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The first detailed examination of flow injection-capillary electrophoresis (FI-CE) active parameters and their interactions via response surface methodology (RSM) is presented. Specifically, RSM in the form of a Box-Behnken design was implemented to effectively predict the significance of capillary length, voltage and injection volume on the optimization of an in-house built FI-CE analyzer. Initial studies were performed assessing peak height and peak shape of the model compound N,N-dimethylformamide. Optimum model conditions were then derived and used in the model separation of two small molecules, nicotinamide adenine dinucleotide, reduced form (NADH) and benzenesulfonamide. By implementing the RSM approach, detailed examination of active FI-CE parameters was possible, including the ability to reveal a significant interactive effect. This work is not only highly significant for advancing FI-CE developments, but instructive for investigators actively exploring other coupled analytical techniques and associated experimental parameters.

This work expands the knowledge of the use of chemometric response surface methodology (RSM) in optimizing conditions for competitive binding partial filling ACE (PFACE). Specifically, RSM in the form of a Box-Behnken design was implemented in flow-through PFACE (FTPFACE) to effectively predict the significance of injection time, voltage, and neutral ligand (neutral arylsulfonamide) concentration, [L(o)], on protein-neutral ligand binding. Statistical analysis results were used to create a model for response surface prediction via contour and surface plots at a given maximum response (DeltaRMTR) to reach a targeted K(b) = 2.50 x 10(6) M(-1). The adequacy of the model was then validated by experimental runs at the optimal predicted solution (injection time = 2.3 min, voltage = 11.6 kV, [L(o)] = 1.4 microM). The achieved results greatly extend the usefulness of chemometrics in ACE and provide a valuable statistical tool for the study of other receptor-ligand combinations.

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.

This work presents the first known use of response surface methodology (RSM) in electrophoretically mediated microanalysis. This concept is demonstrated by examining the optimization of reaction conditions for the conversion of nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide, reduced form by glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) in the conversion of glucose-6-phosphate to 6-phosphogluconate. Experimental factors including voltage, enzyme concentration, and mixing time of reaction at the applied voltage were selected at three levels and tested in a Box-Behnken response surface design. Upon migration in a capillary under CE conditions, plugs of substrate and enzyme are injected separately in buffer and allowed to react at variable conditions. Extent of reaction and product ratios were subsequently determined by CE. The model predicted results are shown to be in good agreement (7.1% discrepancy difference) with experimental data. The use of chemometric RSM provides a direct relationship between electrophoretic conditions and product distribution of microscale reactions using CE, thereby offering a new and versatile approach to optimizing enzymatic experimental conditions.

Multiple-injection affinity capillary electrophoresis (MIACE) was used to determine binding constants ( K b ) between vancomycin, ristocetin, and teicoplanin from Streptomyces orientalis , Nocardia lurida , and Actinoplanes teichomyceticus , respectively, and fluorenylmethoxycarbonyl (Fmoc)-(Gly, Ala, Val, and Phe)- d -Ala- d -Ala peptides. In this technique, separate plugs of sample containing non-interacting standards, peptide one, buffer, and peptide two, were injected into the capillary column and electrophoresed. Peptides migrate through the column at similar electrophoretic mobilities but remain as distinct zones due to the buffer plug between peptides. The electrophoresis is then carried out in an increasing concentration of antibiotic in the running buffer. Continued electrophoresis results in a shift in the migration time of the peptides upon binding to the antibiotic. Analysis of the change in the relative migration time ratio (RMTR) of the resultant complexes relative to the non-interacting standards, as a function of the concentration of antibiotic yields a value for K b . MIACE is a versatile technique that can be used to measure affinity constants between ligands of similar relative molecular mass and charge without the need of separate binding experiments. The findings described, herein, demonstrate the advantages of using MIACE to estimate binding parameters between ligands and receptors.

