Use of Capillary Zone Electrophoresis To Evaluate the Binding of Anionic Carbohydrates to Synthetic Peptides Derived from Human Serum Amyloid P Component Niels H. H. Heegaard’ and Frank A. Robey Peptide and Immunochemistry Unit, Laboratory of Cellular Development and Oncology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892
Caplllary zone electrophorerlr wa8 uwd to atudy Interactlonr betweenantonic carbohydrate@ and aynthetk peptklwderked from the heparln-blndlng reglon of human wrum amylold P component. The method Involver quantltatlon of unbound peptide@ after a chargedependent electrophoretlcwparatlon of the peptldr-carbohydrate mlxture. The concentratlonrof free peptlde were determined by extrapolating the obtalned peak area@of the peptide Inthe preaencoof ligandto a atandard curve. Wrroclatlon conttantr In the lo4 M range were det~,anddMer~InMndinga?fhttyofvMkwrpeptkle “ c a t l o n r were Illuatrated. The a m y requlrer minute amount@of material (@ample volume la 7-15 nL), and a@long a@the reactant@are soluble at the c h w n condltlona, no modlfkatlonr or apeclal characterktlcs of the lnteractlng mdecukr are needed for their ldentlflcatlon. I t should be poaalble to u w electrophoretic wparatlon In caplllarkr to evaluate the bIndhrg of peptides to any Ilgand a@long ab the dMerencoaIn chargdmam ratio betweenfree and complexed peptld. are of a sutfkknt magnltude as In the peptlde-heparin blndlng demonatrated here.
INTRODUCTION Synthetic peptides which represent active sites of parent proteins are useful for studies of structure-function relationships, as experimental toola, and as potential drugs. A full understanding of peptide-ligand interactions includes the characterization of the binding phenomenon itaelf, but this is often unrealized because, to the best of our knowledge, there are no simple binding assays for peptides in solution. Binding studies often attempt to quantitate bound or free ligand after reaching an equilibrium for the ligand-receptor interaction.’ The majority of the methods require labeling or special characteristics of the interacting compounds, or they depend on secondary reagents for the quantitation. The resolution and sensitivity offered for the direct evaluation of synthetic peptides by capillary zone electrophoresis (CZE)2 encouraged us to investigate this method for binding studies where bound and free peptides can be separated on the basis of net charge differences. The application is illustrated here with peptides which are derived from the heparin-binding region of human serum amyloid P component (SAP).e4 The heparin-binding characteristics and specificity in CZE of various derivatives of the Addreas correspondence to this author at Bldg. 30,Room 211,NIH, Bethesda, MD 20892. Phone: (301)496-4779. Fax: (301)402-0823. (1)Klotz, I. M. In Protein function. Apractical approach; Creighton, T. E., Ed.; IRL Press: Oxford, England, 1989; pp 25-54. (2)Jorgenson, J. W.;Lukacs, K. D. Science 1983,222,266-272. (3)Dhawan, S.;Fields, R. L.; Robey, F. A. Biochem. Biophys. Res. Commun. 1990,171,1284-1290. (4)Loveless, R.W.; Floyd-OSullivan,G.; Raynes, J. G.; Yuen, C.-T.; Feizi, T. EMBO J. 1992,11, 813-819. 0003-2700/92/0364-2479$03.00/0
active peptide were investigated. The binding assay is dependent on an electrophoretic separation of free peptide from complexes between ligand and peptide. The method should be generally applicable to studies of noncovalent interactions for any peptide-ligand system where the chargel mass ratio of the peptide-ligand complex is different from that of the free peptide.
