Anal Bioanal Chem (2005) 383: 625–631DOI 10.1007/s00216-005-0047-0

ORIGINAL PAPER

Dinora Chinchilla . Jose Zavaleta . Karla Martinez .Frank A. Gomez

Multiple-injection affinity capillary electrophoresis to estimatebinding constants of receptors to ligands

Received: 1 April 2005 / Revised: 18 July 2005 / Accepted: 21 July 2005 / Published online: 28 September 2005# Springer-Verlag 2005

Abstract Multiple-injection affinity capillary electropho-resis (MIACE) is used to determine binding constants (Kb)between receptors and ligands using as model systemsvancomycin and teicoplanin from Streptomyces orientalisand Actinoplanes teichomyceticus, respectively, and theirbinding to D-Ala-D-Ala peptides and carbonic anhydraseB (CAB. EC 4.2.1.1) and the binding of the latter toarylsulfonamides. A sample plug containing a non-inter-acting standard is first injected followed by multiple plugsof sample containing the receptor and then a final injectionof sample containing a second standard. Between eachinjection of sample, a small plug of buffer is injected whichcontains an increasing concentration of ligand to effectseparation between the multiple injections of sample.Electrophoresis is then carried out in an increasing con-centration 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 their re-spective ligand. Analysis of the change in the relative mi-gration time ratio (RMTR) or electrophoretic mobility (μ)of the resultant receptor–ligand complex relative to thenon-interacting standards, as a function of the concentra-tion of ligand yields a value for Kb. The MIACE techniqueis a modification in the ACE method that allows for theestimation of binding affinities between biological inter-actions on a timescale faster than that found for standardACE. In addition sample volume requirements for thetechnique are reduced compared to traditional ACE assays.These findings demonstrate the advantage of using MIACEto estimate binding parameters between receptors andligands.

Keywords Multiple-injection affinity capillary .Electrophoresis . Vancomycin . Teicoplanin .Carbonic anhydrase B . Binding constants

Abbreviations MIACE: Multiple-injection affinitycapillary electrophoresis . Van: Vancomycin . Teic:teicoplanin . RMTR: relative migration time ratio

Introduction

Currently there is a major need to develop analytical tech-nologies that can aid in the screening of drugs for manyhealth-related problems. For example, Alzheimer’s disease,AIDS-HIV, and cancer are but a few of the many humandiseases for which a rapid screening technique would ex-pedite the development of a cure and the saving of humanlives. Over the past ten years advances in molecular bi-ology have yielded great insights into the many molecularinteractions involved in human disease. Such interactionsare critical in understanding the functions and molecularmechanisms of biological systems and the roles these in-teractions play in health and human disease. Because of thelarge array of receptors and ligands that have been found,and those yet discovered, it is critical to develop analyticaltechniques that can estimate the level and extent of in-teraction between biological species.

During the past decade affinity capillary electrophoresis(ACE) has emerged as a useful and sensitive technique forstudying bimolecular non-covalent interactions and fordetermining binding and dissociation constants of formedcomplexes [1–25]. Since the initial reports in 1992 docu-menting the use of CE to study receptor–ligand inter-actions, ACE has been successfully used to examine a widearray of interactions including protein–drug, protein–DNA,peptide–carbohydrate, peptide–peptide, DNA–dye, carbo-hydrate–drug, and antigen–antibody [26–30]. For example,Kaddis et al. recently used ACE to determine bindingconstants for the activator fructose-6-phosphate (F6P) andsubstrate ATP to the recombinant wild-type (WT) Rhodo-bacter sphaeroides adenosine 5′-diphosphate-(ADP)-glu-

D. Chinchilla . J. Zavaleta . K. Martinez . F. A. Gomez (*)Department of Chemistry and Biochemistry,California State University,5151 State University Drive,Los Angeles, CA 90032-8202, USAe-mail: fgomez2@calstatela.eduTel.: +1-323-3432368Fax: +1-323-3436490

cose pyrophosphorylase (ADPGlc PPase) [1]. McKeon etal. used ACE to determine the binding between heparin-like glycosaminoglycans and the heparin-binding domain(96–110) of amyloid precursor protein (APP) [2]. Finally,Varenne et al. used ACE to examine the binding offucoidan, an anticoagulant polysaccharide of marine ori-gin, to antithrombin [3]. The underlying principle of ACEis that the electrophoretic mobility of a receptor (μR)changes upon binding to a ligand that is present in theelectrophoresis buffer. The change in μ (or other parameterif an alternate form of analysis is used) as a function ofligand concentrations allows binding constants to be de-termined via Scatchard analysis [15].