Binding constants were determined for the activator fructose-6-phosphate (F6P) and substrate adenosine 5'-triphosphate (ATP) (in the presence and absence of F6P) to the recombinant wild-type (WT) Rhodobacter sphaeroides adenosine 5'-diphosphate-(ADP)-glucose pyrophosphorylase (ADPGlc PPase) using affinity capillary electrophoresis (ACE). In these binding studies, the capillary is initially injected with a plug of sample containing ADPGlc PPase and noninteracting standards. The sample is then subjected to increasing concentrations of F6P or ATP in the running buffer and electrophoresed. Analysis of the change in the migration times of ADPGlc PPase, relative to those of the noninteracting standards, as a function of the varying concentration of F6P or ATP yields a binding constant. The values obtained were in good agreement with kinetic parameters obtained from steady state activity assays. The method was extended to examine the F6P binding constants for the R33A and R22A enzymes and the ATP binding constants for the R8A enzyme in the presence and absence of F6P. The R33A enzyme has been shown by activity assays to be insensitive to F6P activation, indicating a defect in binding or in downstream transmission of the allosteric signal required for full activation. ACE indicated no apparent binding of F6P, supporting the former hypothesis. The R22A enzyme was shown by activity assays to have a approximately 15-fold decrease in apparent affinity for F6P compared to that of WT while ACE indicated an affinity comparable to that of WT; potential reasons for this discrepancy are discussed. The R8A enzyme as measured by activity assays exhibits reduced fold-activation by F6P compared to that of WT but increased apparent affinity for ATP in the presence of F6P. The ACE results were in good agreement with the activity assay data, confirming the increased affinity for ATP in the presence of F6P. This method demonstrates the quantitative ability of ACE to study different binding sites/ligand interactions in allosteric enzymes.

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.

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.

This work demonstrates the use of multiple-step ligand injection affinity capillary electrophoresis (ACE) using two model systems: vancomycin from Streptomyces orientalis and carbonic anhydrase B (CAB, EC 4.2.1.1). In this technique a sample plug of receptor and non-interacting standards is injected by pressure and electrophoresed in a buffer containing a given concentration of ligand. The sequence is repeated for all concentrations of ligand generating a single electropherogram containing a series of individual sample plugs superimposed on environments of buffer containing increasing concentrations of ligand. Analysis of the change in the relative migration time ratio, RMTR, relative to the non-interacting standards, as a function of the concentration of the ligand, yields a value for the binding constant. A competitive assay using the technique is also demonstrated using neutral ligands for CAB. These values agree well with those estimated using other binding and ACE techniques. Data demonstrating the quantitative potential of this method are presented.

This work evaluates the use of mobility ratios (M) to estimate binding constants of proteins to ligands using affinity capillary electrophoresis (ACE). This concept is demonstrated using two model systems: vancomycin (Van) from Streptomyces orientalis and carbonic anhydrase B (CAB, EC 4.2.1.1). A plot of change in M (deltaM) over the concentration of ligand [L] versus deltaM yields a more useful representation of the Scatchard plot in capillary electrophoresis (CE) than traditional plots of the change in mobility delta mu over [L] versus delta mu in a wide set of circumstances, especially when comparing electropherograms obtained in the presence of substantial variations in electroosmotic flow. Altering the voltage and/or capillary length of the CE system produced only small variations in M, but much larger changes in the more standard measures of migration used by the mu form of analysis. The use of M in the Scatchard analysis offers a new approach to estimating binding constants of ligands to proteins using ACE.

This 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 of electrophoresis.

This paper demonstrates the use of surface plasmon resonance to study adsorption (either reversible or irreversible) of detergents on PDMS surfaces in real time. The surface plasmon resonance measurements can directly provide information about the adsorption/desorption processes of detergents on the surface revealing the durability of the adsorbed layer and the anticipated degree of the EOF. Hydroxypropyl methylcellulose very strongly adsorbs onto PDMS and can be considered both a semipermanent layer and stable semipermanent coating. Adsorbed SDS or CTAB layers were stable for several minutes upon rinsing the surface with solution not containing the detergent. It was shown that SDS coated onto PDMS in microchips has the potential to afford similar separations in PDMS as found in conventional fused silica capillaries.

A miniaturized capillary electrophoresis system coupled to a surface plasmon resonance (SPR) sensor on a microfluidic platform fabricated from PDMS is detailed. A previously described split-flow injection technique is first utilized to manipulate sample into the microfluidic chip, followed by separation within the fused-silica capillary and final off-capillary detection of analytes via SPR. Instead of using commercial SPR flow cells requiring relatively large detection volumes, samples of less than 1 nL volume are utilized. The interface between the CE system and SPR sensor made it possible to detect minute volumes of sample with minimal dispersion. The flow cell has the potential to be applicable to miniaturized flow-injection (FI) systems where submicroliter volumes of sample are frequently only available for analysis. The components present in solution, but not bound to the sensor surface, were also investigated. The sensitivity of the CE-SPR system was similar to that found in UV-spectrometric instruments and nonchromophoric components could also be measured.