EXPERIMENTAL SECTION Chemicals. Heparin (sodium salt) and hydrogen peroxide (30%)were from Fisher Scientific (Fair Lawn, NJ). Chemicals for HPLC and 2-propanol were from J. T. Baker, Inc. (Phillipsburg, NJ). All chemicals for peptide synthesis came from Applied Biosystems, Inc. (Foster City, CA) except that Bachem, Inc. (Torrance, CA) supplied N-a-BOC-Ng-p-tosyl-D-ar~nine. Aldrich (Milwaukee,WI) provided the 2,2’-dithiodipyridine, and Tris and ammonium sulfate were from Schwarz/Mann Biotech (Cleveland, OH); acetic acid, calcium chloride, formic acid, and sodium chloride came from Mallinckrodt (Paris, KY) and Sigma (St.Louis, MO) provided a-D(+)-mannose1-phosphate,sodium salt. Synthesis of Peptides. Peptides (see Table I) were synthesized as C-terminal amides on an Applied Biosystems 430A automated solid-phase peptide synthesizer using t-BOC chemi s t r ~ Deprotection .~ and release of peptides from the resin were accomplished by treatment with anhydrous hydrogen fluoride containing approximately 10% (v/v) anisole for 2 h at 0 O C . The crude peptides were purified by preparative HPLC on a Vydac (Hesperia, CA) CIScolumn and lyophilized. The amino acid composition of the purified peptides was confirmed by amino acid analysis after acid hydrolysis in 6 N HC1 at 120 OC for 18 h. Dimerization of the cysteine-containingpeptide (SAP-1)took place in dilute TBS (1.5 mM NaCl, 0.05 mM Tris/HCl, pH 7.4) by oxidation of 2 mg/mL with H202 as previously de~cribed.~ S-pyridylationswas performed by reacting 0.5 mg/mL of peptide with 5 mM 2,2’-dithiodipyridine in 0.1 % (v/v) aqueous trifluoroacetic acid for 1 h at room temperature. The pyridylated peptide, which had a longer retention time on the CIScolumn than the unmodified SAP-1peptide and displayed an absorbance at 280 nm was purified by preparative HPLC and lyophilized. Apparatus. CZE was performed with an Applied Biosystems 270A capillary electrophoresis system equipped with a fusedsilica capillary (totallength 72 cm, detector at 50 cm, internal diameter 50 pm) (Applied Biosystems or Polymicro Techniques, Phoenix, AZ). The system was connected to a Spectra-Physics SP4400integrator. The electrophoresisbuffer was 2OmMsodium citrate, pH 2.5 (from Applied Biosystems). CZE Procedure. Electrophoresistook place for 15 min at 20 kV (the current during runs was between 17 and 20 pA and the temperature between 30.1 and 30.4 “C according to the display of the instrument). The anode, unless otherwise indicated, was at the sample application end of the capillary. Vacuum injections for 2.5 or 5 s were used. A 5-5 injection corresponds to a sample (5)Merrifield, R. B. J.Am. Chem. SOC.1963,85,2149-2154. (6) Carlsson, J.; Svenson, A.; RydBn, L. In Solid phase methods in protein sequence analysis; Previero, A., Coletti-Previero,M.-A., Ma.; Elsevier/North-HollandBiomedicalPress: Amsterdam, 1977;pp 29-37.
Q 1992 American Chemlcal Society
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, 1992
Table I. Peptides. Used for Binding Assays name structure SAP-dimer S-pyr-SAP-1
&
Glu-Lys-Pre Leu-Gln-Asn-Phe-Thr-Leu-Cys-Phe-Arg
I
SAP-1 dimer
(Glu-Lys-Pr~Leu.Gln-Asn-Phe-Thr-Leu-Cys-Phe.Arg)*
SAP-l(D) dimer
(Glu-Lys-Pr~Leu-Gln-Asn-Phe-Thr-Leu-Cys-Phe-Arg(D))~
scrambled SAP-1 dimer
(Thr-Arg-Leu-Phe-Pro-Lys-Glu-Cys-Leu-Asn.Gln-Pha)~
Without heparin
IB
Scrambled SAP-dimer
All synthesized as C-terminal amides. Molecular weight of all dimers is 2982 while S-pyr-SAP-1is 1601.
(7) Scatchard, G. Ann. N.Y. Acad. Sci. 1949, 51, 660-672.
