Tài liệu Báo cáo Y học: Short peptides are not reliable models of thermodynamic and kinetic properties of the N-terminal metal binding site in serum albumin doc

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Short peptides are not reliable models of thermodynamicand kinetic properties of the N-terminal metal binding sitein serum albuminMagdalena Sokolowska1, Artur Krezel1, Marcin Dyba1, Zbigniew Szewczuk1and Wojciech Bal1,21Faculty of Chemistry, University of Wroclaw, Poland;2Institute of Biochemistry and Biophysics, Polish Academy of Sciences,Warsaw, PolandA comparative study of thermodynamic and kinetic aspectsof Cu(II) and Ni(II) binding at the N-terminal b inding site ofhuman and bovine serum albumins (HSA and BSA,respectively) and short peptide analogues was performedusing potentiometry and spectroscopic t echniques. It wasfound that while qualitative aspects of i nteraction (spectraand structures o f complexes, o rder of reactions) c ould bereproduced, t he quantitative parameters (stability a nd rateconstants) could not. The N-terminal site in HSA is muchmore similar to BSA than to short p eptides reproducing theHSA sequence. A v ery strong influence of phosphate ions onthe kinetics of Ni(II) interaction was found. This studydemonstrates the limitations of short peptide modelling ofCu(II) and Ni(II) transport by albumins.Keywords: serum albumin; copper(II); nickel(II); bindingconstants; rate constants.Human s erum albumin (HSA) is t he most abundant proteinof blood serum, at concentration of 0 .63 mM(% 4%) [1].It is a v ersatile carrier protein, involved i n the transport ofhormones, vitamins, fatty acids, xenobiotics, drugs andmetal ions, including physiological Ca2+,Zn2+,Co2+andCu2+, as well as toxic Cd2+and Ni2+[1–3]. This variety offunctions is made possible by t he presence of many bindingsites on the surface of the HSA molecule, includinghydrophobic pockets of various sizes a nd shapes andcoordination domains equipped with sets of donor groupsappropriate for particular metals. Among the latter, theN-terminal binding site for Cu2+and N i2+ions has beencharacterized particularly w ell. It is composed of the firstthree amino-acid residues o f the HSA sequence, Asp-Ala-His, and the resulting square-planar complex exhibits aunique coordination mode with deprotonated amidenitrogens of Ala and His residues, in addition to theN-terminal amine and the His imidazole donor (theso-called 4N complex, s ee Fig. 1) [4–7]. Structural s tudieson various peptide analogues in the solid state [8–10] and insolution [11,12], as well as numerous spectroscopic worksconfirmed that such coordination style is a common featureof peptides having N-terminal sequences of the X-Y-Histype (reviewed in [13]). As such, it is shared by manymammalian a lbumins, which differ from HSA at positions 1and/or 2, but not 3 (e.g. bovine serum albumin, BSA,contains the s equence A sp-Thr-His) [ 14–17]. In a lbuminsfrom several species, i ncluding dog (DSA) and pig (PSA),the H is3 r esidue is replaced by Tyr. This, and an y othermutation r emoving His from position 3, results in a lack ofaffinity and specificity for Cu(II) and Ni(II) binding at theN-terminus [7,16,18,19].Recently, we have reported the existence o f the secondspecific binding site for Cu(II) in HSA and BSA, which alsoshares spectroscopic similarities with a PSA site [20]. W enamed it Ômultimetal binding siteÕ, because it can bindNi(II), Z n(II) and C d(II) with sim ilar affinities. B ased oninformation from113Cd NMR studies [21] and HSAcrystallography [2,22], t his site was located a t the interfaceof domains I a nd II of HSA and BSA, where His67 andHis247 are present on the protein surface, adjacent to eachother. This site is at a distance of % 16.5 A˚from Ser5, t hefirst N-terminal residue seen in electron density maps. Forsimplicity, the N-terminal site will be labelled Ôsite IÕ and themultimetal binding site Ôsite IIÕ throughout the text. Theanalysis of binding constants obtained from CD-monitoredmetal ion titrations indicated that site II may havephysiological relevance for Ni(II), Zn(II) and Cd(II). Thisfinding is of particular interest for the yet unrecognizedprocess of blood transport of toxic and carcinogenic nickel.It has been established that the Ni(II) complex at site Iprovides the a ntigenic moiety i n nickel allergy [23,24], butlittle is known about the redistribution of nickel from bloodCorrespondence to W. Bal, Faculty of Chemistry, University ofWroclaw, ul. F. Joliot-Curie 14, 50-383 Wroclaw, Poland.Fax: + 48 71 328 2348, Tel.: + 48 71 3757-281,E-mail: wbal@wchuwr.chem.uni.wroc.plAbbreviations:HSA,humanserumalbumin;BSA,bovineserumalbumin; 4N complex, c omplex with f o ur-nitrogen co ordination of th ecentral metal ion.Definitions of constants: b ¼ [MiHjLk]/([M]i[H]j[L]k), overallcomplex stability constant; *K ¼ b(MH-jL)/b(HnL), the equilibriumconstant of actual complex formation: M + HnL ¼ MH-jL+(n+j)H+cK ¼ [McL]/([M] [cL]), conditional affinity c onstant,wherecL contains all protonation forms at a given pH;iKM¼cK forthe metal binding at the i-th site of serum a lbumin,i ¼ 1 or 2, corresponding to site I or II, M is Cu(II) or Ni(II) [20];Kr¼2KCu/2KNi; relative affinity constant at site II; kobs¼ apparent1st order kinetic constant.(Received 11 July 2001, revised 16 November 2001, accepted 9 January2002)Eur. J. Biochem. 