High-Affinity Binding Site for Copper(II) in Human and Dog Serum

J. Phys. Chem. B 1999, 103, 5591-5597

5591

High-Affinity Binding Site for Copper(II) in Human and Dog Serum Albumins (an EPR Study) M. Valko,* H. Morris, and M. Mazu´ r Department of Physical Chemistry, SloVak Technical UniVersity, SK-812 37 BratislaVa, SloVakia, and School of Pharmacy and Chemistry, LiVerpool John Moores UniVersity, Byrom Street, LiVerpool L3 3AF, United Kingdom

J. Telser Chemistry Program, RooseVelt UniVersity, 430 South Michigan AVe., Chicago, Illinois 60605-1394

E. J. L. McInnes and F. E. Mabbs EPSRC Multi-Frequency EPR Centre, Department of Chemistry, UniVersity of Manchester, Manchester M13 9PL, United Kingdom ReceiVed: December 7, 1998; In Final Form: April 27, 1999

Spectroscopic studies have been performed to investigate the high-affinity binding site for copper in human serum albumin (HSA) and dog serum albumin (DSA). A new approach based on exposure to albumin of the copper in the form of a well-characterized histidine (his) chelate has been adopted. This technique has been shown to minimize interaction at the lower affinity sites. The analysis of the S-band EPR spectrum of [Cu(his)2] at pH 7.3 revealed the major component is a complex formed with two histidines in a histamine-like coordination. Detailed analysis of S-band and X-band EPR and optical spectra of [Cu(II)-HSA] revealed that copper forms a complex with HSA involving R-NH2 terminal, two deprotonated peptide nitrogens (NH of Ala2, and NH of His3), and the imidazole nitrogen of His3 in a square planar arrangement. The spectral data were found to be independent of pH in the range 4.5-9.0 and did not confirm axial Asp1 carboxylate chelation. The EPR study of [Cu(II)-DSA] complex at pH 7.3 confirmed the presence of two bonded nitrogens which substantiate the absence of strategically located His3. It has been suggested that residues of nonnitrogen origin localized in the main body of DSA may be involved in copper binding, which would explain the protection from the Sanger reaction.

Introduction The plasma proteins comprise the major part of the solids in blood plasma. Of the three major plasma protein groups, fibrinogen, albumin, and globulin, albumin is present in the highest mass concentration. Serum albumins have many physiological functions. In addition to their role in transport, distribution, and metabolism of fatty acids and many drugs,1 they also play an essential role in the transport and metabolism of various trace metals, particularly copper.2 Metal specificity, number of binding sites, intrinsic binding constants, and the chemical nature of the ligands at the sites are parameters of major consequence. Although the physicochemical data regarding human3,5 (HSA), bovine4 (BSA), and dog3,5 (DSA) serum albumin are available, there is still disagreement about the number, affinity, and chemical nature of copper(II) sites, as summarized in Table 1. Using equilibrium dialysis, Sarkar and his group concluded that there was no specific copper binding site in dog serum albumin.5a In contrast, Giroux and Schoun4d reported an apparent constant for copper binding to DSA which was 3 orders of magnitude lower than that for BSA. Contrary to both * Corresponding author. Department of Physical Chemistry, Slovak Technical University, Radlinskeho 9, SK-812 37 Bratislava, Slovakia. Fax: +421-7-524 93 198. E-mail: [email protected].

these reports, Saltman’s group5c has recently concluded, again by equilibrium dialysis, that DSA does have a specific affinity for copper; however, the nature is still not clear. In addition, aspartyl carboxylate chelation to copper in the N-terminus of HSA is still controversial.3s We believe that ambiguity about copper(II), zinc(II), and, in general, any metal ion binding to serum albumin is a consequence of the techniques by which metals are presented to albumin. Metals added as salts to neutral solution can hydrolyze to form metal-hydroxy and -oxy polymers.6 Such polymeric metal ions need not necessarily aggregate to form a visible precipitate and may either bind nonspecifically to albumin or be kinetically inert.3r To obtain accurate information about metal binding sites, metal polymerization and hydrolysis must be eliminated. The central tenet of our approach is the presentation of the metal to the albumin in the form of a well-characterized, low molecular weight chelate, such as Cu2+-histidine (1:2). The presence of such chelating ligands excludes metal binding to low-affinity sites; however, high affinity sites can still compete effectively. This method thus provides precise insights as to the amino acid ligands involved in specific, high-affinity metal binding, without interference from nonspecific, lowaffinity metal binding sites. In this work, we present a combined multifrequency electron paramagnetic resonance (EPR) and optical spectroscopic study

10.1021/jp9846532 CCC: $18.00 © 1999 American Chemical Society Published on Web 06/16/1999

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TABLE 1. Reported Data for the Binding of Copper(II) to Bovine, Human, and Dog Serum Albumins author

methoda

pH

number of binding sites

Klotz and Gurme Tanford Rao and Lal Ryall Giroux and Schoun Saltman et al.

Bovine Serum Albumin ED 4.8 P 4.9 ED 6.5 ED 7.4 U 7.5 ED 7.0-8.0

Laussac and Sarkar Lau and Sarkar Saltman et al.

Human Serum Albumin NMR 7.5 ED,S,T 7.5 ED 7.5

Dog Serum Albumin Appleton and Sarkar ED 7.5 Masuoka and Saltman ED 7.0 Rakhit and Sarkar EPR 6.5-11.0

16 17 16 1b 1b 1 1 1 0e 1 >1

log10 K

ref

4.3c 3.7c 4.88c 12.04c 13.2c 11.12d

f g h i j k

l 16.18c m 11.18d k

10.17d

n o p

Metal Chelate-Albumin Systems. For a given experiment, unless otherwise stated, the protein concentration was held constant (1mM). Metal-chelate albumin mixtures were left at 35 °C for 30 min to equilibrate prior to EPR and optical measurements. EPR Spectra Simulations. The X-band EPR spectra were simulated on an IBM-compatible computer using either program QPOW7a-c developed by Prof. R. L. Belford (University of Illinois, Urbana) or program POWW7d-g written by Prof. P. H. Rieger (Brown University, Providence). The S-band EPR spectra were simulated on an IBM-compatible PC computer using a progam developed in our laboratory.8 MO Calculations. MO calculations with full geometry optimization were performed using the Hyperchem package9 within the ZINDO/1 (Zerner intermediate neglect of differential overlap) method.10 Results and Discussion

a

P, polarography; ED, equilibrium dialysis; U, ultrafiltration; S, spectrophotometry; T, titration; EPR, electron paramagnetic resonance; NMR, nuclear magnetic resonance. b Protein and metal were combined at 1:1 ratio prior to equilibrium. c log10 of the reported apparent binding constant. d log10 of the intrinsic stoichiometric constant. e The absence of specific copper binding site was concluded. f Klotz, I. M.; Curme, H. G. J. Am. Chem. Soc. 1948, 70, 939. g Tanford, C. J. Am. Chem. Soc. 1952, 74, 211. h Rao, M. S. N.; Lal, H. J. Am. Chem. Soc. 1958, 80, 3222. Rao, M. S. N.; Lal, H. J. Am. Chem. Soc. 1958, 80, 3226. i Ryall, X. Competitive Dialysis Studies of Metal-Protein Equilibria. Ph.D. Thesis, Australian National University, Canberra, 1974. j Giroux, E. I.; Schoun, J. J. Inorg. Biochem. 1981, 14, 359. k Masuoka, J.; Hegenauer, J.; Van Dyke, B. R.; Saltman, P. J. Biol. Chem. 1993, 268, 21533. l Laussac, J.-P.; Sarkar, B. Biochemistry 1984, 23, 2832. m Lau, S.-J.; Sarkar, B. J. Biol. Chem. 1971, 246, 5938. n Appleton, D. W.; Sarkar, B. J. Biol. Chem. 1971, 246, 5040. o Masuoka, J.; Saltman, P. J. Biol. Chem. 1994, 269, 25557. p Rakhit, G.; Sarkar, B. J. Inorg. Biochem. 1981, 15, 233.

of copper(II)-HSA and copper(II)-DSA interactions, where copper(II) is presented to albumin in the form of histidine and glycine chelates, respectively. Experimental Section Instrumentation. The X-band EPR spectra were recorded on a Bruker SRC-200 D spectrometer coupled to a Aspect 2000 and equipped with a variable temperature unit. Line positions were measured accurately using internal field markers generated by an NMR gaussmeter, while the microwave frequency was measured by a microwave frequency counter. A 100 kHz magnetic field modulation (peak-to-peak amplitude ≈ 3 G) was used. The S-band EPR spectra were recorded on a Bruker ESP 300 E spectrometer equipped with a variable temperature unit. The UV-vis spectra were measured with a 2101-S UV-vis Shimadzu spectrophotometer using a 1 cm cell at ambient temperature (25 °C). The spectra of the metal chelate-albumin were corrected for the absorption of albumin in the absence of chelate. The pH was measured using a glass electrode connected to Digi-Sense pH/mV/ORP meter (Cole-Parmer, Vernon Hills, IL). Chelate Solutions. All metal salts, histidine, and glycine were obtained from Sigma Chemical Co. Chelate solutions were prepared by mixing the chelatating agent with the corresponding metal salt in Hepes buffer (pH 7.3). The metal-to-chelator ratio was 1:2 throughout the work. Albumin Preparations. Stock solutions of HSA and DSA (crystallized and lyophilised, Mr ) 65 000, 1 mM) were prepared by dissolving albumin in Hepes buffer (25 mM NaCl, 30 mM Hepes) at pH 7.3. The solutions were stored at low temperature until use.

Since the majority of the amino acid complexes in blood plasma are formed by Cu2+ and histidine,11 it is rational to emulate the in vivo system by presenting copper to HSA in the form of a histidine chelate. 1:2 Copper(II)-Histidine Complex: X- and S-Band EPR Spectra. The correct structure of the Cu2+-histidine (1:2) complex has been the subject of considerable debate in the literature,12 as the histidine molecule presents three potential coordination sites in aqueous solution. The carboxyl group (pKa ) 1.9), the imidazole nitrogen (pKa ) 6.1), and the amino nitrogen (pKa ) 9.1) all become available for complexation as pH increases. The alternatives proposed for the Cu2+-histidine (1:2) complex are a binding mode either with four equatorially coordinated nitrogen ligands (two imidazole and two amino) or one in which a carboxylate oxygen replaces one of these nitrogens.12 The low-temperature X-band EPR spectrum of the Cu2+histidine (1:2) complex at pH 7.3 is presented in Figure 1A. The spectrum shows three well-resolved low-field parallel lines with a hyperfine splitting of 554 MHz and g|| ) 2.237 (Table 2) (the fourth parallel line is obscured by the perpendicular region). The intense perpendicular band reveals a slight rhombic distortion with g values of 2.044 and 2.047. The fluid solution X-band EPR spectrum shows an isotropic quartet, with partially resolved hyperfine structure on the high-field line (Figure 1, inset I). Interpretation of the high-field part of the X-band spectrum is complicated by several factors, such as the possibility of rhombic hyperfine interactions, 63/65Cu nuclear quadrupole interactions, and g and A strain.13 These difficulties can be overcome by the use of multiple microwave frequencies, particularly at lower frequencies (for example S-band), as has been specifically shown by Hyde and Froncisz in their EPR studies of copper(II)-chelates.13e EPR measurements at lower frequencies can be advantageous for the enhanced resolution of ligand hyperfine splitting due to a combination of (i) an increased admixture of electronic and nuclear spin functions which increases transition probabilities,13a (ii) the reduction of g strain,13b and (iii) the reduction of experimental line width. S-band EPR is indeed successful here in increasing resolution, as shown in the second-derivative high-field part of the frozen solution spectrum (Figure 1B, inset II), which clearly shows nine resolved lines with splitting of 32 MHz and the expected intensity ratios. This suggests the existence of a species with four equivalent nitrogens directly bonded to copper, which would correspond to a major abundance of complexes formed

Binding Site for Copper(II) in Serum Albumins

Figure 1. EPR spectra of a copper(II)-histidine (1:2) complex in PBS at pH 7.3. (A) First-derivative spectrum (X-band, 9.45 GHz) of a frozen solution (100 K). (Inset I) High-field copper band of the first-derivative spectrum (X-band) at room temperature. (B) First-derivative spectrum (S-band, 3.87 GHz) of a frozen solution (100 K). (Inset II) second derivative high-field band. E, experiment; S, computer simulation using parameters summarized in Table 2.

with both histidines in a histamine-like (4N) coordination, in agreement with work of Basosi et al.12a We note that the match between experiment and simulation (Figure 1) for both frequencies (X-band and S-band) was achieved using a single set of parameters (Table 2). Inclusion of additional fit parameters, such as A rhombicity, nuclear quadrupole coupling,13c,d and g and/ or A strain line width effects,13b could in principle improve the fit, but in practice, is not warranted here given the good match between experimental and simulated spectra. Very recently, a simple new technique based on Fourier transform analysis of CW-EPR spectra has been applied in our laboratory.14 This technique has been shown to be very effective in determining the number (in an odd or even sense) of nitrogen atoms directly bonded to Cu(II). FT analysis of the X-band CWEPR fluid solution spectrum of the [Cu(His)2] complex (pH 7.3) revealed, in addition to a major abundance of histamine-like (4N) complexes, a minor amount of glycine-like complexes with three bonded nitrogens (3NO). Similar EPR results, but in an immobile phase, were reported by Antholine et al.12b and by Goodman et al.12c The above results indicate that upon freezing there is a switch of ligands from a mixture of histamine-like and glycine coordination in the mobile phase to exclusively histamine-like coordination in the immobile phase. The low-temperature X-band EPR data with g|| ) 2.237 and visible absorption maximum at a relatively long wavelength of 645 nm (Figure 2A) are in agreement with apical chelation by