The successful application of artificial neural networks toward the prediction of product distribution in electrophoretically mediated microanalysis is presented. To illustrate this concept, we examined the factors and levels required for optimization of reaction conditions for the conversion of nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide, reduced form by glucose-6-phosphate dehydrogenase in the conversion of glucose-6-phosphate to 6-phosphogluconate. A full factorial experimental design examining the factors voltage, enzyme concentration, and mixing time of reaction was utilized as input-output data sources for suitable artificial neural networks training for prediction purposes. This approach proved successful in predicting optimal values in a reduced number of experiments. Model validation addressing the extent of reaction and product ratios were subsequently determined experimentally in replicate analyses, with results shown to be in good agreement (

Papers detailing the use of affinity capillary electrophoresis (ACE) in examining binding parameters between biological species are summarized in this review. The works cited in this review were published during 2007 and were drawn from the major chemical and biochemical journals and especially the major analytical chemistry literature.

The coupling of on-column derivatization of small molecules to affinity capillary electrophoresis (ACE) has only been realized during the past 5 yr. In this technique, multiple zones of reagent(s) and ligand or receptor are injected into the capillary column. Upon electrophoresis, zones of sample overlap, yielding product. Continued electrophoresis results in the product overlapping with receptor (or ligand, if the receptor was derivatized), thereby causing a shift in migration time of the compound in question. Subsequent Scatchard analysis using noninteracting standards realizes a binding constant. Herein, we describe the use of on-column-ligand and receptor derivatization coupled to partial-filling ACE (PFACE) to probe the binding of vancomycin (Van) from Streptomyces orientalis and teicoplanin (Teic) from Actinoplanes teicomyceticus to D-Ala-D-Ala terminus peptides.

Voltage gradient partial-filling affinity capillary electrophoresis (VGPFACE) is used to determine binding constants between carbonic anhydrase B (CAB, E.C.4.2.1.1) and arylsulfonamides, and vancomycin (Van) from Streptomyces orientalis and teicoplanin (Teic) from Actinoplanes teicomyceticus and D-Ala-D-Ala terminus peptides. Two variations of VGPFACE are described herein. In the first technique, the capillary is partially filled with ligand at increasing concentrations followed by a sample containing receptor and two noninteracting standards and electrophoresed in buffer using a voltage gradient that increases from 0 to 25 kV over the duration of the experiment. Upon continued electrophoresis, zones of solution overlap, and equilibrium is established between the ligand and receptor, causing a shift in the migration time of the receptor with respect to the noninteracting standards. This change in migration time is utilized for estimating a binding constant (K(b)). In the second technique, voltage gradient partial-filling multiple-injection ACE (VGPFMIACE), a multiple-injection sequence is used whereby the capillary is partially filled with ligand at increasing concentrations, a noninteracting standard, three or four separate plugs of receptor each separated by small plugs of buffer, and a plug containing a second noninteracting standard; this is then electrophoresed in buffer with a similar voltage gradient. Upon continued electrophoresis, a similar equilibrium is established and a value for K(b) is obtained for the interaction. The VGPFACE technique expands the functionality and potential of ACE as an analytical tool to examine various receptor-ligand interactions.

The development and experimental optimization of a novel flow injection-capillary electrophoresis (FI-CE) analyzer employing UV-visible fiber optic detection is described. The analyzer incorporates a miniature charge-coupled device (CCD) spectrometer and operates in a graphical programming environment. Data from experimental optimization studies and small molecule separations involving affinity capillary electrophoresis (ACE) and indirect detection of anions are presented. Future directions in terms of instrument automation and incorporation into a microfluidic format are also discussed.

A critical review of recent developments in the use of chemometric experimental design based optimization techniques in capillary electrophoresis applications is presented. Current advances have led to enhanced separation capabilities of a wide range of analytes in such areas as biological, environmental, food technology, pharmaceutical, and medical analysis. Significant developments in design, detection methodology and applications from the last 5 years (2002-2007) are reported. Furthermore, future perspectives in the use of chemometric methodology in capillary electrophoresis are considered.

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.