269, 1323–1331 (2002) Ó FEBS 2002to organs in which it can exert procarcinogenic lesions [25].In order to approach the issue of Cu (II) and Ni(II) exchangeby albumin, we characterized the binding parameters andperformed parallel kinetic studies using HSA and BSA andthree simple analogues of the N-terminal binding site. Thesewere: Asp-Ala-His-NH2(DAHam) and Asp-Ala-His-Lys-NH2(DAHKam), which represent the native HSAsequence and Val-Ile-H is-Asn ( VIHN), t he N-terminalpeptide o f another blood serum protein, d es-angiotensino-gen [11]. The structure of the Ni(II) complex of the lattercontains a specific steric shielding, resulting in a particularlyslow kinetics of Ni(II) dissociation. Somewhat surprisingly,we found that, despite the identical mode of coordination,important thermodynamic and kinetic p arameters of Cu(II)and Ni(II) interactions could not be reproduced quantita-tively by short peptides. The present paper presents theresults of our studies.MATERIALS AND METHODSMaterialsNiCl2and CuCl2were purchased from Fluka. HNO3,KNO3, EDT A, dimethylg lyoxime and ethanediol w ereobtained from A ldrich. Tris/HCl, mono- and disodiumphosphates were purchased from Merck. Homogeneous,high purity defatted HSA and BSA [6] a nd Val-Ile-His-Asn(VIHN) peptide were obtained from Sigma. Peptide Asp-Ala-His-NH2(DAHam)wasagiftofHenrykKozlowski,Faculty of Chemistry, University of Wroclaw. Stocksolutions of NiCl2and CuCl2were standardized gravimet-rically with dimethylglyoxime and complexometrically withEDTA, respectively. Concentrations of stock solutions ofHSA and BSA were estimated spectrophotometrically at279 n m [6] and by Cu(II) titrations (see be low). Purities ofboth peptides were determined by potentiometric titrationsto exceed 98%.Peptide synthesisThe N-Fmoc-protected amino acids and Fmoc Rinkamide MBHA resin were obtained from Nova Biochem(Calbiochem-Novabiochem AG, La¨ufelfingen, Switzer-land). Benzotriazol-1-yloxytris(dimethylamino)phospho-nium hexafluorophosphate (BOP) was purchased fromChem-Impex International (Chem-Impex International,Wood Dale, IL, USA). Trifluo roacetic acid, piperidine,N,N-dimethylformamide (DMF) and N,N-diisopropyleth-ylamine (DIPEA) were obtained f rom Riedel – de Hae¨n(Riedel-de Hae¨n GmbH, Seeize, Germany). Acetic anhy-dride (Ac2O) was obtained from POCh (POCh S.A.,Gliwice, Poland). Triisopropylsilane (TIS) was o btainedfrom Lancaster ( Lancaster Synthesis GmbH, Mu¨hlheimam Main, Germany). Acetonitrile (HPLC grade) wasobtainedfromJ.T.Baker(J.T.Baker,Deventer,theNetherlands).The peptide Asp -Ala-His-Lys-NH2was synthesized byFmoc strategy on solid support [26–28] using Rink a mideMBHA resin. Fmoc protection groups were removed by25% p iperidine i n DMF. The N-Fmoc-amino acids(3 equiv.) were co upled by BOP (3 equiv.)/DIPEA (6 equiv.)procedure [27]. Coupling reaction was monitored by Kaiser(ninhydrin) test [27,28]. After coupling reactions aceticanhydride (3 equiv.)/DIPEA (6 equiv.) in DMF was used f orcapping of unreacted peptides chains. C leavage w as effectedusing a mixture of trifluoroacetic acid, H2O, and TIS(v/v/v ¼ 95/2.5/2.5) over a period of 2.5 h , followed byprecipitation with diethyl ether [28]. The crude peptides werepurified by preparative HPLC on the Alltech Econosil C1810 U column (Alltech Associate, Inc., Deerfield, IL, USA),5-lm particle size , 2 2 · 250 mm, eluting with 0.1%trifluoroacetic acid/water at a flow rate of 7 mLÆmin)1withdetection at 223 nm. Fractions collected across the mainpeak were assessed by HPLC analysis on Beckman Ultra-sphere ODS C 18 column (Beckman Instruments, Inc.,Fullerton, CA, USA), 5-lm particle size, 4.6 · 250 mm,eluting with 0.1% trifluoroacetic acid/water (solvent A) and0.1% trifluoroacetic a cid/80% acetonitrile/water ( solventB), using a gradient of 0% B to 100% B over 60 min at flowrate of 1 mLÆmin)1and d etection at 223 nm. Correc tfractions were pooled and lyophilized to yield with solid ofpurity exceeding 99% as assessed by HPLC analysis of thefinal m aterials. I dentity and purity of peptide was confirmedby mass spectrometry, utilizing a Finnigan MAT TSQ 700(Finnigan MAT, San Jose, CA, USA) mass spectrometerequipped with a Finnigan electrospray ionization source.The m/z values found/calculated were 468.8/469.2(M + H)+and 234.9/235.1 (M + 2H)2+.PotentiometryPotentiometric titrations of VIHN, DAHKam, their com-plexes with Cu(II), as well as the DAHKam complexwith Ni(II) in the presence of 0.1MKNO3were performedat 25 °C over the pH range 3–11.5 (Molspin automatictitrator) with 0 .1MNaOH as titrant. Changes in pH weremonitored with a combined glass-Ag/AgCl electrode(Russell) calibrated daily in hydrogen ions concentrationsby HNO3titrations [29]. S ample v olumes o f 1.5 mL, withpeptide concentrations of 1 mMand peptide molar excessover metal ion of 1.1–1.5 were used. The titration data wereanalysed using theSUPERQUADprogram [30]. Standarddeviations computed bySUPERQUADrefer to random errorsonly.CD spectroscopyThe spectra were recorded at 25 °ConaJascoJ-715spectropolarimeter, over the range of 240–800 nm, using1 c m c uvettes. The spectra are expressed in terms ofFig. 1. Scheme of 4N coordination mode in XYH peptides, M is Cu(II)or Ni(II).1324 M. Sokolowska et al. (Eur. J. Biochem. 269) Ó FEBS 2002De ¼ el) er,whereeland erare molar absorptioncoefficients for left and right circularly polarized light,respectively. 1 mMpeptide solutions and peptide molarexcess over metal ion of 1.1 we re used for pH titrations,while 0.5 mMpeptide samples were used for kineticmeasurements. Concentrations of albumin samples were0.5 mMin protein, with varied metal ion amounts. Thealbumin samples for titrations and metal exchangekinetics measurements were kept at pH 7.4 (100 mMsodium phosphate buffer). The kinetics of metal bindingto peptides and their exchange was studied in 100 mMTris/HCl and in 100 mMphosphate buffers, both atpH 7.4.UV–Vis spectroscopyThe kinetics of Ni(II) binding to DAH-am a nd substitutionby Cu(II) in 100 mMphosphate buffer, pH 7.4 at 25 °Cwasstudied on a Beckman DU-650 spectrophotometer, usingmonitor wavelength of 420 nm, and sampling interval of5 s . For control purposes the spectra were also recorded inthe range of 300–900 nm before and after reaction. In aseparate experiment, a titration of DAHK-am with Ni(II)was performed, also monitored at 420 nm. All otherexperimental details were analogous to those used in CDspectroscopy.EPRThe X-band EPR spectra of Cu(II) complexes of VIHNand DAHKam were obtained at 77 K (liquid nitrogen) ona Bruker ESP-300 spectrometer, using Cu(II) concentra-tions of 3 mMand Cu(II)-to-peptide ratios of 1 : 1.Ethanediol aqueous solution (30% v/v) was used as solventfor these measurements to ensure homogeneity of t hefrozen samples.RESULTSComplexation of Cu(II) and Ni(II) by model peptidesand albuminsAmong the systems under s crutiny in this w ork, the Ni(II)complexes of VIHN [11] and the DAHam complexes ofCu(II) and Ni(II) [31] were studied previously. Tables 1 and2 thus present only the novel data: protonation constantsfor DAHKam and VIHN, and stability constants (log avalues) of Cu(II)-VIHN, Cu-DAHKam and Ni-DAHKamsystems. The parameters of CD and EPR spectra of allmajor complexes present at pH 7 .4 are provided i n Table 3.Table 1. Protonation constants (log b values) f or peptides at I =0.1M(KNO3) and 25 °C. Standard deviations on the l ast digits are given inparentheses.Species DAHKam VIHNHL 10.52(2) 7.92(2)H2L 18.05(2) 14.48(2)H3L 24.32(2) 18.37(3)H4L 27.16(3)Table 3. Parameters of CD and EPR spectra of 4N complexes of peptides and albumins at pH 7.4 and 25 °C.CompoundCD Ni(II) CD Cu(II) EPR Cu(II)k (nm) De (M)1Æcm)1) k (nm) De (M)1Æcm)1)Ai(Gs) giVIHN 475 ()1.33) 552 ()0.72) 206 2.18407 (+0.65) 477 (+0.34)271 (+1.35) 315 (+1.33)261 (+1.56) 275 ()2.50)DAHama475 ()1.66) 561 ()0.95) 205 2.18409 (+1.05) 485 (+0.53)263 (+1.32) 306 (+1.40)270 ()2.79)DAHKam 475 ()1.90) 567 ()0.46) 200 2.19410 (+1.61) 489 (+0.48)267 (+1.04) 308 (+0.72)270 ()1.99)HSAb476 ()1.38) 565 ()0.54) 207 2.18410 (+1.19) 486 (+0.49)307 (+0.96)BSAb479 ()1.79) 559 ()0.94) 200 2.18410 (+1.11) 480310(+0.40)(+1.42)aEPR data from [31].bEPR data from [20].Table 2. Stability constants (log b values) o f Ni(II) and Cu(II) com-plexes of peptides at I =0.1M(KNO3) and 25 °C. Standard devi-ations on the last digits are given in parentheses.Species DAHKam-Ni DAHKam-Cu VIHN-CuMH2L 21.48(3) 23.15(6)ML 10.04(3) 14.18(3)MH-1L 4.84(1) 9.88(2)MH-2L )5.21(1) )0.32(3) )1.15(1)Ó FEBS 2002 Peptides fail to model N-terminal albumin site (Eur. J. Biochem. 269) 1325The CD spectra for DAHam co mplexes at pH 7.4 werere-measured to assure full correspondence with kineticexperiments. Figure 2 presents potentiometric speciationdiagrams for Cu(II)-VIHN, Cu-DAHKam and Ni-DAH-Kam syste ms, with relative CD intensities of the d–d bandsof major 4N complexes overlaid (taken as Deextof the higherenergy component minus Deextof the lower-energy compo-nent). The e xcellent agreement between t hese two inde-pendent measures of complex formation confirms thevalidity of the results.CD spectra of albumins were found to be in goodagreement with previous determinations, p erformed in theabsence of buffers [20]. The application of 100 mMphos-phate buffer at pH 7.4 (which conserves native conforma-tions of the p roteins) for albumin studies resulted in weak,but noticeable competition for Ni(II) binding at site I andCu(II) binding at site II. No evidence of formation ofternary complexes was found. Also, no precipitation ofmetal phosphates o r hydroxides occurred. Titration curveswere obtained from the corresponding CD spectra, whichallowed for calcu lations of appropriate conditional a ffinityconstants. This is illustrated in Fig. 3 for Ni(II) binding atsite I of HSA. Because of t he slowness of Ni(II) binding atsite I (see below), but not at site II, the equilibration ofreaction at each point of Ni(II) titrations had to be a ssuredby recording the spectra periodically. Quantitation of sites Iand II (and thus of albumin concentrations) could beobtained f rom C u(II) titrations, as described in our previouspaper [20]. In agreement with previous reports [20,32], thedeficit of site I (25%) was found for HSA, but not for BSA.The binding constants for Ni(II) at site II were obtainedfrom Kr, relative constants, describing Cu(II)/Ni(II) com-petition at site II. For BSA this constant was m easured bythe method described previously [20], based on titratingCu(II) out of site II by Ni(II). This approach failed for HSA,which partially precipitated at higher excess o f Ni(II).Therefore, this constant was calculated from kinetic experi-ments (see below). The2KNivalue for BSA was obtainedwith site I occupied by Cu(II), and thus could be deriveddirectly from fitting the titration curves. The values of2KNiconstants were applied to calculate relative occupancies ofsites I and II in the course of Ni(II) titrations. Finally,ÔintrinsicÕ protein constants were calculated w ith the use ofliterature value s of protonation and stability constants forphosphate complexes [33] These constants a re presented inTable 4 .An analogous titration was performed for Ni(II) com-plexation by DAHKam, in 100 mMphosphate, pH 7.4,using a bsorption spectra. This t itration yielded a linearincrease of complex concentration up to the saturation, thusallowing for determination of ligand concentration, but n otfor stability constant calculations. This b ehaviour is indic-ative of a higher binding constant, making phosphatecompetition negligible.The kinetics of Ni(II) binding to model peptides andalbumins at pH 7.4 was also monitored by CD spectro-scopy. I n t hese experiments, the equimolar amounts ofNi(II) were added t o buffered p eptide or protein solutions inone portion, with subsequent periodical recording of theresulting CD spectra. The peptides were studied in both T risand phosphate buffers, to find out whether the buffercomponents would affect the reaction rate. The reactionendpoint was not affected, because Cu(II) and N i(II)binding capabilities of both buffers at pH 7.4 are almostidentical to each other: log values of conditional affinityconstants (cK) of Tris complexes with Cu(II) and Ni(II),Fig. 2. Speciation diagrams for VIHN-Cu(II) (A), DAHKam-Cu(II)(B) and DAHKam-Ni(II) (C), calculated for 0.5 mMconcentrations ofpeptides and metal ions. The intensities of CD bands of 4N complexes(constructed by adding intensities at extremes of d–d bands andnormalized to molar fractions) are overlapped as d symbols.Fig. 3. Titration of site I in HSA with Ni(II) ions at pH 7.4 in 100 mMphosphate buffer. d, experimental points constructed by addingintens it ies at extr e mes of d–d bands, 475 and 410 nm . Lines are fit tothe conditional binding constant of Ni(II) at site I.1326 M. Sokolowska et al. (Eur. J. Biochem. 269) Ó FEBS 2002calculated from d ata in [34], are 3.4 and 1 .9, respectively, v s.3.1 and 2.0 for analogous phosphate complexes [33].In all cases 1st order kinetic curves were seen. Table 5presents the corresponding constants kobs, obtained by least-square fitting of the curves generated usin g several reporterwavelengths, c orresponding to spectral extrema. Examplesof the spectra and kinetic plots are given for Ni(II) bindingto DAHKam and HSA (Fig. 4 ).Finally, the reaction of Ni(II) removal from site I by C u(II)was studied for peptides [saturated at N i(II)-to-peptide 1 : 1]and for albumins [in the presence of 1 .5-fold molar excess ofNi(II) over site I, to assure its saturation]. The total amountsof Cu(II) and Ni(II) were matched in these measurements. Ina s eparate experiment, HSA saturated with Cu(II) at bothsites was the source of Cu(II) competing for DAHKamsaturated w ith N i(II). The spectra and kinetic plots for HSAreaction are s hown in F ig. 5 .DISCUSSIONComplex formation by model peptidesPotentiometric titrations and parallel CD and EPR spec-troscopic measurements confirm that m ajor complexesformed by peptides studied are typical 4N co mplexes ofthe structure presented in Fig. 1 . For VIHN and DAHamTable 4. Binding constants (log v alues) for Cu(II) and Ni(II) complexes of albumins i n 0.1Mphosphate buffer, pH 7.4, at 25 °C. Standard deviationson the last digit are g iven in parentheses.Albumin log1KNilog2KNialog2KCulog Krlog(1KNi/2KNi)HSA 6.8(3) 4.9(3) 7.1(2) 2.18(5) 1.9(3)BSA 6.69(8) 4.60(5) 6.20(3) 1.63(5) 2.09(8)aDerived from Krdetermined experimentally using1KNi.Table 5. Values of apparent 1 st order k inetic constants kobs(s)1) for Ni(II) binding a nd Ni(II) fi Cu(II) exchange for model peptides and albumins in100 mMTris and phosphate buffers at 25 °C. Standard deviations on the last digits are given in parentheses.Compoundkobs(Ni + HnL fi NiH-jL) kobs(NiH-jL+CufiCuH-jL + Ni)Tris Phosphate Tris PhosphateVIHN 3.18(7) · 10)41.17(3) · 10)37(3) · 10)72.1(2) · 10)6DAHam 1.72(5) · 10)33.2(2) · 10)21.17(3) · 10)61.90(3) · 10)3DAHKam 5.8(1) · 10)32.1(1) · 10)29.2(8) · 10)73.0(1) · 10)5BSA 2.56(7) · 10)37.5(3) · 10)5HSA 2.7(1) · 10)31.57(8) · 10)4HSA fi DAHKam 3.2(2) · 10)5Fig. 4. Kinetics of Ni(II) binding to DAHKam and HSA at pH 7.4 in 100 mMphosphate buffer. Left panel, kinetic plots (d,experimentalpointsconstructed by adding intensities at extremes of d–d bands, 47 5 and 410 nm, lines are fits to 1st order kinetics). Right p anel, the original CD spectraof Ni(II)-DAHKam (top) and Ni(II)-HSA (bottom).Ó FEBS 2002 Peptides fail to model N-terminal albumin site (Eur. J. Biochem. 269) 1327they are represented by the MH-2L formula, where M isCu(II) or Ni(II). For DAHK there a re two such complexes,MH-1L and MH-2L, differing by the protonation state ofthe lysine amine, which is not involved in coordination. ForVIHN only the 4N species were detected, while potentio-metric titrations indicated the presence of minor complexesMH2L a nd ML for DAHKam. The actual existence of suchcomplexes in XYH peptides i s controversial [13,35], e.g. noCD signature could be found for them. As indicated byFig. 2, these complexes, even i f existing at low pH, are notpresent at pH 7.4, and therefore they were not taken underconsideration for kinetic experiments.Spectroscopic data p resented in Table 3 (positions of CDspectral extrema for Cu(II) and Ni(II) complexes, and EPRparameters for Cu(II) species) indicate that 4N complexes ofall three peptides are very similar to each other. Inparticular, the parameters for VIHN complexes do no tdeviate systematically from those of DAHam andDAHKam. This means that the side chain carboxylate ofAsp1 does not have a direct effect on metal coordination(in agreement with previous ob servations [6,7]). A slightredshift of d –d bands accompanied by a subtle decrease ofdelocalization of the unpaired d electron of t he Cu(II) ion inthe DAHKam complex, c ompared to DAHam may be dueto a tiny deviation from tetragonal symmetry caused by aninteraction b etween the p rotonated Lys side chain and theHis ring, observed previously in NMR studies of the Ni(II)complex of HSA [6].Due to different protonation patterns, the stabilityconstants of particular complexes of model peptidescannot be compared d irectly. There are two ways ofcircumventing this obstacle. One, allowing for compari-sons of complexes with similar coordination modes anddifferent p rotonation stoichiometries, uses t he values of*K, the equilibrium constant of the actual complexformation reaction:M þ HnL ¼ MHÀjL þðnþjÞHþThis constant represents the overall ability of ligand L toform a given complex.The other m ethod is to calculate the conditional affinityconstant at a given pH value,cK, corresponding to thefollowing formal reaction, which ignores ligand protona-tion:M þcL ¼ McLwherecL is t otal ligand concentration. This con stant isuseful for comparing stabilities of metal complexes withdissimilar or not fully characterized ligands, such asproteins, for which the accurate protonation informationis unavailable. Such comparisons are, however, limited to aparticular pH value.Both sets of constants are given in Table 6 for our modelpeptides and for related compounds. The cases of highestand lowest affinities were se lected from literature data. Thebinding affinities for t he model peptides a re in the middle ofthe range of values f or both Cu(II) and Ni(II). Note t hat t hevariation of side chain substituents can result in changes ofcomplex stabilities by up to six orders of m agnitude, withoutaffecting the binding mode.Kozlowski et al. have recently proposed to correlate thestabilities of 4N complexes of Xaa-Yaa-His peptides,expressed using *K constants, with the average basicitiesof the nitrogen donors of the peptide [37]. The constantsmeasured in this work fall, however, below the correlationline p roposed by them. This indicates that, while thebasicities of nitrogen donors, partially dictated by sidechains, i s an important factor in complex stability, theouter sphere (steric) interactions also need to beconsidered.Comparison of Cu(II) and Ni(II) binding betweenmodel peptides and albuminsAffinity for Ni(II) at site I can be compared betweenalbumins on one hand and DAHam and DAHKam on theother. Much higher values were f ound for t he complexes ofmodel p eptides. This fact was confirmed by an a ttempt totitrate DAHK-am with Ni(II) in 100 mMphosphate,analogously to albumins. The titration curve was linear,Fig. 5. Kinetics of Ni(II) substitution at site I ofHSA by C u(II) at pH 7.4 in 100 mMphosphatebuffer. Left panel, kinetic p lots of the loss ofNi(II) complex (h, De at 410 nm), formationof Cu(II) complex (s, De at 307 nm), andbuffering of Cu(II) at site II (d, De at 690 nm).Right panel, the original CD spectra.The arrows indicate directions of changesat particular wavelengths.1328 M. Sokolowska et al. (Eur. J. Biochem. 269) Ó FEBS 2002indicating that the Ni(II) was bound to DAHK-am sostrongly that competition from phosphate was negligible.ThecK values for Ni(II) complexes of HSA and BSA arestill within the range provided by XYH peptides, but at itslower end (Tables 4 and 6). No direct measurements ofCu(II) affinities at site I have been reported so far, butestimates based on equilibrium dialysis and other indirectapproaches, reviewed in [20], yield the log1KCuvalue of12–13, confirming the trend found for Ni(II). We can onlyspeculate on the reason of these differences, which might b edue to different basicities of nitrogen donors at the proteinsurface, limited accessibility of the binding site due toshielding from the bulk of the protein, or some conform-ational interactions. The metal-free DAHK sequence inHSA has not been visualized in electron density maps,apparently due to its mobility in the crystals [1,22]. This doesnot necessarily exclude interactions of some kind betweenthe site I complex and other parts of the protein in solution,which are in fact suggested by CD spectra (see below).The comparison of CD sp ectra of complexes also pointstoward slight differences in the conformation of the chelaterings. The characteris tic alternate pattern of the d–d bandsin the C D s pectra is dictated by the c onformation o f the six-membered chelate ring involving the His residue donors(Fig. 1 ). This c onclusion is a direct consequence of thepresence of the same kind of spectrum for 4N complexes ofGGH, where the a carbon of the His residue is the solesource of chirality [10]. However, while positions of thecomponent d– d bands and of CT transitions are relativelyconstant, their absolute and relative in tensities depend quitestrongly on the n onbonding substituents in positions 1, 2,and even 4 (Table 3). Moreover, the comparison with thespectra of albumin c omplexes clearly indicates the influenc eof the whole protein, which can only be t ransferred via thelimitation of conformational freedom of the complexmoiety. The CD spectra of HSA complexes are intermediatebetween those of DAHam and DAHKam, suggesting thatthe conformation of the chelate system in the protein is alsointermediate between these two models.The Cu(II) stabilities at site II were measured directly, bytaking advantage from the presence of weakly competingphosphate ions. The Cu(II)/Ni(II) competition at site II wasalso studied. These experiments yielded binding valuesclearly lower from those obtained previously in the absenceof buffer [20]. The2KCuvalue decreased by % 0.5 log units,while the Krvalues increased by 1–1.5 log units (with Krvalue for HSA still distinctly higher from that for BSA).This translates i nto a hundredfold decrease of Ni(II) affinityat site II in 100 mMphosphate buffer. It is possible thatclustered histidines (His67 and His247, presumably provi-ding metal binding at site II and the neighbouring His242)bind phosphate ions, thereby providing another level ofcompetition for metal ion binding.Kinetics of Ni(II) binding and Cu(II)/Ni(II) exchangeThe data presented in Table 5 demonstrate that the processof Ni(II) binding has a uniform character f or modelpeptides and for albumins. In all cases the apparent 1storder kinetics was found for this bimolecular reaction. Thesame reaction order was seen previously for the reversereaction of acid decomposition of complexes, studied indetail for the Ni(II) complex of GGH [36,39]. The reasonfor this is the common slow step of the rearrangement ofNi(II) ion, between the high spin octahedral and the lowspin square planar forms. The latter is present in the 4Ncomplex, w hile the former in a ll other substrates/products ineither case [13,40].VIHN formed the most sluggish c omplex in both buffers,due to the additional step of side-chain folding [11]. TheDAHKam complex exhibited the highest rate of formationin Tris, while DAHam reacted faster in phosphate. Thissuggests an assistant role of the Lys side chain in Ni(II)anchoring to DAHKam in Tris and its nonparticipation inphosphate, likely due to the blocking by phosphate ions,which would thereby compete with Ni(II). All the kobsvalues for peptides were increased i n the phosphate buffer.The increase was t he most distinct f or DAHam. Themechanism of c atalysis of acid decomposition of nickelamine complexes by various compounds, including phos-phates, was s tudied in detail [41]. In line with electrostaticconsiderations presented there, this rate enhancement islikely due to the facilitated anchoring of n eutral NiHPO4tonitrogen donors of the peptide, compared to a positivelycharged Ni(II)–Tris complex.The rates of Ni(II) complexation by albumins in phos-phate are 10-fold lower from t hose for DAHam a ndDAHKam. This indicates that the metal-free DAHKTable 6. Logarithmic values o f *K andcK constants for model peptides and other XYH pep tide analogues, representing the high-end and the l ow-end ofaffinity series. The values of c onstants were calculated from appropriate s tability constants, using f ormulae provided in the Materials a nd methodssection.PeptideLog *KaLogcKCu(II) Ni(II) Cu NiVIHNb)15.63 )19.75 13.0 8.8DAHamc)14.79 )20.02 13.7 8.5DAHKam )14.44 )19.48 13.8 8.7GGHd)16.43 )21.81 12.4 7.0GGHiste)17.14 )22.65 11.7 6.2HmSHmSHamf)11.05 )16.45 16.0 10.6HP21)15(RTHG-)g)13.13 )19.29 14.5 8.5alog *K ¼ log b(MH-jL) – log b(HnL), j and n ¼ 2, except for DAHKam, where j ¼ 1 and n ¼ 3.bNi(II) data from ref [11].c[31].d[36].eglycylglycylhistamine, [9].fa-hydroxymetylseryl-a-hydroxymetylserylhistidinamide; Cu(II) data from [37]; Ni(II) data from [38].gN-Terminal 15-peptide of human protamine 2, [35].Ó FEBS 2002 Peptides fail to model N-terminal albumin site (Eur. J. Biochem. 269) 1329sequence in albumin is partially sh ielded from solution bythe rest of the protein. There is no correlation between thecomplex stability and the rate of its formation.The Ni(II) for Cu(II) exchange rates f or peptides are o fthe order of 10)6s)1in Tris (again somewhat slower forVIHN, in accordance with the steric shielding of Ni(II)-Nbonds [11,42]). These rates are markedly slower from thatfound for pure acid decomposition of the Ni(II)-GGHcomplex given in [39] (kd¼ 8 · 10)5s)1). This, inconjunction with 1st order kinetics, suggests that thereaction of Ni(II) for Cu(II) exchange in T ris proceeds v iaNi(II) complex dissociation (slow step), followed by therapid formation of the Cu(II) species, and there is littleassistance from the buffer components. There is no accel-eration for DAHKam, compared to DAHam, in accord-ance with the lack of interaction between the Lys a mine andNi(II), once the 4N complex is formed.The situation is quite different for phosphate solutions.The rate for DAHKam i s now much lower from those ofalbumins, and the reaction of DAHam is much faster.The reaction rates for albumins and DAHam are higherfrom the value for pure acid GGH dissociation. Thespread of rate constant values for the exchange r eaction inphosphate is more than three orders of magnitude,compared to just one order for Ni(II) binding. Thesefacts indicate that phosphate ions play a very specific r olein Ni(II) dissociation and Cu(II) binding, different foreach peptide. Note that the participation o f phosphate ismore likely to be of outer sphere character, because thepresence of isodichroic points b etween the s pectra ofsubstrate (NiH-jL) and product (CuH-jL) in reactionmixtures points against a substantial formation of aternary complex with mixed coordination by either metalion.The major difference between peptide a nd albuminexperiments i s in t he form of existence of Cu(II). While itwas p resent initially a weak phosphate complex in peptideexperiments, it was bound at site II in quasi-steady state inalbumin experiments (Fig. 5). This fact is confirmed bycalculations of the occupancy of site II by Cu(II) and Ni(II)in the course of reaction, which yielded values of KrforBSA identical to that obtained from direct titrations(log Kr¼ 1.65 ± 025 vs. 1.63 ± 0.05, respectively).Despite this fact, the values of kobsfor HSA and BSA,very similar t o e ach other, are intermediate between thosefor DAHam and DAHKam. This shows that the mechan-ism o f m etal binding at site I i n a lbumin cannot be modelledreliably by short peptides. The relatively fast rate ofexchange of Ni(II) for Cu(II) suggests the presence ofintramolecular Cu(II) transfer phenomenon in albumin. Itseems that a n unstructured (metal-free) site I cannot reactaccording to this putative mechanism, because the Ni(II)binding reaction [which was in f act Ni(II) transfer from thekinetically labile site II to site I] was tenfold slower for thealbumins than for both DAHam and DAHKam (Table 5).The possibility of an intermolecular interaction was exclu-ded by the experiment in which the target molecule was t heexternal DAHKam Ni(II) complex, with site I of HSAsaturated with Cu(II). The rate constant measured in thisexperiment was identical, within the experimental error,with that obtained in the absence of albumin, and five timeslower from that obtained with HSA alone. The similarity o frates between HSA and BSA suggests that this process maybe common for a lbumins possessing site I . However, arather vague theory that the spectroscopic and kinetic (butnot even thermodynamic) properties of site I in HSA areequally well (poorly) modelled by DAHam and DAHKampeptides is as much as can be inferred from studies usingpeptide models for site I.CONCLUSIONSOur s tudy demonstrated that the N-terminal s ite i n HSA ismuch more similar to that of BSA than to short peptidesreproducing the HSA sequence. The albumins bind Cu(II)and N i(II) distinctly weaker than the model p eptides. A verystrong influence of phosphate ions on Cu(II) and Ni(II)binding at site II, as well as on kinetics of Ni(II) binding andsubstitution by Cu(II) at site I was found, but no structure–activity relationships between the binding sequence andreaction rate could be e stablished. Our results clearlydemonstrate that short peptides cannot be reliably usedfor i nterpretation and modelling of C u(II) and Ni(II)transport by albumins. On the other hand, the directthermodynamic and kinetic characterization of Ni(II)binding at site I in HSA and BSA was obtained. Thesedata can be very useful in further studies of the toxicolo-gically relevant Ni(II)-albumin c omplex. It would be alsointeresting to follow the indirect effects of physiologicallyrelevant Ca2+binding (which occurs at separate sites in theprotein [20,21]) on metal ion binding at site II.ACKNOWLEDGEMENTSThe authors wish to thank Prof Henryk Kozlowski and Dr PiotrMlynarz for their kind g ift of peptide DAHam a nd for sharing the dataon its complexes prior to publication.REFERENCES1. Carter, D.C. & Ho, J.X. (1994) Structure of s erum a lbumin. Adv.Protein. Chem. 45, 153–203.2. He, X M. & Carter, D.C. (1992) Atomic structure and c hemistryof human serum albumin. Nature 358, 209–214.3. Peters,T. Jr (1985) Serum albumin. Prot. Chem. 37, 161–245.4. Glennon, J.D. & Sarkar, B. (1982) Nickel (II) transport in humanblood serum. Biochem. J. 203, 15–23.5. Laussac, J.P. & Sarkar, B. (1984) Characterization of the copper(II) and nickel (II)-tran sport site o f human seru m albumin. S tudiesof copper (II) and nickel (II) bin ding to peptide 1–24 of humanserum albumin by13Cand1H NMR spectroscopy. Biochemistry23, 2832–2838.6. Sadler, P.J., Tucker, A. & Viles, J.H. (1994) Involvement of alysine residue in the N-terminus Ni2+and Cu2+binding site ofserum albumins. Comparison with Co2+,Cd2+,Al3+. Eur. J.Biochem. 220, 193–200.7. Valko, M., Morris, H., Mazu´ r, M., Telser, J., M cInnes, E.J.L. &Mabbs, F.E. (1999) High-affinity binding site for copper (II) inhuman and dog serum albumins (an EPR study). J. Phys. Chem. B103, 5591–5597.8. Camerman, N., Camerman, A . & Sarkar, B . (1976) Moleculardesign to mimic the copper (II) transport site of human albumin.The crystal and molecular structure of copper(II)-glycylglycyl-L-histidine-N-methyl amide mo noaquo c omplex. Can. J. Chem.54, 1309–1316.9. Gajda, T., Henry, B., Aubry, A. & Delpuech, J J. (1996) Protonand metal ion interactions with glycylglycylhistamine, a serumalbumin mimicking pseudopeptide. Inorg. Chem. 35, 586–593.1330 M. Sokolowska et al. (Eur. J. Biochem. 269) Ó FEBS 200210. Bal, W., Djuran, M .I., Margerum, D .W., Gr ay, E.T. Jr,Mazid, M.A., Tom, R.T., Nieboer, E. & Sadler, P.J. (1994)Dioxygen-induced decarboxylation and hydroxylation of[NiII(glycyl-glycyl-L-histidine)] occurs via NiIII: X-ray crystalstructure o f [NiII(glycyl-glycyl-a-hydroxy-D,L-histamine)]3H2O.J. Chem. Soc., Chem. Comm. 1889–1890.11. Bal, W ., Chmurny, G.N., Hilton, B.D., Sadler, P.J. & Tucker, A.(1996) Axial hydrophobic fence in highly stable Ni(II) co mp lex ofdes-angiotensinogen N-terminal peptide. J. Am. Chem. 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Inorg.Biochem. 28 , 431–439.18. Rakhit, G. & S arkar, B. (1981) Electron spin resonance study ofthe c opper (II) complexes of human and dog serum albu mins andsome peptide analogs. J. Inorg. Biochem. 