A Robust Method for Determining DNA Binding Constants Using Capillary Zone Electrophoresis* 1

A Robust Method for Determining DNA Binding Constants Using Capillary Zone Electrophoresis* 1
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  See discussions, stats, and author profiles for this publication at: A Robust Method for Determining DNABinding Constants Using Capillary ZoneElectrophoresis  Article   in  Analytical Biochemistry · November 1998 DOI: 10.1006/abio.1998.2791 · Source: PubMed CITATIONS 37 READS 36 2 authors , including:Lenore M MartinUniversity of Rhode Island 22   PUBLICATIONS   85   CITATIONS   SEE PROFILE All content following this page was uploaded by Lenore M Martin on 23 January 2017. The user has requested enhancement of the downloaded file.  A Robust M ethod for Determining DNA Binding ConstantsUsing Capillary Zone Electrophoresis Chunze Li and Lenore M. Martin Department of Biomedical Sciences, College of Pharmacy, University of Rhode Island,41 Lower College Road, Kingston, Rhode Island 02881-0809  Received J anuary 13, 1998 Capillaryzoneelectrophoresis(CZE or CE)withon-lineUV detectionwasutilizedtomeasurethebindingconstants between purified calf thymus DNA and alibraryofdesignedtetrapeptideswhichhadbeencon-structed using unnatural amino acids with thiazolering side chains. Mixtures containing a constantamountofatetrapeptide,theneutral marker (mesityloxide),and varyingconcentrations of DNA werepre-pared and equilibrated at 8°C for 12 h. CE was thenutilized to separate unbound tetrapeptides from theDNA–peptidecomplex.TheUVabsorbanceofthepeak representing unbound tetrapeptide decreased incre-mentallyasaresultofincreasingtheconcentrationof DNA in the equilibrium mixture. The absorbance of the peak corresponding to the unbound tetrapeptidewasobtaineddirectlyfromtheelectropherogramandused in the calculation of the DNA–peptide bindingconstants.Thebindingconstantfor eachtetrapeptideto calf thymus DNA was obtained from the negativeslope of a Scatchard plot and a comparison of thebinding constants for different peptides showed thatthetetrapeptidesin thelibrary haveDNA-bindingaf-finitiesrangingfrom10 2 to10 6 M  1 .  ©1998 Academic Press Key Wor ds:   capillary electrophoresis; DNA; bindingconstants;peptides;ethidiumbromide;thiazoles. To enhance our ability to screen combinatorial peptidelibraries for DNA-binding affinity, we found it valuable todevelop a fast and efficient method for the determinationof DNA binding constants using capillary electrophoresis(CE). 1 In this paper, we introduce a new CE method,distinct from affinity capillary electrophoresis (ACE),which we used to screen for DNA-binding activity in alibrary containing 15 synthetic tetrapeptides. We haveadapted an experimental procedure used for the quanti-fication of a stable DNA–ligand complex so that the sta-bility of the complex during the analysis is no longer arequirement for our assay.Under our conditions, the macromolecule (DNA) comi-grates with the electroendoosmotic flow. If the DNA–ligand complex dissociates during a run, the intensity ofthe ligand peak still reflects the concentration of freeligand at time zero, since the ligand migrates toward thedetector faster than does either the DNA or the DNA–ligand complex. We focus on the analysis of the ligand tobe screened, in a background of macromolecule, and nobuffer additives are required. In our experiments, weobserved that the peak areas of cationic tetrapeptidesdecreased as a result of increasing the amount of purifiedcalf thymus DNA present in the initial equilibrium mix-tures. By switching the polarity of the electrodes duringelectrophoresis, the method should also work for nega-tively charged ligands.ACE represented the adaptation to CE of a techniqueused to study macromolecule–ligand interactions ingels. Using slab gel electrophoretic methods such as“electrophoretic retardation” (1) or “affinity electro-phoresis” (2), many DNA-binding proteins have beensuccessfully identified. Since the charge-to-mass ratioof a DNA–ligand complex is generally quite differentfrom that of the unbound DNA and ligand separately,the complex migrates separately from the free ligands.Compared with traditional gel electrophoresis, ACEexhibited short analysis time, low-volume sample re-quirements, a high efficiency, and convenience in quan-tification using the on-column UV detector (3). Thediverse applications of CE in the analysis of a verywide range of structurally different compounds, includ-ing small biomolecules, makes it a more practical ap-proach for the study of molecular interactions thanslab gel electrophoresis. 1 Abbreviations used: CE, capillary electrophoresis; ACE, affinitycapillary electrophoresis; BOC,  tert  -butyloxycarbonyl; EOF, electroen-doosmotic flow; bs, binding sites; bp, basepair; HF, hydrogen fluoride.72  0003-2697/98 $25.00Copyright © 1998 by Academic PressAll rights of reproduction in any form reserved. ANALYTICAL BIOCHEMISTRY  263, 72–78 (1998) ARTICLE NO . AB982791  There are two methods currently used for ACE, eachbased on the stability of the complex. If a complex isfairly stable during the time required for electrophore-sis (typically 10 min or more), then assessment of bind-ing constants may be performed as follows (i.e., if thehalf-life for dissociation,  t  1/2  ln 2/ k   1 , then more thanone-half of the complex will dissociate during the run if k   1  0.07 s  1 ): Mixtures with different ratios of DNAand ligand are loaded into the capillary and run usingcapillary electrophoresis to separate the free ligandfrom the DNA–ligand complex (4). As the intensity ofthe ligand peak decreases, that of the complex peakgenerally increases. The binding constants may be cal-culated from either the decreases in the ligand peak orthe increases in the complex peak intensities. Thismethod is ideal for strong binding (  10 8 M  1 ) but isnot generally applicable.If the complex dissociates very quickly comparedwith the time required for electrophoresis, binding con-stants will not be accurately measured via the firstmethod. Instead, the intensity of the DNA-ligand com-plex peak will decrease due to dissociation as the com-plex migrates through the capillary. Hummel andDryer pioneered a method, later widely adapted for CE(5–12), which prevents the dissociation of the complexduring the separation by including the macromoleculein the running buffer. Weaker binding constants maybe determined using this method, based on the changein the migration time of the sample as the concentra-tion of macromolecule in the buffer is increased. Thismethod is widely used in CE, but requires a largequantity of the macromolecule to obtain adequate data.In addition, incorporating the macromolecule as a com-ponent of the buffer may lead to unexpected effects onthe electrophoretic separation.Our method of screening for DNA-binding activity in apeptide library is different from the two methods de-scribed above because it does not depend upon knowledgeof binding kinetics nor does it depend upon a detailedknowledge of the nature of the peptide–DNA complex. MATERIALS AND METHODS Apparatus  A Dionex capillary electrophoresis instrument (CES-1)with both positive and negative voltage sources and anon-column UV and fluorescence detector was purchasedfrom Dionex Corp. (CA). Fused silica capillaries of 50   mi.d. were obtained from Polymicro Technologies Inc.(Phoenix, AZ). The capillary was initially conditioned bywashing it with 1 N sodium hydroxide for 30 min, fol-lowed by a 15-min wash with 0.1 N sodium hydroxide.Then it was extensively rinsed with deionized water andrunning buffer before the capillary was put into use.Samples were injected using a gravity injection methodwith a relative height difference of 50 mm between theinlet and outlet vials for 10 s. The electropherogramswere recorded and analyzed using a Model 1022 PersonalIntegrator (Perkin–Elmer Corp., CT). Water (18.3 M  )was obtained from a Millipore MilliQ water-purificationsystem (Millipore Corp., MA) (Scheme 1). Chemicals  Tris–HCl, Tris base, mesityl oxide (used as a neutralmarker in CE), and calf thymus DNA (double-stranded,10 mg/mL, sonicated to 580–830 bp, purified by phenol/chloroform extraction and ethanol precipitation)were ob-tained from Fluka Chemie AG (Switzerland). Potassiumchloride was from Sigma (St. Louis, MO), and magne-sium chloride was from Fisher Scientific (NJ ). Argon gas(prepurified grade) was from Linde Specialty gases andnitrogen gas was from Corp Brothers, Inc. (RI). The tet-rapeptides used were synthesized in this lab using thesolid-phase method (13, 14) on a 4-methylbenzhy-drylamine resin (15) and the unnatural amino acids  D -3-(4-thiazolyl)alanine  1 and  L -3-(4-thiazolyl)alanine  2 (ob-tained from Synthetech, Inc., Albany, OR) (Fig. 1) on asimultaneous multiple peptide synthesizer (16). Peptidesyntheses were carried out using the  tert  -butyloxycar-bonyl (BOC)protecting group strategy; BOC deprotectionand amino acid coupling reactions were monitored by theninhydrin reaction (17). The high hydrogen fluoride (HF)method of cleavage of the product peptides from the solidsupport (18) was performed without adding any scaven-ger. The purity of the peptide products was confirmed to SCHEME 1.  The design of the experiment. The sample is injectedat the right end of a 50-  m capillary using gravity (hydrodymanic)injection, and then the capillary end is placed into the anode buffersolution and the voltage is applied. All samples in an uncoatedcapillary (negatively charged surface) migrate toward the detectorand the anode (apparent flow, app) due to the electroendoosmoticflow (eof). The eof is usually greater than the electrophoretic mobility(ep) at pHs greater than 4. The order of migration seen in theelectropherogram (view from the detector) will then correspond tothe order shown, left to right: cations, then neutral species, followedby anions. 73 DETERMINING DNA BINDING CONSTANTS  be greater than 90% by RP-HPLC (C18, Vydac, 5   m,0.1% trifluoroacetic acid in H 2 O/CH 3 CN) and capillaryzone electrophoresis (15 mM Tris (1.6 g Tris–HCl/L and1.6 g Tris base/L), 10 mM MgCl 2 , 1 mM dithiothreitol, 30mM KCl, pH 7.9, at 20°C), and products were identifiedby  1 H NMR analysis. The crude products were used forCE binding studies without further purification. Tet-rapeptide No. 4, LDDD-NH 2 , was not available in suffi-cient quantities for testing. The sequences of the tet-rapeptides used to test our CE method are shown inTable 1. Procedure  Mixtures containing different ratios of the tetrapep-tides to purified calf thymus DNA (42%GC) (19) wereprepared in a buffer of 1.5 mM Tris and Tris–HCl, 3.0mM KCl, and 1.0 mM MgCl 2  at pH 7.9 and were equil-ibrated at 8°C (to prevent bacterial growth) for 12 h.Each sample in a series contained the same amount ofa given tetrapeptide (6.2    10  7 to 5.7    10  8 M) anda neutral marker (mesityl oxide, 0.01%, v/v), and theconcentration of DNA was varied (from 1.75   10  4 to8.76    10  3 M). In a typical experiment the stocksolutions were prepared as follows: the peptides weredissolved in water at a concentration of 1 mg/mL, neu-tral marker was mixed with water (1  L in 1 mL), DNAstock solutions were prepared ranging from 10 mg/mLto 0.1 mg/mL, and the running buffer was diluted (1  Linto 7   L). Samples were prepared by mixing severalmicroliters of the peptide stock solution with eitherdilute buffer or a known concentration of DNA dis-solved in dilute buffer to achieve identical peptide con-centrations in the incubation mixture.CE was utilized to separate the unbound tetrapep-tides from the DNA–tetrapeptide complexes at roomtemperature (  20°C) based on their different electro-phoretic mobilities (Figs. 2 and 3). The conditions forelectrophoretic separation of each peptide from itsDNA–peptide complex, using an uncoated capillary,were as follows: length to the detector ( L D ), 70.51 cm;total capillary length ( L T ), 75.81 cm; applied voltage,30 kV; measured current, 80   A. The running buffer(15 mM Tris and Tris–HCl, pH 7.9 at 20°C, 10 mMMgCl 2 , 30 mM KCl), was chosen for comparison withCE studies of the binding of transcription factors withDNA (20, 21). The concentration of the buffer used inthe incubation mixture was 10 times less than that ofthe running buffer to facilitate sample stacking duringelectrophoresis. Sample stacking sharpens analytebands by slowing the mobility at the front of the sam-ple band when the sample makes the transition intothe running buffer of higher ionic strength. All buffersolutions were degassed via sonication and vacuum,and were filtered through 0.22-  m-pore-size sterile fil-ters prior to analysis. Since the sample size was small(10   l), the temperature at which the binding con-stants were sampled was effectively 20°C. The concen-tration of unbound tetrapeptide in each sample wasquantified by UV absorbance at maximum absorbance(238 nm) in the detector of the CE. The binding con-stant of each tetrapeptide was determined by plottingthe results of a series of electrophoretic runs. Eachsample was analyzed in duplicate to check the repro-ducibility of the method. FIG.1.  The chemical structures of the two amino acid monomersused to construct the enantiomeric peptide library. TABLE 1 The Stoichiometric Binding Constants of the 15 TetrapeptidesCalculated from the Scatchard Plots a  ID No.Peptidesequence  K  a (1) (M  1 ) b  K  a (2) (M  1 ) c  12 LLLL-NH2 2.1  10 6 2.1  10 6 9 DLLL-NH2 4.2  10 5 2.0  10 3 11 LLDL-NH2 1.9  10 5 1.0  10 3 8 LDLD-NH2 5.5  10 4 1.3  10 3 15 LLLD-NH2 5.2  10 4 7 LDLL-NH2 2.8  10 4 2 DDDL-NH2 2.4  10 4 6 DDLD-NH2 2.3  10 4 3 LDDL-NH2 2.0  10 4 14 DLLD-NH2 1.8  10 4 10 DLDL-NH2 1.5  10 4 13 DLDD-NH2 1.4  10 4 16 LLDD-NH2 1.4  10 4 1 DDDD-NH2 2.5  10 3 5 DDLL-NH2 1.7  10 3 Note.  The peptides are ordered so that  K  a (1) ranges from highestaffinity to lowest. Peptides are separated into three different cate-gories, having high, medium, and low binding constants, respec-tively. a  Nonlinear Scatchard plots were not fitted. Instead, the data wasobserved to be biphasic and was assumed to represent two hypothet-ical binding sites having high and low affinities. b  K  a (1) is the stoichiometric equilibrium binding constant nearsaturation. c  K  a (2) is the binding constant at high [DNA]/[peptide] ratios. 74  LI AND MARTIN  The method was tested using ethidium bromide(1.20    10  3 M) and calf thymus DNA at concentra-tions ranging from 1.25    10  4 to 1.25    10  2 M, forcomparison with the results from our peptides. RESULTS By convention (22), the net migration of all species ina positive polarity capillary zone electrophoresis exper-iment is toward the cathode in an uncoated capillary.Our peptides were positively charged at pH 7.9, and sothe elution of the peptides past the detector was quitefast, because it represented the sum of the electro-phoretic mobility toward the cathode and electroen-doosmotic flow (EOF). High EOF is observed in un-coated capillaries at buffer pHs greater than 4, due tothe unmasking of negative charges on the wall of thecapillary. At pH   4, the velocity of the EOF becomesgreater than most electrophoretic migration velocitiesand dominates the separation. Neutral molecules donot have any electrophoretic mobility and thus migrateat the same velocity as the EOF. Anions such as DNAand the peptide–DNA complexes actually electro-phoretically migrate against the EOF (toward the an-ode and the injector), but in our experiments they weredragged along by the EOF toward the cathode as well.The molar concentrations of unbound tetrapeptidesin equilibrium mixtures with DNA were readily deter-mined using the UV absorbance intensity of the pep-tide peaks in the electropherograms. The amount ofunbound tetrapeptide remaining after equilibrationwith DNA in a given mixture was calculated using theheight of the peak. In CE, peak heights are more uni-versally quantifiable than peak areas due to the vary-ing speeds (mobilities) at which different analytes passthrough the detector. DNA molarity is expressed perbase pair using the extinction coefficients determinedby LePecq and Paoletti for calf thymus DNA (23). Theextinction coefficient of the tetrapeptides was deter-mined to be 6495 cm  1 M  1 at the wavelength of max-imum absorptivity (238 nm). The binding constantswere then determined from the best-fit lines on ourplots (Fig. 4) (24–26). Data Analysis T  p  DNA bs º ComplexEquation [1] shows the ratio of the concentration of apeptide–DNA complex to the concentration of unboundbinding sites (bs) on duplex DNA, and the concentra-tion of unbound tetrapeptide at equilibrium. K  a  [Complex][DNA bs ] f  [ T  p ] f [1] FIG. 2.  Electropherogram showing an injection of DNA alone(above), peptide 7 alone (middle), and DNA in a complex with peptide7 (below) monitored at 260 nm (resulting in decreased sensitivity).The CE runs were performed at 30 kV with a field strength of 396V/cm at pH 7.85 in 15 mM Tris–HCl, 30 mM KCl, 10 mM MgCl 2 . FIG. 3.  Electropherogram showing the peptide 12 peak intensitydecrease which occurs after equilibration with purified calf thymusDNA, plotting the absorbance at 238 nm (the maximum absorbance ofthe peptide) as a function of migration time to the detector. The tops ofthe peptide peaks are aligned to facilitate comparison of peak heights.The CE runs were performed at 30 kV with a field strength of 396 V/cmat pH 7.85 in 15 mM Tris–HCl, 30 mM KCl, 10 mM MgCl 2 . 75 DETERMINING DNA BINDING CONSTANTS
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