This work is an overview of our use of affinity capillary electrophoresis (ACE) to estimate binding constants between D-Ala-D-Ala terminus peptides and the glycopeptides vancomycin (Van) from Streptomyces orientalis, teicoplanin (Teic) from Actinoplanes teicomyceticus, and ristocetin A (Rist) from Nocardia lurida. In these studies, modifications in the ACE technique, including partial-filling ACE (PFACE), flow-through PFACE (FTPFACE), on-column ligand derivatization ACE (OCLDACE), on-column receptor derivatization ACE (OCRDACE), multiple-step ligand injection PFACE (MSLIPFACE), and multiple-injection ACE (MIACE), are described and used to determine binding constants of peptides to antibiotics. The findings described herein demonstrate the advantages of ACE in estimating binding parameters between antibiotics and small peptides over other analytical techniques.

Multiple-injection affinity capillary electrophoresis (MIACE) is used to determine binding constants (K b) between receptors and ligands using as model systems vancomycin and teicoplanin from Streptomyces orientalis and Actinoplanes teichomyceticus, respectively, and their binding to D-Ala-D-Ala peptides and carbonic anhydrase B (CAB. EC 4.2.1.1) and the binding of the latter to arylsulfonamides. A sample plug containing a non-interacting standard is first injected followed by multiple plugs of sample containing the receptor and then a final injection of sample containing a second standard. Between each injection of sample, a small plug of buffer is injected which contains an increasing concentration of ligand to effect separation between the multiple injections of sample. Electrophoresis is then carried out in an increasing concentration of ligand in the running buffer. Continued electrophoresis results in a shift in the migration time of the receptor in the sample plugs upon binding to their respective ligand. Analysis of the change in the relative migration time ratio (RMTR) or electrophoretic mobility (μ) of the resultant receptor–ligand complex relative to the non-interacting standards, as a function of the concentration of ligand yields a value for K b. The MIACE technique is a modification in the ACE method that allows for the estimation of binding affinities between biological interactions on a timescale faster than that found for standard ACE. In addition sample volume requirements for the technique are reduced compared to traditional ACE assays. These findings demonstrate the advantage of using MIACE to estimate binding parameters between receptors and ligands.

Afffinity capillary electrophoresis (ACE) is a new analytical technique that has been shown to be an efficient and accurate tool in studying biomolecular noncovalent interactions and determining binding and dissociation constants of formed complexes. ACE uses as its basis the change in migration time of a receptor upon binding to a ligand found in the electrophoresis buffer. Subsequent Scatchard analysis using noninteracting markers realizes a binding constant. Herein, ACE and three modifications in the technique, partial-filling ACE (PFACE), flow through PFACE (FTPFACE), and multiple-step ligand injection ACE (MSLIACE) are used to probe the binding of ristocetin A (Rist A) and vancomycin (Van) from Streptomyces orientalis to D-Ala-D-Ala terminus peptides and carbonic anhydrase B (CAB, E.C.4.2.1.1) to arylsulfonamides.

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 (μ) 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.

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 (μ) 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.

This work evaluates the use of a competitive binding assay using flow-through partial-filling affinity capillary electrophoresis (FTPFACE) to estimate binding constants of neutral ligands to a receptor. We demonstrate this technique using, as a model system, carbonic anhydrase B (CAB, EC 4.2.1.1) and arylsulfonamides. In this technique, the capillary is first partially filled with a negatively charged ligand, a sample containing CAB and two noninteracting standards, and a neutral ligand, then electrophoresed. Upon application of a voltage the sample plug migrates into the plug of negatively charged ligand (L(-)) resulting in the formation of a CAB-L(-) complex. Continued electrophoresis results in mixing between the neutral ligand (L(0)) and the CAB-L(-) complex. L(0) successfully competes out L(-) to form the new CAB-L(0) complex. Analysis of the change in the relative migration time ratio (RMTR) of CAB relative to the noninteracting standards, as a function of neutral ligand concentration, yields a value for the binding constant. These values are in agreement with those estimated using other binding and ACE techniques. Data demonstrating the quantitative potential of this method is presented.

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.

The use of capillary electrophoresis and indirect detection to quantify reaction products of in-capillary enzyme-catalyzed microreactions is described. Migrating in a capillary under conditions of electrophoresis, plugs of enzyme and substrate are injected and allowed to react. Capillary electrophoresis is subsequently used to measure the extent of reaction. This technique is demonstrated using two model systems: the conversion of fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate by fructose-biphosphate aldolase (ALD, EC 4.1.2.13), and the conversion of fructose-1,6-bisphosphate to fructose-6-phosphate by fructose-1,6-bisphospatase (FBPase, EC 3.1.3.11). These procedures expand the use of the capillary as a microreactor and offer a new approach to analyzing enzyme-mediated reactions.