Although ACE has been shown to be effective inestimating binding parameters of ligands to receptors, incases where only small quantities of material are available,analysis by traditional ACE techniques is made difficult. Inaddition, expeditious analysis of the interaction in questionmay also be desirable particularly when combinatorialapproaches to drug design are utilized. In cases where bothconditions are needed, modifications in the standard ACEtechnique are warranted.

In two previous studies we described multiple-pluginjection sequences that allowed for the estimation ofbinding constants between receptors and ligands [14, 22].In the first study, a series of four plugs containing receptorand standards were injected and exposed to an increasingconcentration of ligand in the electrophoresis buffer [22].Four binding constants for the same receptor–ligand in-teraction were subsequently obtained. A drawback to thistechnique was that the number of injections was limited astoo short a delay between injections caused unwantedoverlap of adjacent plugs of sample thereby making sepa-ration and differentiation of peaks difficult. In the secondstudy, a more elaborate plug sequence was demonstratedutilizing partial-filling techniques [14]. Here, a sample ofreceptor and standards were multiply injected each sepa-rated by an increasing concentration of ligand. The wholesequence of ligand concentrations could be visualized inone electropherogram and a shift in the receptor migrationtime was observed thereby allowing for the estimation ofa binding constant. This technique, too, had a drawbackin that a long capillary was needed for the separation thuslengthening the time required for the experiment. In addi-tion, timing was critical since receptor and markers wererequired to migrate into the zone of ligand by the pointof detection.

Herein we describe a new technique called multiple-injection affinity capillary electrophoresis (MIACE) usingas model systems vancomycin (Van) and teicoplanin (Teic)(Fig. 1a) and carbonic anhydrase B. In this techniquemultiple injections of sample containing receptor, non-interacting standards, and buffer are injected. The sampleof buffer and running buffer contain an increasing con-centration of ligand that, upon interaction with the sample,causes a shift in migration time of the receptor due tocomplexation with the ligand. Subsequent Scatchard anal-ysis yields a value for Kb. The present study requires onlyone set of standards and can accommodate multiple in-

jections of receptor. The number of receptor injections canbe increased and is dependent on the capillary length andapplied voltage.

Fig. 1 a Structures of vancomycin (top), teicoplanin (bottom), andD-Ala-D-Ala ligands 1–6 used in this study. b Ligands 7 and 8 usedin this study

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Materials and methods

Chemicals and reagents

All chemicals were of analytical grade. Vancomycin, D-Ala-D-Ala, N-acetyl-D-Ala-D-Ala (5), N,N′-diacetyl-Lys-D-Ala-D-Ala (6), nicotinamide adenine dinucleotide (NAD),and 4-carboxybenzenesulfonamide (CBSA) (7) were pur-chased from Sigma Chemical Company (St. Louis, MO,USA) and were used without further purification. Teico-planin·HCl was purchased from Advance Separation Tech-nologies Inc. (Whippany, NJ, USA) and was used withoutfurther purification. Fmoc-Gly-NHS, Fmoc-Ala-NHS, Fmoc-Phe-NHS, and Fmoc-Val-NHSwere purchased from BachemCalifornia Inc. (Torrance, CA, USA). Mesityl oxide (MO)was purchased from Calbiochem (San Diego, CA, USA).Stock solutions of Van (0.2 mg mL−1), Teic (1.0 g L−1), andCBSA (0.5 g L−1) were each prepared by dissolving inbuffer (20 mM phosphate buffer; pH 7.5). Stock solutionsof the N-protected amino acids (4 mM) were prepared bydissolving the compounds in buffer. 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 preparedbased on literature procedures [31]. Compound 8 was agift of S. Mallik (North Dakota State University) [32].