SAP-1 dimer
7-
0
volume of approximately 18 nL on the basis of the Poiseuille equation and the pressure specifications given by the manufacturer. Detection was at 200 nm (range, 0.004; rise time, 0.05). Between runs the capillary was washed for 1 min with 0.1 M NaOH followed by 3 min with electrophoresisbuffer from a buffer vial different from the one used for running buffer. Electrophoresis buffer was changed after every eight runs or after peak tailing was observed. Because the temperature regulation was not completely accurate and because of differences in the number of ions in the system, there were current fluctuations which resulted in small variations in migration times. The values of the areas were corrected for these small differences,and standard curves of peak areas versus peptide concentration were produced for each peptide. The correlation coefficients for these standard curves all were above 0.99. The time for equilibration of the binding reaction between heparin and the peptides was estimated by incubating 0.2 mgi mL SAP-1 dimer with 1.2 X 10-7 M heparin (final volume: 55 pL). CZE was then performed at 1, 3 , 4 , 17, and 21 h. After 3 h, the integrated area of the peptide peak was constant. Accordingly, all samples were incubated overnight before being analyzed. All incubations took place at ambient temperature. Different peptide concentrations in a volume of 100 p L were mixed with 10 pL of 10 mg/mL mannose 1-phosphate in dilute TBS corresponding to a final monosaccharide concentration of 3.5 mM. The solutions were then analyzed by CZE as described above. For experiments involving heparin, different concentrations of peptides in a volume of 50 pL were incubated with either 5 pL of dilute TBS or a constant amount of heparin in dilute TBS (5 p L of 0.1 mg/mL heparin corresponding to a final concentration of 6.0 X M heparin). The areas of peaks from samples incubated in the presence of ligand were taken as a measure of free peptide. The amount of bound peptide was then determined by subtraction from the total amount of peptide obtained from the control samples incubated without ligand. The integrated, migration time-corrected areas were converted to molar concentrations by means of the standard curves for each peptide. Bindingcurveswere constructed by plotting bound peptide as a function of total peptide' and according to the Scatchard equation.7 Each data point of the binding curves corresponds to the mean f the standard deviation of the results of three to four incubations. The possibility of dissociation during the vacuum-injection step was tested by allowing 40 p L of the SAP-1 dimer (0.25 mg/ mL in dilute TBS) to react with 10 p L of heparin (0.1 mg/mL in dilute TBS) or with 10 pL of dilute TBS overnight. Then, electrophoresis was performed using 1,2.5,5, and 10-s injection times and the ratios between the integrated peptide-peak areas from samples with heparin and the controls were calculated. It was found to be constant (0.205 f 0.04). When CZE was performed at a neutral pH, the peptides of this study interacted strongly with the capillary walls and could not be recovered consistently. The use of an electrokinetic injection mode instead of vacuum injection was discouraged by the fact that the ratios between bound and free peptide for the
,
'I
With heparin
I
I[ I
I
1 1
0 5 10 15 min Figure 1. CZE demonstration of the binding specificity of the regular and the scrambled version of the heparin-binding peptide. A mixture of the SAP-1 dimer and the scrambled SAP-1 dimer (cf. Table I) was incubatedin(A) the absence or (B) the presenceof heparin. Scrambled SAP-1 dimer (25 pL, 0.1 mg/mL in dilute TBS) and SAP-1 dimer (25 pL, 0.1 mg/mL in dilute TBS) were incubated with 10 pL of dilute TBS (A) or 10 gL of heparin (0.1 mg/mL in dilute TBS) (B) overnight at room temperature before being analyzed by CZE, as described in the Experimental Section.
incubations were highly dependent on the injection time (not shown).
same
RESULTS AND DISCUSSION The synthetic peptides used in this study (Table I) are derived from an active peptide from SAP3 which inhibits the binding of heparin to the intact p r ~ t e i n .The ~ cysteinecontaining syntheticpeptides were protected against oxidative degradation either by dimerization (SAP-1 dimer, S A P - ~ ( D ) dimer, and scrambled SAP-1dimer) or by S-pyridylation of the monomer (8-pyr-SAP-1). An S-carboxymethylated SAP-1 peptide had previously been found not to bind heparin in solid-phase assays (unpublished observations). The only difference between the three dimers used in this study was the use of D-arginine for the SAP-UD)dimer and a randomly scrambled sequence for the scrambled SAP-1 dimer using the amino acid residues of the SAP-1 dimer. CZE was applied to examine how these structural differences influenced the ability of the peptides to bind heparin. Since heparin is a polyanionic compound it was expected that the migration of a heparin-peptide complex in an electric field would be different from that of the free peptide. In the presence of heparin, the SAP-1 dimer peak disappeared completely from a 15-min CZE analysis while the scrambled peptide was only slightly affected (Figure 1). Heparin alone was not detected a t 200 nm after 1hr a t 20 kV, (after reversing the polarity, Le., with the cathode at the sample application end, a broad heparin peak appeared after approximately 6 min at 20 kV). At pH 2.5, the electroendosmotic flow toward the cathode is minimal and therefore, heparin, which is anionic a t this pH due to its sulfation, does not pass the detector. This also appears to happen to a heparin-peptide complex. At lower concentrations of heparin, the peptide peak did not disappear completely and a proportionality between the amount of heparin and the decrease in the size of the SAP-1 dimer peak could be demonstrated (Figure 2). It was clear from the pilot experiments shown in Figures 1 and 2 that different peptides of the same overall charge had different affinities for heparin. As described in the Exper-
ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, 1992 2481
4.200 i o 5
-
3.150
-
i 2.100lo5-
1.050
I 3.3 pM Hep.