J. Phys. Chem. B, Vol. 103, No. 26, 1999 5593 a donor atom (group) of greater ligand field strength than water, as proposed very recently by ESEEM analysis.15 [Cu(His)2]-HSA Interaction. The low-temperature X-band EPR spectrum of [Cu(His)2]-HSA (1:1) at physiological pH is presented in Figure 3A. The spectrum is obviously different from the copper carrier (Cu(His)2, Figure 1) and corresponds to a single paramagnetic species, based on the well-defined spectral features. In addition, the absorption maximum exhibits a marked shift to λmax ) 535 nm (Figure 2B), in comparison with the copper carrier (λmax ) 645 nm, Figure 2A). This documents that the strong binding site competes effectively with two histidines as copper ligands but the multiple lower affinity binding sites do not. The spectrum is therefore assigned to copper(II) bound to the high-affinity site of HSA with no features attributable to any residual [Cu(His)2]. To exclude the posssibility of displacement of one histidine in the reaction with HSA, while the second histidine remains coordinated to copper, we have investigated the EPR spectra of [Cu(his)] presented to albumin. Although the EPR spectrum of [Cu(his)] is different (fluid solution EPR data: giso ) 2.142, ACu iso ) 193 MHz) compared to [Cu(his)2] (see Table 2), once HSA is introduced, the same spectrum for [Cu(II)-HSA] is obtained.16 The combination of EPR and electronic absorption spectral changes unambiguously illustrates that the carrier chelate has completely released the copper, which is instead bound to a single site in HSA. All spectral data are summarized in Table 2. Resolution of the superhyperfine EPR (X-band) structure on the perpendicular band was further enhanced in the secondderivative spectrum (Figure 3, inset I). This band is characterized by a slightly asymmetric nonet with relative intensities 1:4:10: 16:19:16:10:4:1 and a splitting of 44 MHz (14.5 G), typical of coupling to four nitrogen atoms.17 The same reasons as in the case of copper-histidine described above directed us to perform S-band measurements. A well-resolved spectrum was obtained, as documented in Figure 4. We note, that its simulation (see Figure 4, inset) using the same set of parameters (Table 2) confirmed the results obtained from measurements at X-band. The peptides of HSA contain many functional groups that are potential ligands for copper(II). These include the R-NH2; the NH2’s of Lys4, 12, 20, ...; the COO- of Asp1, 13, ..., and of Glu6, 16, 17, ...; the imidazole nitrogens of His 3, 9, ...; and the amide nitrogens of the peptide groups of Ala2, 8, .... Investigations with single tripeptide molecules, e.g., glycylglycyl-L-histidine and glycylglycyl-L-histidine N-methylamide, designed to mimic the Cu(II) transport site of HSA, give evidence that the binding site involves primarily the first three NH2 terminal residues.18 At physiological pH, specific metal donor binding sites such as NH2 terminal, carboxylate COOof Asp1, and imidazole of His3 are actually deprotonated.19 The relatively symmetrical perpendicular EPR band with nine superhyperfine lines can be attributed to the arrangement of four equivalent nitrogens around copper(II). This suggests that copper(II) forms a complex with HSA involving an R-NH2 terminal, two deprotonated peptide nitrogens (NH of Ala2 and NH of His3) as well as the imidazole nitrogen N(1) of the same His3. Laussac and Sarkar20 have performed a 13C and 1H NMR study of peptide1-24 of HSA. They concluded that there is axial Asp1 COO- chelation for copper(II). However, for the [Cu(II)-HSA] complex, 13C NMR resonances are almost unobservable due to the large paramagnetic broadening, and consequently, apical chelation by Asp1 COO- should be considered with caution. Analysis of the optical spectrum presented in Figure 2B along with EPR data should definitively

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TABLE 2. Parameters Used to Simulate EPR Spectra at X- and S-Bands and Optical Data for [Cu(his)2], [Cu(gly)2], [Cu-HSA], and [Cu-DSA] Complexesa,b complex

g1(g⊥)

g2

g3(g||)