Apparatus

The capillary electrophoresis (CE) system used in thisstudy was a Beckman Model P/ACE 5510 (Fullerton, CA,USA). The capillary tubing (Polymicro Technology, Inc.,Phoenix, AZ, USA) used for the experiment was uncoatedfused silica with an internal diameter of 50 μm, length frominlet to detector of 40.5 cm (49 cm for Teic and compounds1 and 2), and a length from detector to outlet of 6.5 cm (11cm for Teic and compounds 1 and 2). The conditions usedin CE were as follows: for Van and Teic, voltage, 25 kV;current, 6.8 μA for Van, 7.9 μA for Teic; detection, 200nm; temperature, 23.0±0.1°C; for CAB, voltage, 30 kV;current, 7.5 μA; detection, 200 nm; temperature, 23.0±0.1°C. Data were collected and analyzed with BeckmanSystem Gold or 32 Carot software.

Procedures

For Van, the capillary was first equilibrated with buffer(192 mM glycine/25 mM Tris; pH 8.3) containing in-creasing concentrations of peptide (0–100 μM). Separateplugs of sample solution (3.6 nL each) containing themarker mesityl oxide (MO), five plugs of Van, and secondmarker (CBSA) were then introduced by pressure injectioneach separated by a plug of buffer (18-s injection). Theelectrophoresis was carried out using Tris-glycine bufferwith increasing concentrations of ligand at 25 kV for6.0 min to complete the detection of all species. Exper-imental conditions for Teic and CAB were similar exceptonly three plugs of receptor were introduced and exper-

iments were run for 7.0 min to Teic. For CAB, horse heartmyoglobin (HHM) was used as a marker and was intro-duced directly after introduction of the first marker MO.

Forms of analysis

Two forms of analysis, one using two markers and the otherusing a single marker, were used to estimate Kb. In thedual marker form of analysis, Kb is estimated using twonon-interacting standards, which we term the relative migra-tion time ratio (RMTR) (Eq. (1)) [17].

RMTR ¼ tp � ts0� ��

ts0 � tsð Þ (1)

Here, tp, ts, and ts′ are the measured migration times of theantibiotic peak (e.g., Van), and two non-interacting stan-dard peaks (CBSA andMO), respectively. A Scatchard plotcan be obtained via Eq. (2).

�RMTRP;A

�L½ �¼ Kb�RMTRmax

P;A � Kb�RMTRP;A (2)

Here,ΔRMTRP,A is the magnitude of change in the RMTRas a function of the concentration of peptide. Equation 2allows for the estimation of Kb on a relative time scaleusing two non-interacting standards and corrects forchanges in EOF that occur in the capillary column due tovariations in viscosity and Joule heating.

In the second form of analysis, Kb is estimated usingone non-interacting standard which relates changes in theelectrophoretic mobility (μP,L) of, for example, Van oncomplexation with the ligand (L) present in the buffer toKb. Analysis of the magnitude of the change in mobility,ΔμP,L as a function of the concentration [L] of ligandyields Kb (Eq. (3)).

��P;L ¼ �P;L � �P

¼ lcld�V 1

�tP;L � 1

�tm;L

� �� 1=tP � 1=tmð Þ� � ð3Þ

Here, ΔμP,L is the change in mobility of Van as a functionof the concentration of peptide; tP,L and tm,L are themeasured migration times of Van and a non-interactingstandard (e.g., MO or CBSA) at the concentration ofpeptide, respectively; lc (cm) is the total length of thecapillary; ld (cm) is the length of capillary from the inletend of the capillary to the detector; tm (s) is the measuredmigration time of the non-interacting standard; tP is themeasured migration time of the Van; and V is the voltageacross the capillary. The values of ΔμP,A obtained usingEq. (3) over a range of concentrations of peptide are thenused for Scatchard analysis (Eq. (4)).

��P;A

�A½ � ¼ Kb��max

P;A � Kb��P;A (4)

In general, either Eq. (2) or (4) can be used for ACEstudies although we have found that the use of two markers

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(Eq. (2)) yields a more accurate value for Kb especiallywhen EOF varies from run to run.

Results and discussion

In the first series of experiments, we examined the bindingof D-Ala-D-Ala terminus peptides to Van by using theMIACE technique. Van-group antibiotics are glycopep-tides that kill bacterial cells by inhibiting peptidoglycanbiosynthesis [33–40]. They function by binding to theterminal D-Ala-D-Ala dipeptide (binding also occurs toD-Tyr-D-Ala and D-Tyr-D-Ser terminus peptides) of bac-terial cell wall precursors, thereby impeding further pro-cessing of these intermediates into peptidoglycan.

In the MIACE technique, a plug of sample (0.5 psi at 3 s)containing the non-interacting standard mesityl oxide(MO) was first injected into the capillary column followedby five plugs (0.5 psi at 3 s) of sample containing Van(Fig. 2). Between each injection of Van was placed a smallplug (0.5 psi at 18 s) of buffer to aid in the separationof all Van peaks. A final plug (0.5 psi at 3 s) of thesecond non-interacting standard, 4-carboxybenzenesulfon-amide (CBSA), was then injected and electrophoresed.

Upon electrophoresis individual plugs of sample migratethrough the capillary column to afford seven peaks (five forVan and two for the standards) at the point of detection.Figure 3 shows a representative series of electrophero-grams of Van in a capillary filled with increasing con-centrations of 1 at 200 nm. The peaks for Van are notbaseline resolved but can easily be differentiated from eachother. As the concentration of 1 was increased (0–300 μM)in the running buffer the peaks for Van shifted to longermigration times as the Van–1 complexes are more negativethan Van itself. The inverted peaks to the right of the Vanpeaks are due to the dilution of ligand 1 in the runningbuffer upon complexation to Van. These negative peaks arecommonly observed in ACE studies and are particularly

pronounced when the ligand or receptor in the runningbuffer are chromophoric and/or when high concentrationsof the ligand/receptor are used for the binding assay. Dueto the higher mass of the newly formed complex uponincreasing the concentration of ligand 1, the height of thepeaks for Van increase in comparison to the MO marker.Analysis of the change in the relative migration time ratio(RMTR) or electrophoretic mobility (μ) of the resultantcomplex relative to the non-interacting standards, as a

Fig. 2 Schematic of a multiple-injection affinity capillary electrophoresis (MIACE) experiment

Fig. 3 Representative set of electropherograms of Van (filled dia-monds, triangles, squares, and circles, and open squares) in 192mM glycine/25 mM Tris buffer (pH 8.3) containing various con-centrations of ligand 1 using the MIACE technique. The total anal-ysis time in each experiment was 6.0 min at 25 kV (current 6.8 μA)using a 40.5-cm (inlet to detector), 50-μm I.D. open, uncoatedquartz capillary. MO (open circles) and CBSA (open triangles) wereused as internal standards

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function of the concentration of ligand (or receptor), yieldsa value for Kb.

Figure 4 shows Scatchard plots of the data for the fiveVan peaks using Eq. (2); all five Van peaks result in similar

Scatchard plots. In this experiment three repetitions wererun at each concentration of ligand and the third repetitionwas used for the analysis. In our previous work with ACEwe have observed that the initial repetitions tend to yield

Fig. 4 Scatchard plots of the data for Van and ligand 1 according to Eq. (2)

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binding constants with smaller values for R2 [1, 4, 6]. Weattribute this to the capillary not having reached equilib-rium with the ligand in the running buffer. Table 1 sum-marizes the binding data obtained for Van and ligand 1.The average binding constant was determined to be 22.3×103M−1. In our previous work we determined a value forKb of 41.6×103M−1 [19]. This study, though, was con-ducted at a pH of 7.5 and in phosphate buffer. It shouldbe noted that previous studies have shown that pH affectsthe value of Kb obtained by both ACE and other assaytechniques.

We then determined the binding affinities of Van toligands 2–6 by using a similar injection sequence and theresults are summarized in Table 2. The binding constantsobtained using MIACE were comparable to those deter-mined using standard ACE techniques [4, 17–19].

We also examined the binding of several D-Ala-D-Ala terminus peptides to Teic. Like Van, Teic is a linearheptapeptide, cross-linked between residues 1 and 3, 2 and4, and 4 and 6 by diphenyl ether bridges and betweenresidues 5 and 7 by a biphenyl bridge. Teic is a mixture offive closely related analogues, designated T-A1–1 throughT-A1–5 [6, 18]. They differ by approximately 20 molecularmass units because of the variation of the carbon length andsubstituent groups of the hydrophobic acyl side chain(hydrophobic tail) that is attached to a 2-amino-2-deoxy-β-D-glucopyranosyl moiety. This hydrophobic tail gives Teicits unique characteristics. Table 2 lists the binding data forTeic and ligands 1, 2, and 5. In general, the values for Kb

obtained by MIACE parallel the values found in previousACE studies [4, 17–19].

We then examined the binding of arylsulfonamides tothe enzyme CAB. CAB is a zinc protein of the lyase classthat catalyzes the equilibration of dissolved carbon dioxideand carbonic acid. It is strongly inhibited by sulfonamide-containing molecules. Using a similar series of injectionsas that used for the Van studies, three samples of CAB wereinjected onto the capillary column and their binding toligands 7 and 8 (Fig. 1b) examined. Figure 5 shows arepresentative series of electropherograms of CAB in acapillary filled with increasing concentrations of 7 at 200nm. Compound 7 is negatively charged and when bound toCAB shifts the migration time of the CAB–7 complex to agreater migration time. Table 3 lists the values for Kb forligands 7 and 8 binding to CAB. Negative peaks representthe dilution of ligand in the electrophoresis buffer uponbinding to CAB. The voids are common in ACE techniquesand are more pronounced when the ligand (or receptor) inthe running buffer is highly chromophoric.

Fig. 5 Representative set of electropherograms of CAB (filleddiamonds, squares, and triangles) in 192 mM glycine/25 mM Trisbuffer (pH 8.3) containing various concentrations of ligand 7 usingthe MIACE technique. The total analysis time in each experimentwas 6.0 min at 30 kV (current 7.5 μA) using a 40.5-cm (inlet todetector), 50-μm I.D. open, uncoated quartz capillary. MO (opencircles) and HHM (open triangles) were used as internal standards

Table 2 Experimental values of binding constants Kb (103M−1) of

ligands 1–6 to Van and Teic obtained by Eqs. (2) and (4)

Antibiotic Ligand Kb

Teic 1 144.0a

Van 2 15.3b

Teic 2 21.1c

Van 3 40.2Van 4 25.9Van 5 3.7d

Teic 5 20.7Van 6 43.0aPrevious estimates [4, 19]: Kb=220×10

3 M−1 (pH 8.3),Kb=21.8×10

3 M−1 (pH 7.5)bPrevious estimate [19]: Kb=174.5×10

3 M−1 (pH 7.5)cPrevious estimate [18]: Kb=185.1×10

3 M−1 (pH 7.5)dPrevious estimate [17, 19]: Kb=3.6–5.0×10

3 M−1 (pH 8.3)

Table 1 Experimental values ofbinding constants Kb (10

3M−1)of ligand 1 to Van obtained byEq. (2)

aPrevious estimates [19]:Kb=41.6×10

3 M−1 (pH 7.5)

Van peak Kb R2

1 21.4 0.9502 22.5 0.9603 23.0 0.9224 25.1 0.8905 19.6 0.871

Table 3 Experimental values of binding constants Kb (105M−1) of

ligands 7 and 8 to CAB obtained by Eqs. (2) and (4)

Ligand Kb

7 4.84a

8 4.47aPrevious estimates [14]: Kb=0.72–2.0×10

6 M−1 (pH 8.3)

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Conclusion

The present study demonstrates the ease of using multiple-injection ACE (MIACE) to estimate binding constantsbetween receptors and ligands. Vancomycin, teicoplanin,and carbonic anhydrase B were used as model systems andestimates of binding constants of these receptors to theirrespective ligands were derived. In this technique multipleinjections of sample containing receptor were subjected toan increasing concentration of ligand in the running buffer.Scatchard analysis was applied to the change in migrationtime upon formation of complex to afford multiple bindingconstants comparable to those obtained using other ACEtechniques and traditional assay methods. The advantagesof MIACE are several. One, smaller quantities of ligand areneeded to conduct the studies in comparison to other assaytechniques. Two, purification of the sample prior to injec-tion is not necessary as long as the component to beanalyzed can be separated from other species. Three, mul-tiple binding constants can be obtained in a series of ACEexperiments thus shortening the amount of time requiredto conduct the assay. Finally, the commercial availabilityof automated instrumentation, and high reproducibility ofdata, make it experimentally convenient. This work servesas a foundation for using MIACE to probe the binding ofother receptor–ligand combinations. Future work will focuson expanding the versatility of the technique by modifyingthe plug-plug injection sequence to other systems.

Acknowledgements The authors gratefully acknowledge finan-cial support for this research by grants from the National ScienceFoundation (CHE-0136724 and DMR-0351848), and the NationalInstitutes of Health (R15 AI055515–01). K.M. was a Partnership forResearch and Education in Materials (PREM) High School Fellow.

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