0
*
-
l e
I
Total S-pyridyl SAP.1
0 '
1o
.~
to
-~
0 001
M
BoundlFree
1
a
4.0 pM Hep 0.3 . I
0
15 min
Flgurr 2. Effect of Increasing amounts of heparin on the SAP-1 dimer peak area in CZE. This figure Illustratesthat the amount of free peptlde Is dependent on the amount of llgand added. The SAP-1 dimer was Incubated with different amounts of heparln (given on the figure) and then analyzed by CZE. A 20-pL aliquot of SAP-1 dimer (1 mg/mL in dilute TBS) was incubated with the stated concentrations of 30 pL of heparin In dilute TBS overnight at room temperature and analyzed by CZE. Small differences In migration tlmes were due to small current varlatlons (see the Experlmental Section).
imental Section above, experiments using different injection times (from 1 to 10 s) of samples of peptides and peptides incubated with heparin did not lead to different ratios of free to total peptide and it was therefore concluded that the vacuum-injectionstep did not interfere with the equilibrium. It cannot be ruled out completely that the electrophoresis itself disturbed the equilibria but since the peak representing free peptide was always sharp and symmetrical, dissociation of peptide from the complexes did not appear to take place during electrophoresis. Thus, it seemed possible to use CZE to determine the amount of free peptide at different ratios between peptide and ligand and, therefore, to use the technique for quantitative binding studies. The approach was first tested with a simple charged monosaccharide, mannose 1-phosphate,which had been found to inhibit the binding of SAP to the neoglycolipid from Hansenula holstii pentamannose phosphate4 and which interacted with the SAP-1peptide in experiments (not shown) similar to the one in Figure 2. Fixed amounts of mannose 1-phosphate were incubated with varying amounts of the S-pyridylatedSAP-1monomer under the conditionsdescribed in the Experimental Section. The data yielded a saturable binding curve (Figure 3) and a linear Scatchard plot,' suggesting one class of binding sites with a Ka of 9.5 X M. This experiment demonstrated the feasibility of using CZE for binding studies in a simple system where a monomeric peptide is interacting with a monosaccharide. A more complex situation is illustrated in Figure 4 where the results of the CZE assays of the interaction of S-pyrSAP-1 with heparin are shown. The experiments were conducted in an approximately 100-1500 molar excess of peptides over heparin (average M , = 150008). Again, the binding was saturable and the Scatchard plot (Figure 4) yielded a biphasic curve which suggested the presence of (8) Hardingham, T.E.;Fosang, A. J. FASEB
J. 1992,6, 861-870.
0.2 :
:
0.1
a
i,,,
0
1.125
0
Bound (x105)
I
2.250
3.375
4500
M
Flgure 3. (A, Top) CZEhrived binding curve for the Interaction of Spyr-SAP-1 with mannose 1-phosphate. (6, Bottom) Scatchard plot of the same data with a line fltted to the points uslng linear regression. Various concentrations of peptlde were Incubated In a total volume of 110 pL with a flnal concentratlon of 3.5 mM mannose l-phosphate. CZE analyses were performed as outilned in the Experimental Section except that the voltage was 25 kV for 10 mln. The amount of bound peptkle was then plotted as a function of the total amount available by convertingthe integrated peak areas to concentrations on the basis of the Spyr-SAP-1 standard curve (not shown). BoundlFree
2.5
1
ji
1.5
1
1 i
a
0.5
* *
a
* 1.o
Flgure
2.0
3.0
Bound (x105)
a 4.0
50
60
70
M
Scatchard plot for the lnteractlon of the S-pyrldylated SAP-1 with heparin. Varlous concentrations of peptides were Incubated in a total volume of 55 pL wRh a constant amount of heparin (final concentratlon: 6 X lo-' M). Analyses were performed as outlined in the Experimental Section. After the CZE, the Intqrated peak areas were converted to concentrations by using the values derived from the standard curves of each peptide (not shown). 4.
CZEderived
various binding sites on heparin with different affinities that may be subject to cooperative effects upon peptide binding.'
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, 1992
The dissociation constant for a first set of binding site(s) was estimated on the basis of the Scatchard plot (visual decomposition of the curve into two straight lines) to be just below 1 X 10-5 M, while the affinity of a second set of binding site(s) was 6 X M. For the SAP-1dimer interacting with heparin (not shown) the system showed saturability while, for the S A P - ~ ( Ddimer, ) no saturation plateau was reached within the experimental range (not shown). Due to the complexity of the possible binding sites in heparin and the potential for cooperative effects in the binding of ligands to a dimeric peptide, an estimation of the binding constant was not possible. However, the results suggested that the affinity of the S A P - ~ ( Ddimer ) for heparin is at least 10-100-fold less than the heparin affinity of the SAP-1dimer. This indicates that L-arginine plays an important role in the binding of SAP-1 to heparin. The results indicate that heparin subunits8 contain more than one sugar entity capable of binding the SAP-1 peptide and that this happens with different affinities and/or cooperative effects. Additionally, the CZE analysis of various peptide modifications demonstrated specific peptide reactivities. Alterations such as the use of a D form of the C-terminal arginyl residue or scrambling the sequence in otherwise identical peptides decreased the affinity considerably and indicated the specificity of the interactions and of the CZE procedure itself. The binding of the peptides to heparin is partly chargedependent with the positively charged arginyl residue interacting with the strongly anionic multiple sulfate groups of heparin. The low ionic strength conditions of the assayswould tend to amplify such electrostatic interactions and to weaken hydrophobic forces. However, sequence-specificinteractions of heparin subunits with the Phe-Thr-Leu-Cys-Phe domain also appear to be important in the overall binding because binding of the scrambled peptide to heparin was very weak. A heterogeneous compound such as heparin which has 24 different possible disaccharide structures, with different patterns of sulfation and varying chain lengths8is not ideally suited for binding site characterization because of the complex binding isotherms that are obtained. The peptides presented (9)Gordon, M.J.; Huang, X.; Pentoney, S. L., Jr.; Zare, R. N. Science 1988,242, 224-228. (10)Burgi, D.S.;Chien, R.-L. Anal. Chem. 1991,63,2042-2047. (11)Pentoney, S.L., Jr.; Zare, R. N.; Quint, J. F. Anal. Chem. 1989, 61.1642-1647. (12)Steward, M. W.;Petty, R. E. Immunology 1972,22, 747-756. (13)Velick, S.F.;Parker, C. W.; Eisen, H. N. h o c . Natl. Acad. Sci. U.S.A. 1960,46, 1470-1482. (14)Stone, M. J.; Metzger, H. J. BioE. Chem. 1969,243, 5049-5055. (15)Eisen, H.N.;Karush, F. J. Am. Chem. SOC.1949,71,363. (16)Heegaard, N. H. H.; Bjerrum, 0. J. Anal. Biochem. 1991,195, 319-326. (17)Grossman, P. D.;Colburn, J. C.; Lauer, H. K.; e t al. Anal. Chem. 1989,61,1186-1194. (18)Compton, B.J.; Ogrady, E. A.Anal. Chem. 1991,63,2597-2602.
here also bind DNA and some simple charged carbohydrates (Figure 3 and unpublished observations). The investigation of these interactions by CZE is in progress. The lowest concentration of peptide detectable in this study was ca. lo+ M, which is the same value as reported by others.9 This constitutes a limitation of the present methodology because,for strong interactions, the concentration of unbound peptide might be too low to be detected: binding is saturated even at the lowest concentrations employed, and the receptor concentration has to be lowered so much that the amount of peptide bound is below accurate levels of detection for the CZE system. Preconcentration steps (e.g. based on isotachophoresislo)might be useful to improve the detection limit as long as it can be shown that these procedures do not interfere with the established equilibria. Detection systems which are more sensitivell than UV detection might also be necessary for executing binding studies with very low concentrations of free peptide. Advantages of the use of CZE for binding studies are the exceedingly small sample volumes required (10-18 nL, corresponding to picomole amounts for the peptides in this study), the short time (10-15 min) for each analysis, and the direct quantitation which does not depend on secondary reagents or special characteristics of the interacting molecules. In alternative techniques, quantitation is achieved on the basis of selective precipitation,12 fluorescence shifts after binding,13 size-dependent fractionation in gel filtration14 or dialysis techniques,15 or sizelcharge-dependent separation in affinity electrophoresis procedures.16 None of these techniques offers the resolution and sensitivity of CZE, and most of them require some kind of labeling of the interacting compounds. As mentioned above, none can measure the binding of small molecules such as S-pyridylated SAP-1 and mannose 1-phosphate (Figure 3). It had been suggested previously that CZE might be a convenient method for evaluating noncovalent binding interactions of proteins in their native state" and the present study shows this to be true for the interactions of peptides with their ligands. Affinity measurements by CZE should be widely applicable because the separation in CZE is a function of both molecular size and charge.9J7J8 Thus, any molecular complex would be endowed with electrophoretic features distinguishable from those of the unbound molecules and should, therefore, be specifically measurable.
ACKNOWLEDGMENT The economical support from the Danish Medical Research Council (Grant Nos. 12-9573and 12-1070) to N.H.H.H. has been greatly appreciated. RECEIVED for review May 19, 1992. Accepted July 30, 1992.