15, 233–241.19. Predki, P.F., Harford, C., Brar, P. & Sarkar, B. ( 1992) Furthercharacterization of the N-terminus copper (II) and nickel(II) -binding motif o f proteins. Studies of metal binding to c hickenserum albumin and the native sequence peptide. Biochem. J. 287,211–215.20. Bal, W ., Christodoulou, J., Sadler, P.J. & Tucker, A. (1998) Multi-metal binding site of serum albumin. J. Inorg. Biochem. 70, 3 3–39.21. Sadler, P.J. & Viles, J.H. (1996)1Hand113Cd NMR investigationsof Cd2+and Zn2+binding sites on serum albumin: competitionwith Ca2+,Ni2+,Cu2+and Zn2+. Inorg. Chem. 35, 4490–4496.22. Sugio, S., Kashima, A., Mochizuki, S., Noda, M. & Kobayashi,K. (1999) Crystal structure of human serum albumin at 2.5 Aresolution. Protein Eng. 12 , 439–446.23. Dolovich, J., Evans, S.L. & Nieboer, E. (1984) Occupationalasthma from nick el sensitivity. I. Human serum albumin i n theantigenic determinant. Br.J.Ind.Med.41, 51–55.24. Patel, S.U., Sadler, P.J., Tucker, A. & Viles, J.H. (1993 ) Directdetection of albumin in human blood plasma by1H NMR spec-troscopy. Complexation of nickel (II). J. Am. Chem. Soc. 115,9285–9286.25. Kasprzak, K.S., Jaruga, P., Zastawny, T.H., North, S.L., Riggs,C.W., Olinski, R. & D izdaroglu, M. (1997) Oxidative DNA basedamage and i ts repair in kidneys and livers of n ickel (II) -treatedmale F344 rats. Carcinogenesis 18, 271–277.26. Meienhofer, J., Waki, M., Heimer, E.P., Lambros, T.J.,Makofske, R.C. & Chang, C.D. (1979) So lid phase synthesiswithout repetitive hydrolysis. Preparation o f leucylalanyl-glycyl-valine using 9-fluorenylmethyloxy- carbonylamino acids. Int.J. Peptide Protein Res. 13, 35–42.27. Fields, G.B., ed. (1997) Solid-Phase Peptide S ynthesis. MethodsEnzymol. 289. Academic Press, New York.28. Chan, W.C. & White, P.D., eds. (2000) Fmoc Solid P hase PeptideSynthesis. A P ractical Approach. Oxford University Press, N ewYork.29. Irving, H., Miles, M.G. & Pettit, L.D. (1967) A study of someproblems in determining the stoichiometric proton dissociationconstants of complexes by potentiometric titrations using a glasselectrode. Anal. Chim. Acta 38, 475–488.30. Gans, P., Sabatini, A. & Vacca, A. (1985) SUPERQUAD: animproved general program for computation of formation con-stants from potentiometric data. J. Chem. Soc. Dalton Trans.1195–1199.31. Mlynarz, P., Valensin, D., Kociolek, K., Zabrocki, J ., Olejnik, J. &Kozlowski, H. (2002) Impact of the peptid e seq uence on thecoordination abilities of albumin-like tripeptides t owards Cu2+,Ni2+and Zn2+ions. Potential alb umine-like p eptide c helators.New. J. Chem. 26, in press.32. Chan, B., Dodsworth, N., Woodrow, J., Tucker, A. & Harris, R.(1995) Site-specific N-terminal auto -degradation o f human s erumalbumin. Eur. J. Biochem. 227, 524–528.33. Banerjea, D., Kaden, T. & Sigel, H. (1981) Enhanced stability ofternary complexes in solution through the participation ofheteroaromatic N bases. Comparison of the coordinationtendency of pyridine, imidazole, ammonia, acetate, and hydrogenphosphate toward metal ion nitrilotriacetate complexes. Inorg.Chem. 20, 2586–2590.34. Fischer, B., Haring, U ., Tribolet, R. & Sigel, H. (1979) Metal ion/buffer interactions. Stability of binary and ternary complexescontaining 2-amino-2(hydroxymethyl)-1,3-propanediol (Tris) andadenosine 5¢-triphosphate (ATP). Eur. J. Biochem. 94, 523–530.35. Bal, W., Jezowska-Bojczuk, M. & Kasprzak, K .S. (1997) Bindingof nickel (II) a nd copper (II) to the N-terminal sequence of humanprotamine HP2. Chem. Res. Toxicol. 10, 906–914.36. Hay, R.W., Hassan, M.M. & Quan, C.Y. (1993) Kinetic andthermodynamic studies of the c opper (II) an d nickel (II) com-plexes of glycylglycyl-L-histidine. J. Inorg. Biochem. 52, 17–25.37. Mlynarz, P., Bal, W., Kowalik- Jankowska, T., Stasiak, M.,Leplawy, M.T. & Kozlowski, H. (1999) Introduction ofa-hydroxymethylserine residues in the peptide sequence results inthe strongest peptidic copper (II) ch elator known to d ate. J. Chem.Soc. Dalton Trans. 109–110.38. Mlynarz, P., Gaggelli, N., Panek, J., Stasiak, M ., Valensin, G.,Kowalik-Jankowska, T., Leplawy, M.L., Latajka, Z. &Kozlowski, H. (2000) How the a-hydroxymethylserine residuestabilizes oligopeptide complexes with nickel (II) and copper (II)ions. J. Chem. Soc. Dalton Trans. 1033–1038.39. Bannister, C.E., Raycheba, J.M.T. & Margerum, D.W. (1982)Kinetics of nickel (II) glycylglycyl-L-histidine reactions with acidsand triethylamine. Inorg. Chem. 21, 1106–1112.40. Pettit, L.D., Pyburn, S., Bal, W., Kozlowski, H. & Bataille, M.(1990) A study of the comparative dono r p roperties o f t he term inalamino and imidazole nitrogens in peptides. J. Chem. Soc. DaltonTrans. 3565–3570.41. Read, R.A. & Margerum, D.W. (1982) Kinetics of hydrogenphosphate catalysed chelate ring opening in (ethylenediamine)nickel (II). Inorg. Chem. 22, 3447–3451.42. Raycheba, J.M.T. & Margerum, D.W. (1980) Effect of non-coordinative axial blocking on the stability and kinetic behavior ofternary 2 ,6-lutidine-nickel (II) -oligopeptide complex. Inorg.Chem. 19, 837–843.Ó FEBS 2002 Peptides fail to model N-terminal albumin site (Eur. J. Biochem. 269) 1331 . Short peptides are not reliable models of thermodynamic and kinetic properties of the N-terminal metal binding site in serum albumin Magdalena. Academy of Sciences,Warsaw, PolandA comparative study of thermodynamic and kinetic aspects of Cu(II) and Ni(II) binding at the N-terminal b inding site of human
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