The study of receptor–ligand interactions by affinity capillary electrophoresis (ACE) requires an accurate form of analysis. Here, we examine the use of two noninteracting standards (markers) in the analysis of binding constant data in ACE studies. This concept is demonstrated using two model systems: carbonic anhydrase B (CAB, EC 4.2.1.1) and arylsulfonamides, and vancomycin (Van) from Streptomyces orientalis and the dipeptide N-acetyl- d -Ala- d -Ala. In this procedure a plug of receptor and noninteracting standards is injected, and analysis of the change in the relative migration time ratio of the receptor, relative to the noninteracting standards, as a function of the concentration of the ligand yields a value for the binding constant. The findings described here demonstrate that data from ACE studies can best be analyzed using two noninteracting standards, yielding values comparable to those estimated using other binding and ACE techniques.

This work evaluates the concept of a partial-filling technique in affinity capillary electrophoresis (ACE) using two model systems: vancomycin from Streptomyces orientalis and carbonic anhydrase B (CAB, EC 4.2.1.1). In this technique the capillary is first partially-filled with ligand followed by a sample of receptor and non-interacting standard and electrophoresed. Analysis of the change in the mobility ratio, M, of the receptor, relative to the non-interacting standard, as a function of the concentration of the ligand, yields a value for the binding constant. These values agree well with those estimated using other binding and ACE techniques. Data demonstrating the quantitative potential of this method is presented.

Affinity capillary electrophoresis has been used to determine the two dissociation constants of the complex between anti-DNP rat monoclonal IgG 2b antibody and charged ligands that contained a N-dinitrophenyl group. Singly and multiply charged ligands were used to establish the influence of the charge on the mobility of the complex between Ig and its ligand(s). Zwitterionic buffer additives lessened adsorption of protein to the walls of the capillary. A form of analysis of the binding data is derived that is more useful than Scatchard analysis for certain multivalent systems where cooperativity of binding is in question, but where it is also possible to make plausible assumptions about electrophoretic mobilities of protein and protein-ligand complexes. The uncertainties and assumptions of this analysis are contrasted with those of Scatchard analysis. For this antibody and these monovalent ligands, the dissociation of the ligands from the antibody occurred noncooperatively. The charge on IgG 2b at pH 8.3 is estimated to be -8.0 ± 0.2 ; this value is obtained by analysis of the electrophoretic mobilities of complexes IgG 2b L2, where the ligands L are structurally similar but have different charges (the charges on the ligands were also determined by CE).

The first reported hybrid artificial neural network-genetic algorithm (ANN-GA) approach for the optimization of on-capillary dipeptide derivatization is presented. More specifically, genetic optimization proved valuable in the determination of effective network structure with three defined parameter inputs: (i) phthalic anhydride injection volume, (ii) time of injection, and (iii) voltage, for the maximum conversion of the dipeptide D-Ala-D-Ala by phthalic anhydride. Results obtained from the hybrid approach proved superior to an ANN model without GA optimization in terms of training data and predictive ability. The model developed will likely prove useful for the analysis of other organic-based reaction systems.

Capillary electrophoresis (CE) is an analytical method that is useful for investigating processes that modify the charge of proteins. This paper explores the ability of CE to rationalize charges and electrophoretic mobilities of a simple protein--insulin and its acylated derivatives--as a function of pH. Insulin is a peptide hormone (MW = 5700) that has two alpha-amino groups (G alpha and F alpha) and one epsilon-amino group (K epsilon). Treatment of insulin with acetic anhydride affords seven derivatives that differ in the sites of acetylation of the three amino groups. Analysis of the pH dependence of the electrophoretic mobilities of these derivatives gives pKa values for the two N-terminal ammonium groups: pKa (G alpha) = 8.4; pKa (F alpha) = 7.1. Values of the total charge of insulin estimated from electrophoretic mobility differ from those estimated from values of pKa for its ionizable groups by less than 0.5 unit for both bovine and human insulins over the range of pH from 5.5 to 9.5. Analysis of the concentration dependence of the electrophoretic mobility of insulin yields a lower limit for the association constant for dimerization of insulin of KD > or = 6 x 10(3) M-1 (25 mM tris and 192 mM Gly, pH 8.4). Studies of electrophoretic mobility as a function of pH and extent of acetylation of amino groups rationalize the charge of insulin in detail. The sensitivity of CE to charge permits the quantitative study of electrostatic properties of proteins in solution. Insulin is a useful small-protein model with which to investigate phenomena in electrophoresis.