preparations from normal sheep were assayed, there was little or no competition for the bound peptides. Assays using two of the peptides, peptides spanning amino acid positions 142–154 and 155–178, clearly differentiated scrapie‐positive sheep from normal animals. This assay is a new method that can be used to diagnose scrapie and, possibly, other transmissible spongiform encephalopa

ISSN:0173-0835    

DOI:10.1002/elps.1150190308

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractAffinity capillary electrophoresis (ACE) has been used to investigate the epitope on the human immunodeficiency virus (HIV) core protein p24 recognized by the monoclonal antibody (mAb) 13‐102‐100. The affinity of a series of peptides withN‐ andC‐terminal truncations of the epitope sequence determined by mass spectrometry was studied. The peak area change assay was used for the study of the interactions of the mAb with those peptides, exhibiting tight binding to the mAb, and the migration time shift assay was used to probe the relative affinities of peptides showing weak binding to the mAb. The experimental results show that the monoclonal antibody 13‐102‐100 recognizes the peptide VHPVHAGPIAP with highest affinity. Smaller peptides incorporating only part of the epitope, however, are recognized to some extent in the ACE

ISSN:0173-0835    

DOI:10.1002/elps.1150190309

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractThis work evaluates the concept of a double enzyme‐catalyzed microreactor using capillary electrophoresis (CE). Migrating in a capillary under electrophoresis conditions, plugs of substrate and two enzymes are injected separately in buffer and allowed to react. Extent of reaction and product ratios were subsequently determined by CE. This concept is demonstrated using two model systems: the conversion of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and adenosine monophosphate (AMP) by hexokinase (HK, EC 2.7.1.1) and apyrase (APY, EC 3.6.1.5), respectively, in the conversion of glucose to glucose‐6‐phosphate and inorganic phosphate, respectively, and the conversion of nicotinamide adenine dinucleotide, reduced form (NADH), to nicotinamide adenine dinucleotide (NAD) and back to NADH by lactate dehydrogenase (LDH, EC 1.1.1.27) and glucose‐6‐phosphate dehydrogenase (G6PDH, EC 1.1.1.49), respectively, in the conversion of pyruvate to lactate and glucose‐6‐phosphate (glc‐6‐P) to 6‐phosphogluconate, respectively. These procedures illustrate the use of the capillary as a double microreactor and the ease of quantitation of reaction products under conditions

ISSN:0173-0835    

DOI:10.1002/elps.1150190310

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractDifferent modes of capillary isoelectric focusing (CIEF) with salt mobilization and zwitterionic additive to cathodic mobilizer were applied to characterize the biotin‐binding protein actinavidin and its affinity properties. The analysis is performed in a neutral coated capillary with completely eliminated electroosmotic flow. Synthetic pIstandards with absorption in the visible region were used in all runs and pIdeterminations were based on a fitted nonlinear calibration graph. CIEF of highly purified actinavidin in native conditions was revealed in about 12 peaks in pIrange 6.2–7.5, with three major forms having pI6.80, 6.86, 6.90. CIEF of a mixture of actinavidin and increasing concentrations of two affinity ligands (biotin and biotinylated oligonucleotide) demonstrated drastic changes in the number of protein isoforms. In the latter case it resulted in only one peak (pI5.05) when the ligand was in excess. This method, which can be named affinity CIEF, was found to be well‐suited for studying structural changes, occurring in receptor proteins upon binding with a ligand. CIEF of the protein, performed under denaturing conditions (6Murea in a solution of carrier ampholytes) is also reported. It was revealed in three isoforms with a pImore acidic than that of native actinavidin. It is demonstrated that careful selection of denaturing conditions was necessary for the reproducible re

ISSN:0173-0835    

DOI:10.1002/elps.1150190311

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractCapillary affinity gel electrophoresis was applied to sequence‐specific and base composition‐specific recognition of oligodeoxynucleotides, utilizing the formation of heteroduplexes between a nucleic acid analogue immobilized into the capillary gel and soluble oligodeoxynucleotides with different sequences. Capillary affinity gel electrophoresis using capillaries filled with a conjugated gel of polyacrylamide and a synthetic nucleic acid analogue [poly(9‐vinyladenine)] was effective for the selective separation of hexathymidylic acid from a mixture of four homopolymers of A6, C6, G6, and T6and for the complete resolution of five heteropolymers of hexadeoxynucleotides (TAAAAA, TTAAAA, TTTAAA, TTTTAA, TTTTTA). We also demonstrated that capillary affinity gel electrophoresis was useful for the selective and the sensitive sequence‐specific recognition of sequence isomers of DNA (TTTTAA, TTTTAAT, TTTATA, and

ISSN:0173-0835    

DOI:10.1002/elps.1150190312

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY