Partial-filling affinity capillary electrophoresis …...Partial-filling affinity capillary electrophoresis techniques to probe the binding of glycopeptide antibiotics to D-Ala-D-Ala

Partial-filling affinity capillary electrophoresistechniques to probe the binding of glycopeptide

antibiotics to D-Ala-D-Ala terminus peptides

Jose Zavaletaa, Dinora B. Chinchillaa, Catherine F. Kaddisa, Karla Martineza, Abby Browna, Alvaro Gomeza, AmarisPaoa, Alejandra Ramireza, Sanjay Nilapwarb, John E. Ladburyb, and Frank A. Gomeza

aDepartment of Chemistry and Biochemistry, California State University, Los Angeles, CA, U.S.A.bDepartment of Biochemistry & Molecular Biology, University College London, London, U.K.

Correspondence: Prof. Frank A. Gomez, Department of Chemistry and Biochemistry, California State University,5151 State University Dr., Los Angeles, CA 90032-8202, U.S.A.; e-mail: [email protected]

Abstract

This work is an overview of our use of affinity capillary electrophoresis (ACE) to estimate binding constants between D-Ala-D-Ala terminus pep-tides 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 injectionPFACE (MSLIPFACE), and multiple-injection ACE (MIACE), are described and used to determine binding constants of peptides to antibiotics. Thefindings described herein demonstrate the advantages of ACE in estimating binding parameters between antibiotics and small peptides over otheranalytical techniques.

The development of resistance to antibacterial agentsis an ever-increasing, worldwide problem that

threatens the chemical effectiveness of drugs used inthe treatment of many infectious diseases.1–10 Recentreports document the proliferation of antibiotic-resistantbacterial species that are causing serious health prob-lems, particularly in third-world countries. Historically,the use of glycopeptide antibiotics of the vancomycingroup has been the standard route to treating bacterialinfections (Figure 1a). These molecules are producednaturally by the fermentation of microorganisms andinhibit the growth of Gram-positive bacteria by hinder-ing the biosynthesis of cell wall peptidoglycan. Theybind to the D-Ala-D-Ala portion of peptidoglycan inter-mediates and inhibit the transglycosylation reaction

needed for cross-linking of the cell wall, thereby result-ing in bacteriostatis or bacterial cell death.

In the most serious incidents of bacterial infection,Van, from Streptomyces orientalis, has traditionally beenprescribed because of its effectiveness in treating infec-tions caused by bacteria resistant to other types ofantibiotics. Similar to Van are the antiobioticsteicoplanin (Figure 1b) and ristocetin (Figure 1c) fromActinoplanes teicomyceticus and Nocardia lurida,respectively.5,6 They inhibit cell wall synthesis byimpeding the action of transglycosylases and transpep-tidases. These drugs and their derivatives form the basisfor the development of the next generation of gly-copeptide antibiotics in the treatment of tomorrow’sbacterial infections. Hence, it follows that it is important

Indexing terms

Glycopeptide antibiotics, affinity capillary electrophoresis, vancomycin, teicoplanin, ristocetin, binding constants

Abbreviations

ACE, affinity capillary electrophoresis; Van, vancomycin; Teic, teicoplanin; Rist, ristocetin A; PFACE, partial-filling ACE; FTPFACE,flow-through PFACE; OCLDACE, on-column ligand derivatization ACE; OCRDACE, on-column receptor derivatization ACE; MSLIPFACE, multiple-step ligand injection PFACE; MIACE, multiple-injection ACE; ADP, adenosine 5’-diphosphate; ADPGlc PPase,ADP glucose pyrophosphorylase; HHM, horse heart myoglobin; NAD, nicotinamide adenine dinucleotide; NADH, nicotinamideadenine dinucleotide, reduced form; CAB, carbonic anhydrase B; CAA, carbonic anhydrase A; CBSA, 4-carboxybenzenesulfon-amide; MO, mesityl oxide; NHS, N-hydroxysuccinimide; DADA, D-Ala-D-Ala; EOF, electroosmotic flow; RMTR, relative migrationtime ratio; µ, electrophoretic mobility; ITC, isothermal titration calorimetry

J. CAP. ELEC. AND MICROCHIP TECH. 009 : 5/6 2006 ###### J. CAP. ELEC. AND MICROCHIP TECH. 009 : 5/6 2006

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to examine their mode of action both in vivo and invitro, develop novel synthetic routes to new molecularspecies, and develop new analytical techniques to aidin assessing their extent of interaction to ligands. Thelatter point is of particular importance to analyticalchemists given the huge number of biological interac-tions recently discovered, those yet undiscovered, andthe many drug candidates that have resulted from com-binatorial approaches to rational drug design and devel-opment. Therefore, techniques that can analyze theextent of interaction between a receptor and ligandboth accurately and expeditiously are in great need.

Affinity capillary electrophoresis is a versatile ana-lytical technique that has been used to study a varietyof bimolecular noncovalent interactions and in deter-mining binding and dissociation constants of formedcomplexes.11–49 Since the initial reports in 1992 docu-menting the use of ACE to study biological interactions,the technique has been used successfully to examine amyriad of interactions including protein–drug, pro-tein–DNA, peptide–carbohydrate, peptide–peptide,DNA–dye, carbohydrate–drug, and antigen–anti-body.11–15 For example, Novotny et al. investigated theinteractions between dextran oligomers and small mol-ecules using ACE.16 Kaddis et al. used ACE to examinethe binding of phosphates to the recombinant wild-typeRhodobacter sphaeroides adenosine 5’-diphosphate-(ADP)-glucose pyrophosphorylase (ADPGlc PPase).17

Finally, El-Shafey et al. determined the binding andthermodynamic constants of enediynes with bovineserum albumin using ACE.18

Unlike more established analytical techniques (forexample, size exclusion, equilibrium dialysis, sedimenta-tion, slab gel electrophoresis, and fluorescence quench-ing) used to estimate affinity parameters between recep-tors and ligands that frequently require the separation offree and/or complexed species in an equilibrium mix-ture, ACE uses changes in migration time of a receptor(or ligand) induced upon complexation with a ligand (orreceptor) relative to noninteracting standards in the elec-trophoresis buffer. Subsequent Scatchard analysis yieldsa value for the binding constant. Recent work in our lab-oratories has documented the versatility of ACE tech-niques, particularly those based on partial-filling tech-niques. In partial-filling ACE, only a portion of the cap-illary is filled with ligand (or receptor), unlike in stan-dard ACE, in which the entire capillary is filled with theanalyte in question. As long as an equilibrium is estab-lished between ligand and receptor at the point ofdetection, a Kb can be estimated.

This paper describes our work in developing newACE techniques to estimate binding constants between D-Ala-D-Ala terminus peptides and the glycopeptide antibi-otics Van, Teic, and Rist. Specifically, we describe our use

PARTIAL-FILLING ACE continued


FIGURE 1 Structures of a) vancomycin complexed with N-Ac-D-Ala-D-Ala (5), b) teicoplanin, and c) ristocetin.

a

b

c

of partial-filling ACE (PFACE), flow-through PFACE (FTP-FACE), on-column ligand and receptor derivatization ACE(OCLDACE and OCRDACE, respectively), multiple-stepligand injection PFACE (MSLIPFACE), and multiple-injection ACE (MIACE) in examining binding eventsbetween glycopeptide antibiotics and small peptides(Figure 2). This work demonstrates the versatility of par-tial-filling ACE techniques and their potential in examin-ing a host of biological interactions.

Experimental

Chemicals and reagents

All chemicals were analytical grade. Vancomycinfrom Streptomyces orientalis, N-acetyl-D-Ala-D-Ala, 2; D-Ala-D-Ala, Nα,Nε-diacetyl-Lys-D-Ala-D-Ala, 5; D-Ala-D-Ala-D-Ala; D-Ala-D-Ala-D-Ala-D-Ala; Gly-Ala-Ala-D-Ala-D-Ala;horse heart myoglobin (HHM); nicotinamide adenine di-

nucleotide (NAD); nicotinamide adenine dinucleotide,reduced form (NADH); and carbonic anhydrase B (CAB,EC 4.2.1.1, containing carbonic anhydrase A [CAA] andCAB isozymes, from bovine erythrocytes) were purchasedfrom Sigma Chemical Co. (St. Louis, MO, U.S.A.) and wereused without further purification. Teicoplanin•HCl waspurchased from Advanced Separation Technologies Inc.(Whippany, NJ, U.S.A.) and was used without furtherpurification. Ristocetin A was from Bio/Data Corp.(Horsham, PA, U.S.A.) and was used without further purifi-cation. 4-Carboxybenzenesulfonamide (CBSA) was fromAldrich Chemical Co., Inc. (Milwaukee, WI, U.S.A.) andwas used without further purification. N-succinyl-D-Ala-D-Ala, 1, was synthesized based on literature procedures.14

Mesityl oxide (MO) was purchased from Calbiochem (SanDiego, CA, U.S.A.). Fmoc-Gly-NHS, Fmoc-Ala-NHS, Fmoc-Phe-NHS, and Fmoc-Val-NHS were purchased fromBachem California (Torrance, CA, U.S.A.). Fmoc-Gly-D-Ala-D-Ala, 4; Fmoc-Ala-D-Ala-D-Ala, 2; Fmoc-Phe-D-Ala-D-Ala, 3; and Fmoc-Val-D-Ala-D-Ala, 4, were synthesizedbased on literature procedures.4 Fmoc-Ala-OH was syn-thesized in our laboratories.

For PFACE, stock solutions (1 mg/mL) of CAB andVan (4 mg/mL) were each prepared by dissolving thelyophilized sample in buffer (192 mM glycine-25 mMTris, pH 8.3).

For FTPFACE, stock solutions of CAB (1 mg/mL),vancomycin (4 mg/mL), and HHM (1 mg/mL) wereeach prepared by dissolving the lyophilized protein inbuffer (192 mM glycine-25 mM Tris, pH 8.6).

For OCLDACE, stock solutions of Van (0.200mmol/L), Gly-Ala-Ala-D-Ala-D-Ala (0.01 mol/L), D-Ala-D-Ala-D-Ala-D-Ala (0.01 mol/L), D-Ala-D-Ala (0.01mol/L), D-Ala-D-Ala-D-Ala (0.01 mol/L), NAD (2.5mmol/L), and CBSA (3 mmol/L) were each prepared bydissolving in buffer (20 mM phosphate buffer, pH 7.5).

For OCRDACE, stock solutions of NAD (1mg/mL), Teic (0.4 mg/mL), and Rist (1 mg/mL) wereeach prepared by dissolving the samples in buffer (20mM phosphate, pH 6.9). Stock solutions of acetic anhy-dride and succinic anhydride were prepared by dissolv-ing the compounds in acetonitrile.

For MSLIPFACE, stock solutions of Van (4 mg/mL),bovine CAB (1 mg/mL), and HHM (1 mg/mL) wereeach prepared by dissolving the lyophilized protein inbuffer (192 mM glycine-25 mM Tris, pH 8.3).

For MIACE, stock solutions of Van (0.2 mg/mL),Teic (1.0 mg/mL), and CBSA (0.5 mg/mL) were each pre-pared by dissolving in buffer (20 mM phosphate buffer,pH 7.5). Stock solutions of the N-protected amino acids(4 mM) were prepared by dissolving the compounds inbuffer. Fmoc-Gly-D-Ala-D-Ala (1), Fmoc-Ala-D-Ala-D-Ala(2), Fmoc-Phe-D-Ala-D-Ala (3), and Fmoc-Val-D-Ala-D-Ala (4) were prepared based on literature procedures.



FIGURE 2 Peptide ligands examined in this study.

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Apparatus

The capillary electrophoresis system used in thisstudy was a Beckman model P/ACE 5510 or 2100(Fullerton, CA, U.S.A.). Data were collected and ana-lyzed with Beckman System Gold software. The capil-lary tubing (Polymicro Technologies, Inc., Phoenix, AZ,U.S.A.) was of uncoated fused silica with an internaldiameter of 50 µm. For PFACE, the length of the capil-lary from the inlet to the detector was 50.5 cm, and thelength from the detector to the outlet was 6.5 cm. Theconditions used in CE were as follows: voltage, 25 kV;current, 5.2 µA; detection, 200 nm; temperature, 25 ± 2°C. For FTPFACE, the length of the capillary from theinlet to the detector was 60.5 cm, and the length fromthe detector to the outlet was 6.5 cm. The conditionsused in CE were as follows: voltage, 28 kV; current,5.2–5.8 µA; detection, 200 nm; temperature, 25 ± 2 °C.For OCLDACE and OCRDACE, the length of the capil-lary from the inlet to the detector was 40.5 cm, and thelength from the detector to the outlet was 6.5 cm. Theconditions used in CE were as follows: voltage, 20 kV;current, 22 µA; detection, UV detection at 200, 214, and254 nm; temperature, 25 ± 0.1 °C. For MSLIPFACE, thelength of the capillary from the inlet to the detector was80.5 cm, and the length from the detector to the outletwas 6.5 cm. The conditions used in CE were as follows:voltage, 24 kV; current, 4.0 µA; detection, 200 nm; tem-perature, 25 ± 2 °C. For MIACE, the length of the capil-lary from the inlet to the detector was 40.5 cm (49 cmfor Teic and compounds 1 and 2), and the length fromthe detector to the outlet was 6.5 cm (11 cm for Teic andcompounds 1 and 2). The conditions used in CE wereas follows: for Van and Teic, voltage, 25 kV; current, 6.8µA for Van, 7.9 µA for Teic; detection, 200 nm; temper-ature, 23.0 ± 0.1 °C; for CAB, voltage, 30 kV; current, 7.5µA; detection, 200 nm; temperature, 23.0 ± 0.1 °C.

Procedures

For PFACE, a sample of D-Ala-D-Ala ligand wasvacuum injected into the capillary for 15 sec followedby a sample (3.6 nL) of solution for 3 sec containing0.14 mg/mL of Van and 0.08 mg/mL of MO in buffer.The electrophoresis was carried out using a Tris-glycinebuffer and increasing concentrations of the D-Ala-D-Alaligand (0–1150 µM) for 4.0 min.

For FTPFACE, a sample of 4 was vacuum injectedinto the capillary for 0.18 min (0.10 min for 3) at highpressure followed by a sample (3.6 nL) of solution for3 sec containing 0.035 mg/mL of Van, 0.14 mg/mL ofCAB, 0.14 mg/mL of HHM, and 0.08 mg/mL of MO inbuffer. The electrophoresis was carried out using a Tris-glycine buffer and increasing concentrations of 4(0–1150 µM) for 7.0 min (5.0 min for 3).

For OCLDACE, a sample solution (1.2 nL) (a 1-sectime of injection equates to 1.2 nL of volume of liquid)containing D-Ala-D-Ala terminus peptides, NAD, andCBSA was introduced by pressure injection onto thecapillary equilibrated with buffer (20 mM phosphate;pH 7.5). Separate plugs (2.4 nL each) of Fmoc-aminoacid-N-hydroxysuccinimide (NHS) ester in acetonitrileand buffer (20 mM phosphate, pH 7.5) were next intro-duced by pressure injection and electrophoresed. In thebinding studies, a solution of Van (about 491 nL) atincreasing concentration (0~80 µmol/L) was introducedafter the buffer plug by voltage injection (24 kV, 1.5min) and the electrophoresis run at 24 kV to completedetection of all species.

For OCRDACE, a sample of D-Ala-D-Ala ligandwas vacuum injected into the capillary for 8.0–12.0 sec.Plugs containing buffer (3.6 nL), Teic (1.2–2.4 nL), andacetic and succinic anhydride (1.2–2.4 nL) were thenintroduced by vacuum injection. Electrophoresis wascarried out using 20 mM phosphate buffer (pH 6.9) for5.0 min. A similar procedure with Rist was used, exceptthat the anhydride plug preceded that of the Rist plug.

For MSLIPFACE, the capillary was first filled withbuffer solution that did not contain ligand (solution A) fol-lowed by a sample (7.2 nL; a 1-sec time of injectionequates to 1.2 nL of volume of solution) of solution (solu-tion B) containing 0.10 mg/mL of Van, 0.17 mg/mL ofMO, and 0.33 mg/mL of CAB. The sample was subjectedto electrophoresis in a solution (solution C) containing thefirst concentration (50 µM) of derivatized D-Ala-D-Ala lig-and for 2.0 min at 24 kV. A second sample of solution(14.4 nL) (solution B) containing Van, MO, and CAB wasinjected for 12 sec and subjected to electrophoresis in thenext higher concentration of derivatized D-Ala-D-Ala lig-and (100–1200 µM) for 2.0 min at 24 kV. The process ofsample injection and ligand electrophoresis was repeateduntil all concentrations of ligand were run.

For MIACE, for Van, the capillary was first equilibrat-ed with buffer (192 mM glycine-25 mM Tris, pH 8.3) con-taining increasing concentrations of peptide (0–100 µM).Separate plugs of sample solution (3.6 nL each) containingthe marker MO, five plugs of Van, and second marker CBSAwere then introduced by pressure injection, each separatedby a plug of buffer (18-sec injection). The electrophoresiswas carried out using Tris-glycine buffer with increasingconcentrations of ligand at 25 kV for 6.0 min to completethe detection of all species. Experimental conditions for Teicwere similar, except only three plugs of receptor were intro-duced and experiments were run for 7.0 min to Teic.

Microcalorimetric studies

All the titration experiments were performedusing the VP-ITC system (MicroCal Inc., Northampton,



MA, U.S.A.). In each experiment, 20 aliquots of 15 µL ofFmoc-Phe-D-Ala-D-Ala (DADA) (1.30 mM), Fmoc-Val-DADA (1.20 mM), and Fmoc-Ala-DADA (1.31 mM) wereinjected into 1.463 mL of ristocetin (0.108 mM in 192mM glycine-25 mM Tris buffer, pH 8.3) at 25 °C.Resulting data were fitted after subtracting the heats ofdilution. Heats of dilution were determined in separateexperiments from the addition of Fmoc-Phe-DADA,Fmoc-Ala-DADA, and Fmoc-Val-DADA into buffer.Titration data were fitted using a nonlinear least-squarescurve-fitting algorithm with three floating variables: sto-ichiometry, binding constant (Kb = 1/Kd), and changeof enthalpy of interaction (∆H°).

Results and discussion

In over a decade of work on ACE, we have exam-ined several receptor–ligand combinations and specifi-cally the glycopeptide antibiotic–peptide system. Theuse of this system is amenable to CE given the lack ofadsorption of antibiotic onto the capillary column, read-ily available materials, relative ease of synthesis of pep-tides, and binding constants in the range conducive touse by CE. Although ACE has been demonstrated as aversatile technique in estimating binding parameters,the great demand imposed by the chemical and phar-maceutical industries for robust analytical techniquesutilizing ever-decreasing amounts of materials has war-ranted the development of other methods of analysis.Partial-filling techniques in CE were originally devel-oped in the mid-1990s specifically for the separation ofenantiomers by cyclodextrins.43,44 Unlike standard ACE,which generally requires that the entire capillary befilled with ligand species, in PFACE a much smallerzone of ligand is injected into the capillary column.Until our work in 1999, no other group had extendedthe use of partial filling to receptor–ligand interactionsencompassing proteins and peptides. The following dis-cussion expands on the various partial-filling modes wehave developed to estimate binding constants betweenantibiotics and small peptides.

PFACE

In 1999, we developed partial-filling ACE to exam-ine receptor–ligand interactions (Figure 3a). In thistechnique, the capillary is first partially filled with ligandfollowed by a sample of receptor and noninteractingstandards and electrophoresed. Analysis of the changein the receptor migration time relative to a standardyields a Scatchard plot.

Using PFACE, we examined the interactionbetween Van and N-succinyl-D-Ala-D-Ala, 1, and N-acetyl-D-Ala-D-Ala, 2. Extensive studies on molecularrecognition between Van and various small peptides

with D-Ala-D-Ala terminus and structurally modifiedvariants have been conducted using other techniques.25

It is known that the three amide linkages and the freeterminal carboxyl group are essential for Van binding.In this study, a plug of 1 was first vacuum injected athigh pressure into the capillary for 15 sec followed bya plug of Van and MO and electrophoresed at 25 kV.The concentration of 1 was successively increased from



FIGURE 3 Schematic of a) PFACE and b) FTPFACE.

a

b

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0 to 1150 µM, and each concentration of ligand was runin triplicate twice over two separate days.

Figure 4 shows a representative series of electro-pherograms of Van in a capillary partially filled withvarious concentrations of 1. Upon addition of increas-ing concentrations of 1 in the running buffer, the Vanpeak shifts to greater migration time for any given con-centration. MO is used as a neutral marker in the analy-sis and does not interact with 1 in the running bufferunder conditions of electrophoresis. Ligand 1 is a small,negatively charged molecule and has a more negative

electrophoretic mobility than uncomplexed and com-plexed Van. Hence, it elutes at a greater migration timethan both Van and complexed Van. The complexationbetween 1 and Van resulted in an increasing negativecharge and the complex is detected later than theuncomplexed form. As can be seen in Figure 4, all ofthe electropherograms had the same elution pattern.Minimal variances in the migration times of Van and MOwere observed. Negligible peak broadening wasobserved at the intermediate concentrations, and littlechange in electroosmotic flow (EOF) was observedeven at high concentrations of ligand.

The heights of the ligand plateaus in Figure 4increase due to the increased concentration of 1 partial-ly filled in the capillary. The box-like structure of theligand peak at any concentration of 1 denotes a con-stant concentration of ligand in the capillary. A longenough injection time permits Van and MO to elutewith the box formed by the ligand. Too short a ligandinjection time leads to incomplete overlap of markerand receptor with the ligand box, thereby hinderinganalysis of the migration times. Figure 5a is a Scatchardplot of the data using the mobility ratio M as the basisfor analysis. In this form of analysis, Kb can be estimat-ed using M (Eq. [1]).31 Here, teo and tp are the meas-ured migration times

M = teo/tp + 1 (1)

of a reference peak and the receptor peak, respective-ly. A Scatchard plot can be obtained via Eq. (2). Here,∆MP,L is the

∆MP,L/[L] = Kb∆MP,Lmax – Kb∆MP,L (2)

magnitude of the change in the mobility ratio as afunction of the concentration of ligand. Eq. (2) allowsfor the estimation of Kb on a relative time scale andcompensates for fluctuations in voltage and/or capil-lary length.

Table 1 summarizes the binding data for the lig-ands to Van. These values agree well with previous ACEstudies and with those obtained from other assays forstructurally similar compounds.24,29,35 Of particular inter-est is the amount of ligand used in any given run.Unlike standard ACE techniques in which the receptoris electrophoresed in a running buffer of increasingconcentrations of ligand, the partial-filling ACE tech-nique uses much smaller quantities of material (~18 nLof solution compared to approximately 1 µL for stan-dard ACE techniques) for any given run. The ability toobtain binding parameters using small quantities ofsample is integral in the development and design ofnew drugs and is the major advantage of partial-filling



FIGURE 4 Representative set of electropherograms of Van in0.192 M glycine-0.025 M Tris buffer (pH 8.3) containingvarious concentrations of 1 using the PFACE technique. Thetotal analysis time in each experiment was 4.0 min at 25 kV(current: 5.2 µA) using a 60.5-cm (inlet to detector), 50-µm-i.d. open, uncoated quartz capillary. MO was used as aninternal standard.

ACE over both traditional ACE techniques and otherforms of binding assays.

FTPFACE

As an extension to PFACE, we introduced flow-through PFACE to estimate binding constants of ligands toreceptors (Figure 2b). Like PFACE, the capillary is firstpartially filled with ligand, followed by a sample of recep-tor and noninteracting standards, but here a smaller zoneof ligand is injected. Upon application of a voltage, thereceptor and standards flow into the ligand plug, whereequilibrium is achieved between the receptor and ligand.Continued electrophoresis results in the receptor andstandards flowing through the domain of the ligand plug.

Using peptide 1, a plug of 1 at increasing concen-trations was vacuum injected into the capillary for 0.18min followed by a plug of sample containing Van, CAB,HHM, and MO, and was electrophoresed for 7.0 min.MO and HHM were used as the noninteracting stan-dards for the analysis. The concentration of 1 was suc-cessively increased from 0 to 1150 µM and each con-centration of ligand.

Figure 6 shows a representative series of electro-pherograms of Van with increasing concentrations of 1using the FTPFACE technique. At high concentrations of1, the CAB peak is slightly perturbed, making its use inthe analysis impossible. We attribute this perturbation inthe peak shape of CAB to changes induced by high con-centrations of 1 in the ligand buffer and not to bindingof 1 to CAB, since there was no shift in the migrationtime of the protein. As can be seen in Figure 6, all of theelectropherograms had the same elution pattern. At highconcentrations of 1 (>300 µM), it is evident that someadsorption of ligand onto the capillary is occurring.

Figure 5b is a Scatchard plot of the data for Van.In this form of analysis, Kb is estimated using a dual-marker form of analysis that we term the “relativemigration time ratio” (RMTR) (Eq. [3])22:

RMTR = (tr – ts′)/(ts′ – ts) (3)

Here, the migration time of a receptor (tr) refer-enced to two noninteracting standards (ts and ts′). In thepresent experiments, ts and ts′ are the migration times of



FIGURE 5 a) Scatchard plot of the data for Van according to Eq. (2). b) Scatchard plot of the data for Van according to Eq. (2). c)Scatchard plot of the data for 3 according to Eq. (4) (in with other electropherograms). d) Scatchard plot of Teic and its deriva-tives with 2. e) Scatchard plot of the data for Van according to Eq. (2). RMTR = relative migration time ratio.

a b c

d e

MO and HHM, respectively. A Scatchard plot can beobtained via Eq. (4):

∆RMTRR,L/[L] = Kb∆RMTRR,Lmax – Kb∆RMTRR,L (4)

Here, ∆RMTRR,L is the magnitude of the change inthe relative migration time ratio as a function of the con-centration of ligand. Eq. (4) allows for the estimation ofKb on a relative time scale using two noninteracting stan-dards and compensates for fluctuations in voltage in thecapillary column. Binding constants were obtained fortwo D-Ala-D-Ala ligands. A previous study conducted atpH 5.1 realized binding constants of approximately oneorder larger magnitude.24 It is well known that bindingconstants of Van group antibiotics to peptides are highlydependent on the pH and nature of the solution.24 Thevalues for the binding constants agree well with previousestimates by ACE and other binding techniques conduct-ed at pH values ranging from 5.1 to 8.3.1,5,11,14,20

FTPFACE has several advantages as a method formeasuring biomolecular noncovalent interactions: 1) Itrequires even smaller quantities of receptor and ligandthan traditional ACE techniques. 2) Purification of thesample prior to injection is not necessary as long as thecomponent to be analyzed can be separated from otherspecies. 3) It does not require radiolabeled or chro-mophoric ligands. 4) The commercial availability ofautomated instrumentation and the high reproducibilityof data make it experimentally convenient.

OCLDACE and OCRDACE

A major advantage of CE is that during electrophore-sis multiple zones of solution remain relatively unperturbed



Table 1EXPERIMENTAL VALUES OF BINDING CONSTANTS Kb (104 MOL L–1) OFLIGANDS 1–13 TO GLYCOPEPTIDE ANTIBIOTICS MEASURED BY THEDIFFERENT ACE TECHNIQUES

Kb

Ligand Antibiotic Technique (104 mol L–1) Ref.1 Vancomycin PFACE 1.34 33

FTPFACE 0.91 34MSLIPFACE 0.99 29

2 Vancomycin PFACE 0.39 33ACE 0.5 38FTPFACE 0.36 34MSLIPFACE 0.5 29MIACE 0.37 42PFMIACE 0.72 50

3 Vancomycin OCLDACE 2.26 404 Vancomycin OCLDACE 4.16 40

MIACE 2.2 42PFMIACE 1.95 50MIACE II* 0.98 49

5 Vancomycin MIACE 4.2 426 Vancomycin OCLDACE 1.49 407 Vancomycin OCLDACE 1.51 408 Vancomycin OCLDACE 3.1 409 Vancomycin MIACE 4.0 42

PFMIACE 17.1 50MIACE II 10.2 49

10 Vancomycin MIACE 2.59 42MIACE II 2.43 49PFMIACE 1.91 50

11 Vancomycin OCLDACE 17.5 40MIACE 1.53 42

12 Vancomycin OCLDACE 3.43 4013 Vancomycin OCLDACE 3.67 401 Ristocetin ND**2 Ristocetin ACE 1.52 483 Ristocetin OCLDACE 4.64 244 Ristocetin OCLDACE 0.82 24

ACE 4.14 48MIACE II 1.17 49

5 Ristocetin ACE 3.34 486 Ristocetin OCLDACE 2.19 247 Ristocetin OCLDACE 2.96 248 Ristocetin OCLDACE 2.40 249 Ristocetin ACE 1.64 48

MIACE II 2.55 49ITC 4.69

10 Ristocetin ACE 2.53 48MIACE II 1.3 49ITC 4.0

11 Ristocetin OCLDACE 5.24 24ACE 0.91 48MIACE II 1.71 49ITC 2.93

12 Ristocetin OCLDACE 5.18 2413 Ristocetin OCLDACE 4.72 241 Teicoplanin ACE 3.4 412 Teicoplanin ACE 4.4 41

MIACE 2.1 42PFMIACE 2.5 50

3 Teicoplanin OCLDACE 4.42 244 Teicoplanin OCLDACE 2.18 24

ACE 22.0 41

Table 1 cont.Kb

Ligand Antibiotic Technique (104 mol L–1) Ref.MIACE 14.4 42MIACE II 23.7 49

5 Teicoplanin ACE 26.0 41PFMIACE 7.8 50

6 Teicoplanin OCLDACE 1.79 247 Teicoplanin OCLDACE 16.5 248 Teicoplanin OCLDACE 7.58 249 Teicoplanin MIACE 15.5 42

PFMIACE 6.24 5010 Teicoplanin MIACE II 21.5 49

PFMIACE 12.2 5011 Teicoplanin OCLDACE 18.5 24

ACE 42.0 41MIACE 2.1 42

12 Teicoplanin OCLDACE 13.7 2413 Teicoplanin OCLDACE 3.50 24

*MIACE II: modified technique.**ND: not determined.

with respect to each other as minimal diffusion occurs. Thislack of overlap should not be confused with mixing ofspecies due to differences in electrophoresis mobility. Weused this strength of electrophoresis when we were thefirst to couple on-column derivatization to ACE.

OCLDACE

In the first series of experiments, we examined theon-column derivatization of a single D-Ala-D-Ala termi-nus peptide and its binding to Van using an on-columnsynthesis PFACE technique (Figure 7a). Here, plugs ofD-Ala-D-Ala-D-Ala-D-Ala and two noninteracting mark-ers dissolved in phosphate buffer, Fmoc-Ala-NHS esterdissolved in acetonitrile, buffer, and Van in phosphatebuffer partially filled into the capillary column, areinjected and electrophoresed. NAD and CBSA wereused as markers in the analysis and do not interact withany of the compounds in the running buffer under con-ditions of electrophoresis. Their migration times remainconstant during the course of the binding experiment.

Overlap of the separate zones of species yields the newFmoc-Ala-D-Ala-D-Ala-D-Ala-D-Ala, 3, species. Thezone of Van then migrates into the zone of 3 and adynamic equilibrium is achieved between Van and 3.Fmoc-Ala-NHS ester is dissolved in acetonitile becauseit is readily hydrolyzed in aqueous solution, whichwould hinder the formation of 3. The plug of buffer isintended to prevent early overlap of the Van and reac-tion plugs prior to synthesis of the new Fmoc species.

Figure 8 shows a representative series of electro-pherograms of 3 in a capillary partially filled withincreasing concentrations of Van at 205 nm. Addition ofincreasing concentrations of Van in the running buffershifts 3 to the left (shorter migration time) for all con-centrations of Van. The peak for 3 shifts to the leftbecause the mass of the newly formed complex isgreater than the ligand itself. At the highest concentra-



FIGURE 6 Representative set of electropherograms of Van in0.192 M glycine-0.025 M Tris buffer (pH 8.3) containingvarious concentrations of 1 using the FTPFACE technique.The total analysis time in each experiment was 7.0 min at 28kV (current: 5.8 µA) using a 60.5-cm (inlet to detector), 50-µm-i.d. open, uncoated quartz capillary. MO, CAB, andHHM were used as internal standards.

FIGURE 7 Schematic of a) OCLDACE and b) OCRDACE.

a

b

tion of Van, a 40-second shift in 3 is observed betweenuncomplexed and complexed forms. In these experi-ments, approximately 16.8 pmol of peptide and 74pmol of Van are used in the binding assay. The heightsof the Van plateaus in Figure 8 increase due to theincreased concentration of Van partially filled in thecapillary column. Three other peaks are observed in theelectropherograms and are identified as unreactedFmoc–Ala-NHS ester (A), D-Ala-D-Ala-D-Ala-D-Ala (B),and Fmoc-Ala acid (C). These species do not interferein the PFACE binding studies. Based on the peakheights of A, B, and C, we estimate the yield of 5 to be

approximately 15%. It is apparent for the series of elec-tropherograms that D-Ala-D-Ala-D-Ala-D-Ala has someaffinity for Van since a shift to shorter migration timesis observed. Unfortunately, we were unable to elucidatea binding constant for this interaction. It is also evidentfrom the series of electropherograms that the conver-sion of D-Ala-D-Ala-D-Ala-D-Ala to 3 is constantthroughout the duration of the experiment. Analysis ofthe data at wavelengths other than 205 nm was possi-ble but resulted in reduced peak heights for both 3 andthe noninteracting markers, making PFACE analysis dif-ficult. Figure 5c is the Scatchard plot based on Eq. (4).

To assess the correctness of the on-column ligandderivatization PFACE technique, we conducted a sepa-rate ACE experiment between 3 and Van. Compound 3was initially synthesized off-column and used for theACE studies. A sample of 3, NAD, and CBSA wereinjected onto the column and electrophoresed to obtainthe RMTR for 3. Increasing concentrations (0–80 µM) ofVan were subsequently injected onto the column, there-by inducing changes in the migration time of 3.Measurement of the RMTR due to complexation of 3and Van resulted in a binding constant of 17.7 × 103 M–1.This value is slightly smaller than that obtained usingthe on-column ligand synthesis PFACE technique. A fac-tor that may have contributed to the observed differ-ences in binding constants between the on-columnPFACE and standard ACE techniques is the small varia-tion in Fmoc species formed between runs of the for-mer technique. If too great a deviation in the amount ofpeptide formed on-column occurs, such variations mayinfluence the ratio of bound to unbound peptide and,hence, the migration time of the peptide. Still, webelieve that since multiple electrophoresis runs wereused for the Scatchard analysis, such deviations causedby variable synthetic yields are minimized over thetotality of the analysis.

OCRDACE

Binding constants between the glycopeptidesteicoplanin and ristocetin and their derivatives to D-Ala-D-Ala terminus peptides were determined by on-column receptor derivatization coupled to PFACE orACE (Figure 7b). In this technique, derivatives of theglycopeptides Teic and Rist are first synthesized on-column before analysis by ACE or PFACE. After the col-umn has been partially filled with increasing concentra-tions of D-Ala-D-Ala terminus peptides, a plug of bufferfollowed by two separate plugs of reagents are inject-ed. The order of the reagent plugs containing the antibi-otic and two noninteracting standards and the anhy-dride varies with the charge of the glycopeptide. Uponelectrophoresis, the antibiotic reacts with the anhydride



FIGURE 8 Representative series of electropherograms of Fmoc-Ala-D-Ala-Ala-D-Ala-D-Ala, 3, in 20 mM phosphate buffer(pH 7.5) at 205 nm containing various concentrations of Vanusing the on-column synthesis PFACE technique. The totalanalysis time in each experiment was 5.0 min at 24 kV (cur-rent: 35.4 µA) using a 40.5-cm (inlet to detector), 50-µm-i.d.open, uncoated quartz capillary. NAD and 4-CBSA were usedas internal standards. A–C are explained in the text.

yielding a derivative of Teic or Rist. Continued elec-trophoresis results in the overlap of the derivatizedantibiotic and the plug of D-Ala-D-Ala peptide. Analysisof the change in RMTR of the new glycopeptide relativeto the noninteracting standards, as a function of theconcentration of the D-Ala-D-Ala ligand, yields a valuefor the binding constant.

In these series of experiments, we examined thebinding interaction between Teic and its derivatives to 2.In these studies, PFACE was used to estimate bindingconstants. In PFACE, the capillary is partially filled withpeptide. As long as a dynamic equilibrium is establishedbetween ligand and receptor prior to the point of detec-tion, a binding constant can be estimated. In the presentstudies, a plug containing increasing concentrations of 2was initially injected to partially fill the capillary. Abuffer plug was then vacuum injected into the capillaryto separate the reagents from the ligand plug. The third

injection contained Teic, MO, and NAD as standards.The final injection contained a mixture of succinic andacetic anhydride. Upon electrophoresis, the anhydridesand Teic plugs overlap and react, forming new Teicderivatives. The buffer plug serves as a barrier betweenTeic and the D-Ala-D-Ala terminus peptides so that mix-ing does not occur prior to ACE analysis. The Teicderviatives then migrate into the zone of 2 and a dynam-ic equilibrium is achieved as electrophoresis continues.

Figure 9 shows a representative series of electro-pherograms of Teic and its derivatives. The plug of 2 isobserved as a box in the electropherogram that increas-es in height with increasing concentrations of 1 (0–500µM) in the plug. The glycopeptide, its derivatives, andthe D-Ala-D-Ala terminus peptides used in this studyare all negatively charged at pH 6.9 and therefore eluteafter the neutral marker MO. The difference in chargeneutralized upon acetylation and succinylation dictatesthe difference in mobilities between Teic and its acety-lated and succinylated derivatives. The pKa of the N-ter-minus amine of Teic is 7.1. This equates to an approx-imate charge difference between Teic and its acetylatedand succinylated forms of 0.4 and 1.4, respectively.

Upon addition of increasing concentrations of 2 in therunning buffer, the migration times of Teic and its derivativesshift to greater migration times. The complexation between2 and the Teic derivatives resulted in an increasing negativecharge on the compounds, and the complexes are detectedlater than the uncomplexed form. At the point of saturation,the Teic peaks no longer shift to the right despite increasingconcentrations of 2 in the running buffer.

Further proof that derivatization of Teic occurs isthat Teic and Teic-acetyl-A2-2 elute at the same place,relative to the internal standards, as do Teic-acetyl-A2-2and Teic-succinyl-A2-2, respectively. These results alsodemonstrate that derivatization of Teic has a greatereffect on changing the charge than the mass. Teic-A2-Xexists in much smaller concentrations than the majorform of Teic and upon derivatization yields much small-er peaks for its acetylated and succinylated forms.Figure 5d is a Scatchard plot of the data for Teic and itsderivatives. In this form of analysis, Kb is estimatedusing a dual-marker form of analysis (the relative migra-tion time ratio) (Eq. [1]).25 Table 2 details the binding ofTeic and its derivatives to ligands 2, 4, and 5.

Using similar experimental protocols, we alsoexamined the on-column acetylation and succinylationof Rist. Unlike Van and Teic, Rist possesses an esterifiedC-terminus, thereby making it approximately neutral atthe pH of the study.6,9 Upon derivatization, the Ristderivatives become negatively charged and elute atgreater migration times than Rist (data not shown).Table 3 summarizes the binding data for Rist and itsderivatives to peptides 2 and 5.



FIGURE 9 Representative series of electropherograms of Teicand its derivatives in 20 mM phosphate buffer (pH 6.9) con-taining various concentrations of 2, using on-column recep-tor synthesis coupled to PFACE. The total analysis time ineach experiment was 5.0 min at 20 kV using a 46.5-cm (inletto detector), 50-µm-i.d. open, uncoated quartz capillary.

MSLIPFACE

In MSLIPFACE, a sample plug of receptor andstandards is injected by pressure and electrophoresed ina buffer containing a given concentration of ligand(Figure 10). The sequence is repeated for all concentra-tions of ligand generating a single electropherogramcontaining a series of individual sample plugs superim-posed on environments of buffer containing increasingconcentrations of ligand.

In the first series of experiments, we examined theinteraction of Van and 1 and 2. In this technique, a sam-ple containing Van, MO, and CAB was injected by pres-sure and electrophoresed in a solution of 2 for 2.0 min.The concentration of 2 was sequentially increased from0 to 1150 µM and the process was repeated eight timesuntil all concentrations of ligand were used.

Figure 11 shows the electropherogram of Van gen-erated using the MSLIPFACE technique. Upon addition ofincreasing concentrations of 2 in the running buffer, theVan peak shifts to a greater migration time for any givenconcentration of 2 with respect to the noninteractingmarkers. The complexation between 2 and Van resultedin an increasing negative charge, and the peak for Vancomplexed to the ligand shifts to a longer migration timerelative to the neutral marker MO increasing 2 in the run-ning buffer. As can be seen in Figure 11, a single electro-pherogram is generated using the MSLIPFACE technique.At any one time during the experiment, only three differ-ent ligand concentrations and three sample plugs arecontained in the capillary column. The instrument wasprogrammed in order to ensure that all plugs of samplewere contained in one single electropherogram. The totaltime for the experiment was approximately 27 min.Traditional ACE techniques require in excess of 50 min,depending on the number of repetitions of ligand run,voltage, capillary length, and buffer conditions. Figure 5eis a Scatchard plot of the data for Van. Table 1 summa-rizes the binding data for the two ligands and Van.

Of particular interest is the amount of ligand usedin any given run. Unlike standard ACE techniques, in

which the receptor is electrophoresed in a runningbuffer of increasing concentrations of ligand, the MSLIP-FACE technique uses much smaller quantities of materi-al (1.9 nmol compared to approximately 9.9 nmol forstandard ACE techniques) for any given ACE experi-ment since the capillary is not completely filled with lig-and. In the present experiment, we used an 87-cm-longcapillary to demonstrate the technique. We have usedcapillaries of smaller lengths and, hence, even smalleramounts of ligand can be used.

MIACE

Recently, we expanded on the multiple-injectionconcept by developing multiple-injection affinity capil-lary electrophoresis to determine binding constants (Kb)between receptors and ligands using Van and Teic asmodel systems. In a general form of the technique, aplug of sample containing a noninteracting standard isfirst injected, followed by multiple plugs of sample con-taining the receptor and then a final injection of samplecontaining a second standard. Between each injection ofsample is injected a small plug of buffer containing anincreasing concentration of ligand to effect separationbetween the multiple injections of sample. The elec-trophoresis is then carried out in an increasing concen-tration of ligand in the running buffer. Continued elec-trophoresis results in a shift in the migration time of thereceptor in the sample plugs upon binding to theirrespective ligand. Analysis of the change in the RMTR orelectrophoretic mobility (µ) of the resultant receptor–-ligand complex relative to the noninteracting standards,



Table 2EXPERIMENTAL VALUES OF BINDING CONSTANTS Kb (104 M–1) OFTEIC-A2-2 AND A2-X WITH LIGANDS 2, 4, AND 5 MEASURED BY THEOCRDACE TECHNIQUE

Kb (104 M–1)Antibiotic 2 4 5Teic 37 140 65Teic-A2-X 20 140 130Teic-acetyl-A2-2 13 16 7.9Teic-acetyl-A2-X 6.2 7.6 8.7Teic-succinyl-A2-2 6.2 5.0 12Teic-succinyl-A2-X 3.4 nd 11

ND: not determined.

Table 3EXPERIMENTAL VALUES OF BINDING CONSTANTS Kb (104 M–1) OFRIST AND ITS DERIVATIVES WITH LIGANDS 2 AND 5 MEASURED BY THEOCRDACE TECHNIQUE

Kb (104 M–1)Antibiotic 2 5Rist 4.8 10Rist-acetyl 1.7 3.1Rist-succinyl 0.9 1.8

FIGURE 10 Schematic of MSLIPFACE.

as a function of the concentration of ligand, yields avalue for Kb.

We first examined the binding of 4 to Van usingthe MIACE technique. In the MIACE technique, a plug ofsample (0.5 psi at 3 sec) containing MO was first inject-ed into the capillary column followed by five plugs (0.5psi at 3 sec) of sample containing Van (Figure 12).Between each injection of Van was placed a small plug(0.5 psi at 18 sec) of ligand 4 to aid in the separation ofall Van peaks. A final plug (0.5 psi at 3 sec) of the sec-ond noninteracting standard, 4-CBSA, was then injectedand electrophoresed in a solution of 4.

Upon electrophoresis, individual plugs of samplemigrate through the capillary column to afford sevenpeaks (five for Van and two for the standards) at thepoint of detection. Figure 13 shows a representativeseries of electropherograms of Van in a capillary filledwith increasing concentrations of 4 at 200 nm. Thepeaks for Van are not baseline resolved but can easilybe differentiated from each other. As the concentration

of 4 was increased (0–300 µM) in the running buffer,the peaks for Van shifted to longer migration timessince the Van–4 complexes are more negative than Vanitself. The inverted peaks to the right of the Van peaksare due to the dilution of 4 in the running buffer uponcomplexation to Van. These negative peaks are com-monly observed in ACE studies and are particularly pro-nounced when the ligand or receptor in the runningbuffer is chromophoric and/or when high concentra-tions of the ligand/receptor are used for the bindingassay. Due to the higher mass of the newly formedcomplex upon increasing the concentration of 4, theheights of the peaks for Van increase in comparison tothe MO marker. Analysis of the change in the RMTR orµ of the resultant complex relative to the noninteractingstandards, as a function of the concentration of ligand(or receptor), yields a value for Kb.

Figure 14 shows Scatchard plots of the data forthe five Van peaks using Eq. (2). As can be seen, all fiveVan peaks result in similar Scatchard plots. The average



FIGURE 11 Representative electropherogram of Van in 0.192 Mglycine-0.025 M Tris buffer (pH 8.3) containing various con-centrations of 2 using the MSLIPFACE technique. The totalanalysis time in each experiment was 27 min at 24 kV (cur-rent: 4.0 µA) using an 80.5-cm (inlet to detector), 50-µm-i.d.open, uncoated quartz capillary. MO and CAB (containingCAA and CAB isozymes) were used as internal standards.The number above each set of sample peaks refers to the con-centration of 2 in µM.

FIGURE 13 Representative set of electropherograms of Van(darkened diamond, triangle, square, circle, and opensquare) in 192 mM glycine-25 mM Tris buffer (pH 8.3) con-taining various concentrations of 4 using the MIACE tech-nique. The total analysis time in each experiment was 6.0min at 25 kV (current 6.8 µA) using a 40.5-cm (inlet todetector), 50-µm-i.d. open, uncoated quartz capillary. MO(open circle) and CBSA (open triangle) were used as inter-nal standards.FIGURE 12 Schematic of an MIACE experiment.

binding constant of 4 to Van was determined to be 22.3× 103 M–1.

We then examined the binding of Van to other lig-ands. Using a similar injection sequence, we deter-mined the binding affinities of ligands 2–6 to Van. Table1 summarizes the binding data obtained for Van and lig-ands 2–6. The binding constants obtained using MIACEwere comparable to those determined using standardACE techniques.4,17–19 A similar study was conductedwith Teic, the results of which are shown in Table 1.

A modification in MIACE was recently developedin our laboratories49 (Figure 15). In this technique, twopeptides with similar charge and mass can be separat-ed and their binding to glycopeptide antibioticsassessed simultaneously. Although CE is a very power-ful technique for separating charged species, com-pounds with the same charge and only minimal differ-ences in molecular weight are not always able to beseparated, thereby resulting in a single peak or, at best,peak overlap. In a modification in MIACE, separateplugs of sample containing noninteracting standards,peptide 1, buffer, and peptide 2 are injected into the

capillary column and electrophoresed. Peptides migratethrough the column at similar electrophoretic mobilitiesbut remain as distinct zones due to the buffer plugbetween peptides. The electrophoresis is then carriedout in an increasing concentration of antibiotic in therunning buffer. Continued electrophoresis results in ashift in the migration time of the peptides upon bindingto the antibiotic and a Scatchard plot derived using theRMTR form of analysis. The data obtained using thistechnique (denoted as MIACE II) are listed in Table 1.

We used isothermal titration calorimetry (ITC) toexamine the binding of several ligands to theantibioitics.50 Figure 16 is the ITC sample data for thetitration of 9 to Rist. Table 1 lists the data for the bind-ing of ligands 9–11 and Rist using ITC. As can be seen,there is close agreement between the data from ACEand ITC. It was determined that ligands 9–11 bind toRist with stoichiometry of close to 1.

The advantages of MIACE are several-fold: 1)Smaller quantities of ligand are needed to conduct thestudies in comparison to other assay techniques. 2) Thenumber of receptor injections can be increased and is



FIGURE 14 Scatchard plots of the data for Van and 1 according to Eq. (2).

dependent on the capillary length and applied voltage.3) MIACE allows for the estimation of binding affinitiesbetween biological interactions on a timescale fasterthan that found for standard ACE. 4) Purification of thesample prior to injection is not necessary as long as thecomponent to be analyzed can be separated from otherspecies. 5) Multiple binding constants can be obtainedin a series of ACE experiments, shortening the amountof time required to conduct the assay.

Comparison of the different ACE techniques

When comparing the different ACE techniques,the most important criteria are the amount of sampleused and the speed of analysis. With respect to sam-ple consumption, the FTPFACE technique utilizes theleast amount of sample (ligand) since only a smallplug of solution with ligand is injected rather than alarger zone or entire capillary as found in PFACE andstandard ACE, respectively. OCLDACE and OCRDACEalso use small amounts of ligand, but have an initialderivatization step requiring relatively higher concen-trations of reagents than are typically used in FTP-FACE and PFACE. Hence, although in some cases itmight require less material than FTPFACE and PFACE,it is generally more costly due to the derivatizationstep. MSLIPFACE uses amounts of ligand on par withFTPFACE, but is a more time-consuming techniquerequiring many injections of solution onto the capil-lary. Also, timing is critical in MSLIPFACE since zonesof solution must overlap with some accuracy at thepoint of detection. The MIACE technique is most effi-cient from a time perspective since multiple plugs ofreceptor can be simultaneously analyzed, therebyreducing the need for multiple repetitions at everyconcentration of ligand.

Conclusion

The present study details the measurement ofbinding constants between the glycopeptide antibioticsvancomycin, ristocetin, and teicoplanin and small pep-tides using PFACE, FTPFACE, OCLDACE, OCRDACE,MSLIPFACE, and MIACE. In these techniques, modifica-tions in standard ACE have been developed that utilizesmaller quantities of sample and expedite the analysis ofthe respective interaction. These techniques have poten-tial in the pharmaceutical industry and in industries inwhich the high-throughput analysis of drugs and drugtargets is critical. Future work is focused on expandingthe range of receptor–ligand interactions studied and infurther developing more novel ACE techniques.



FIGURE 15 Schematic of the plug sequence used to separate pep-tides of similar charge and mass using the MIACE II technique.

FIGURE 16 Sample raw data for the titration of 9 into Rist at 25°C. In the top panel, the peaks in the lower data set show aheat produced by 15-µL serial injections of 1.16 mM 9 into1.463 mL of 0.108 mM Rist contained in the sample cell of thecalorimeter. Integration of these peaks produces the bindingisotherm shown in the bottom panel (solid squares). The pointsare fitted to a model for a single independent binding site.

Acknowledgment

We gratefully acknowledge financial support forthis research by grants from the National ScienceFoundation (CHE-0136724, CHE-0515363, and DMR-0351848) and the National Institutes of Health (1R15AI055515-01, 1R15AI65468-01, and GM54939).

©Copyright 2006. ISC Technical Publications, Inc.Manuscript received ???.

References

1. Williams, D.H.; Davies, N.L.; Zerella, R.; Bardsley, B. Noncovalentinteractions: defining cooperativity. Ligand binding aided byreduced dynamic behavior of receptors. Binding of bacterial cellwall analogues to ristocetin A. J. Am. Chem. Soc. 2004, 126, 2042–9.

2. Heck, A.J.R.; Bonnici, P.J.; Breukink, E.; Morris, D.; Wills, M.Modification and inhibition of vancomycin group antibiotics byformaldehyde and acetaldehyde. Chem. Eur. J. 2001, 7, 910–16.

3. Chiosis, G.; Boneca, I.G. Selective cleavage of D-Ala-D-Lac bysmall molecules: re-sensitizing resistant bacteria to vancomycin.Science 2001, 293, 1484–7.

4. Kerns, R.; Dong, S.D.; Fukuzawa, S.; Carbeck, J.; Kohler, J.;Silver, L.; Kahne, D. The role of hydrophobic substituents in thebiological activity of glycopeptide antibiotics. J. Am. Chem. Soc.2000, 122, 12,608–9.

5. Gasper, M.P.; Berthod, A.; Nair, U.B.; Armstrong, D.W.Comparison and modeling study of vancomycin, ristocetin A,and teicoplanin for CE enantioseparations. Anal. Chem. 1996,68, 2501–14.

6. Pearson, A.J.; Heo, J.-N. Approaches to the fully functionalizedDEF ring system of ristocetin A via highly selective ruthenium-promoted S(N)Ar reaction. Org. Lett. 2000, 19, 2987–90.

7. Beauregard, D.A.; Maguire, A.J.; Williams, D.H.; Reynolds, P.E.Semiquantitation of cooperativity in binding of vancomycin-group antibiotics to vancomycin-susceptible and resistant organ-isms. Antimicrob. Agents Chemother. 1997, 41, 2418–23.

8. Shiozawa, H.; Chia, B.C.S.; Davies, N.L.; Zerella, R.; Williams,D.H. Cooperative binding interactions of glycopeptide antibi-otics. J. Am. Chem. Soc. 2002, 124, 3914–19.

9. Lee, J.-G.; Sagui, C.; Roland, C. First principles investigation ofvancomycin and teicoplanin binding to bacterial cell wall termi-ni. J. Am. Chem. Soc. 2004, 126, 8384–5.

10. Allen, N.E.; LeTourneau, D.L.; Hobbs, J.N. Molecular interactionsof a semisynthetic glycopeptide antibiotic with D-alanyl-D-ala-nine and D-alanyl-D-lactate residues. Antimicrob. AgentsChemother. 1997, 41, 66–71.

11. Chu, Y.-H.; Avila, L.Z.; Biebuyck, H.A.; Whitesides, G.M. Usingaffinity capillary electrophoresis to identify the peptide in a pep-tide library that binds most tightly to vancomycin. J. Org. Chem.1992, 58, 648–52.

12. Chu, Y.-H.; Whitesides, G.M. Affinity capillary electrophoresiscan simultaneously measure binding constants of multiple pep-tides to vancomycin. J. Org. Chem. 1992, 57, 3524–5.

13. Baba, Y.; Tsuhako, M.; Sawa, M.; Akashi, M.; Yashima, E. Specificbase recognition of oligodeoxynucleotides by capillary affinitygel electrophoresis using polyacrylamide-poly(9-vinyladenine)conjugated gel. Anal. Chem. 1992, 64, 1920–5.

14. Chu, Y.-H.; Avila, L.Z.; Biebuyck, H.A.; Whitesides, G.M. Use ofaffinity capillary electrophoresis to measure binding constants ofligands to proteins. J. Med. Chem. 1992, 35, 2915–17.

15. Heegaard, N.H.H.; Robey, F.A. Use of capillary zone elec-trophoresis to evaluate the binding of anionic carbohydrates tosynthetic peptides from serum amyloid P component. Anal.Chem. 1992, 64, 2479–82.

16. El-Shafey, A.; Zhong, H.; Jones, G.; Krull, I.S. Application ofaffinity capillary electrophoresis for the determination of bindingconstants and thermodynamic constants of enediynes withhuman serum albumin and histone. Electrophoresis 2002, 23,945–50.

17. Kaddis, J.; Zurita, C.; Moran, J.; Borra, M.; Polder, N.; Meyer, C.R.;Gomez, F.A. Estimation of binding constants for the substrateand activator of Rhodobacter sphaeroides ADP-glucosepyrophosphorylase using affinity capillary electrophoresis. Anal.Biochem. 2004, 327, 252–60.

18. Varenne, A.; Gareil, P.; Colliec-Jouault, S.; Daniel, R. Capillaryelectrophoresis determination of the binding affinity of bioactivesulfated polysaccharides to proteins: study of the binding prop-erties of fucoidan to antithrombin. Anal. Biochem. 2003, 315,152–9.

19. Silverio, C.F.; Azad, M.; Gomez, F.A. On-column derivatizationand analysis of the antibiotics teicoplanin and ristocetin coupledto affinity capillary electrophoresis. Electrophoresis 2003, 24,808–15.

20. Qian, X.-H.; Tomer, K.B. Affinity capillary electrophoresis inves-tigation of an epitope on human immunodeficiency virus recog-nized by a monoclonal antibody. Electrophoresis 1998, 19,415–19.

21. Kiessig, S.T.; Bang, H.; Thunecke, F. Interaction of cyclophilinand cyclosporins monitored by affinity capillary electrophoresis.J. Chromatogr. A 1999, 853, 469–77.

22. Mito, E.; Zhang, Y.; Esquivel, S.; Gomez, F.A. Estimation ofreceptor–ligand interactions by the use of a two-marker systemin affinity capillary electrophoresis. Anal. Biochem. 2000, 280,209–15.

23. Dunayevskiy, Y.M.; Lyubarskaya, Y.V.; Chu, Y.-H.; Vouros, P.;Karger, B.J. Simultaneous measurement of nineteen binding con-stants of peptides to vancomycin using affinity capillary elec-trophoresis-mass spectrometry. J. Med. Chem. 1998, 41, 1201–4.

24. Azad, M.; Brown, A.; Silva, I.; Gomez, F.A. Estimation of bindingconstants between ristocetin and teicoplanin to peptides usingon-column ligand derivatization coupled to affinity capillaryelectrophoresis. Anal. Bioanal. Chem. 2004, 379, 149–55.

25. VanderNoot, V.A.; Hileman, R.E.; Dordick, J.S.; Linhardt, R.J.Affinity capillary electrophoresis employing immobilized gly-cosaminoglycan to resolve heparin-binding peptides.Electrophoresis 1998, 19, 437–41.

26. Colton, J.J.; Carbeck, J.D.; Rao, J.; Whitesides, G.M. Affinity capillaryelectrophoresis: a physical-organic tool for studying interactions inbiomolecular recognition. Electrophoresis 1998, 19, 367–82.

27. Busch, M.H.A.; Kraak, J.C.; Poppe, H. Principles and limitationsof methods available for the determination of association con-stants with affinity capillary electrophoresis. J. Chromatogr.1997, 777, 329.

28. Gomez, F.A.; Avila, L.Z.; Chu, Y.-H.; Whitesides, G.M.Determination of binding constants of ligands to proteins byaffinity capillary electrophoresis: compensation for electroosmot-ic flow. Anal. Chem. 1994, 66, 1785–91.

29. Zhang, Y.; Gomez, F.A. Multiple-step ligand injection affinitycapillary electrophoresis for determining binding constants ofligands to receptors. J. Chromatogr. A 2000, 897, 339–47.

30. Erim, F.B.; Kraak, J.C. Vacancy affinity capillary electrophoresisto study competitive protein–drug binding. J. Chromatogr. B1998, 710, 205–10.

31. Chu, Y.-H.; Avila, L.Z.; Gao, J.; Whitesides, G.M. Affinity capil-lary electrophoresis. Acc. Chem. Res. 1995, 28, 461.

32. Lynen, F.; Zhao, Y.; Becu, Ch.; Borremans, F.; Sandra, P.Considerations concerning interaction characterization ofoligopeptide mixtures with vancomycin using affinity capillaryelectrophoresis-electrospray mass spectroscopy. Electrophoresis,1999, 20, 2462.

33. Heintz, J.; Hernandez, M.; Gomez, F.A. Use of a partial-fillingtechnique in affinity capillary electrophoresis for determining



SusanM

Underline

SusanM

Note

Norberto: received date needed.

binding constants of ligands to receptors. J. Chromatogr. A 1999,840, 261–8.

34. Mito, E.; Gomez, F.A. Flow-through partial-filling affinity capil-lary electrophoresis can estimate binding constants of ligands toreceptors. Chromatographia 1999, 50, 689–94.

35. Brown, A.; Silva, I.; Chinchilla, D.; Hernandez, L.; Gomez, F.A.Partial-filling techniques for affinity capillary electrophoresis toprobe receptor–ligand interactions. LC•GC Europe 2004, 1–7.

36. Villareal, V.; Kaddis, J.; Azad, M.; Zurita, C.; Silva, I.; Hernandez,L.; Rudolph, M.; Moran, J.; Gomez, F.A. Partial-filling affinity cap-illary electrophoresis. Anal. Bioanal. Chem. 2003, 376, 822–31.

37. Kaddis, J.; Mito, E.; Heintz, J.; Plazas, A.; Gomez, F.A. Flow-through partial-filling affinity capillary electrophoresis can esti-mate binding constants of neutral ligands to receptors via a com-petitive assay technique. Electrophoresis 2003, 24, 1105–10.

38. Gomez, F.A.; Mirkovich, J.N.; Dominguez, V.M.; Liu, K.W.;Macias, D.M. Multiple-plug binding assays using affinity capillaryelectrophoresis. J. Chromatogr. A 1996, 727, 291–9.

39. Rao, J.; Colton, I.J.; Whitesides, G.M. Using capillary elec-trophoresis to study the electrostatic interactions involved in theassociation of D-Ala-D-Ala with vancomycin. J. Am. Chem. Soc.1997, 119, 9336–40.

40. Zhang, Y.; Kodama, C.; Zurita, C.; Gomez, F.A. On-column lig-and synthesis coupled to partial-filling affinity capillary elec-trophoresis to estimate binding constants of ligands to a recep-tor. J. Chromatogr. A 2001, 928, 233–41.

41. Silverio, C.F.; Plazas, A.; Moran, J.; Gomez, F.A. Determination ofbinding constants between teicoplanin and d-ala-d-ala terminuspeptides by affinity capillary electrophoresis. J. Liq. Chrom. Rel.Tech. 2002, 25, 1677–91.

42. Chinchilla, D.; Zavaleta, J.; Martinez, K. Multiple-injection affini-ty capillary electrophoresis to estimate binding constants ofreceptors to ligands. Anal. Bioanal. Chem. 2005, 383, 625-31.

43. Amini, A.; Westerlund, D. Evaluation of association constantsbetween drug enantiomers and human alpha 1-acid glycoproteinby applying a partial-filling technique in affinity capillary elec-trophoresis. Anal. Chem. 1998, 70, 1425–30.

44. Tanaka, Y.; Terabe, S. Separation of the enantiomers of basicdrugs by affinity capillary electrophoresis using a partial fillingtechnique and α1-acid glycoprotein as chiral selector.Chromatographia 1997, 44, 119–28.

45. De Lorenzi, E.; Galbusera, C.; Bellotti, V.; Mangione, P.;Massolini, G.; Tabolotti, E.; Andreolim, A.; Caccialanza, G.Affinity capillary electrophoresis is a powerful tool to identifytransthyretin binding drugs for potential therapeutic use in amy-loidosis. Electrophoresis 2000, 21, 3280–9.

46. Chu, Y.-H.; Dunayevskiy, Y.M.; Kirby, D.P.; Vouros, P.; Karger,B.L. Affinity capillary electrophoresis-mass spectrometry forscreening combinatorial libraries. J. Am. Chem. Soc. 1996, 118,7827–35.

47. Handwerger, S.; Pucci, M.; Volk, K.J.; Liu, J.; Lee, M.S.Vancomycin-resistant leuconostoc mesenteroides and lactobacil-lus casei synthesize cytoplasmic peptidoglycan precursors thatterminate in lactate. J. Bacteriol. 1994, 176, 260–4.

48. Azad, M.; Hernandez, L.; Plazas, A.; Rudolph, M.; Gomez,F.A. Determination of binding constants between the antibi-otic ristocetin A and D-Ala-D-Ala terminus peptides by affin-ity capillary electrophoresis. Chromatographia 2003, 57,339–43.

49. Zavaleta, J.; Chinchilla, D.; Martinez, K.; Gomez, F.A. Multiple-injection affinity capillary electrophoresis to examine bindingconstants between glycopeptide antibiotics and peptides. J.Chromatogr. A 2006, 1105, 59-65.

50. Ladbury, J.E.; Chowdhry, B.Z. Sensing the heat: the applicationof isothermal titration calorimetry to thermodynamic studies ofbiomolecular interactions. Chem. Biol. 1996, 3, 791–801.



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Partial-filling affinity capillary electrophoresis …...Partial-filling affinity capillary electrophoresis techniques to probe the binding of glycopeptide antibiotics to D-Ala-D-Ala