Cu ACu 1 (A⊥ )

ACu 2

Cu ACu 3 (A|| )

AN1 (AN⊥)

AN2

AN3 (AN|| )

λmax nm

c

2.044 2.049 2.051 2.034

2.047

2.237 2.258 2.162 2.268

27 16 48 32

27

554 552 625 563

32

32

32

44 43

43

44 38

645 619 535 587

[Cu(his)2] [Cu(gly)2]d [Cu-HSA] [Cu-DSA]e

2.059

32

a Hyperfine and superhyperfine interactions are given in megahertz. b The EPR spectra were recorded at 100 K. c Fluid solution EPR data (XCu d band): giso ) 2.117, ACu iso ) 199 MHz. Nitrogen shf structure not satisfactorily resolved. Fluid solution EPR data (X-band): giso ) 2.120, Aiso ) 196 MHz. Nitrogen shf structure not resolved. e The low-frequency measurements did not enhance the resolution.

Figure 2. (A) Optical spectrum of a copper(II)-histidine (1:2) complex in PBS at pH 7.3 at ambient temperature. (B) Optical spectrum of a copper(II)-HSA (Cu/HSA ) 1:1) complex in PBS at pH 7.3 at ambient temperature. Copper was presented to albumin in the form of a copperhistidine (1:2) complex. The optical spectrum was taken after equilibrating for 30 min.

answer the question concerning the coordination environment of the copper center. The lowest energy absorption (∆) is representative of either the dz2 f dxy transition or the dz2 f dx2-y2 transition, depending upon the orientation of the ligand environment with respect to the x and y axes. ∆ is primarily determined by two factors: (i) the strength of the axial donor (the stronger the donor, the higher in energy the dz2 level will be, leading to a smaller value for ∆ (spectral red shift)); (ii) the displacement of the copper out of the N4 plane (the larger the displacement, the lower in energy the dxy (dx2-y2) level will be, which again leads to a smaller value for ∆). It has been suggested that the π-donating or accepting properties of the axial ligand must also be considered. Specifically, it was suggested that the π-donor capability of the anionic ligands may lead to an increase in energy of the both the dxz and dxy orbitals above that of the dz2 orbital and thereby contributes to the red spectral shift. The observed absorption at 535 nm is sufficiently low to exclude the red shift; thus, carboxylate chelation cannot be considered in describing the high-affinity environment of HSA for copper binding. This conclusion is in agreement with the correlation between absorption maximum and the number and variety of donor atoms bound equatorially, where an absorption

Figure 3. EPR spectra (X-band, 9.46 GHz) of a frozen solution of [Cu(II)-HSA] and [Cu(II)-DSA] complexes in PBS at pH 7.3. (A) First-derivative spectrum of the [Cu(II)-HSA] complex at 100 K. (Inset I) Second derivative perpendicular band. (B) First-derivative spectrum of the [Cu(II)-DSA] complex at 100 K. (Inset II) Second derivative perpendicular band. E, experiment; S, computer simulation using parameters summarized in Table 2.

maximum at 535 nm was assigned to four equatorially coordinated nitrogens only.21 The same conclusion further is supported by an analysis of spin Hamiltonian parameters. The shift in g values that would result from coordination by Asp1 carboxylate oxygen can be understood from the equations17a

g|| ) ge - 8λR2β2/∆||

and

g⊥ ) ge - 2λR2δ2/∆⊥ (1)

assuming axial symmetry and where λ is the spin-orbit coupling constant and R2, β2, and δ2 are the coefficients of the Cu dxy, dx2-y2, and dxz,yz (eg) atomic orbitals in their respective molecular orbitals, all of which should be relatively constant. Thus, only ∆|| (dx2-y2 f dxy) and ∆⊥ (eg f dxy) are variables. Addition of a fifth ligand, also causing displacement from the N4 plane, will shift both electronic transitions to lower energies giving larger g values. In the transition from square planar to square pyramidal geometry, the positive shift of the g values is accompanied by a lowering of the absolute value of the copper hyperfine coupling constants from the value obtained for the planar complexes. The values g|| ) 2.162 and |A||| ) 625 MHz found for the CuHSA complex are at the outer limit of EPR parameters found

Binding Site for Copper(II) in Serum Albumins

J. Phys. Chem. B, Vol. 103, No. 26, 1999 5595

Figure 4. First-derivative EPR spectrum (S-band, 3.87 GHz) of a frozen solution of [Cu(II)-HSA] complex in PBS at pH 7.3: E, experiment; S, computer simulation using parameters summarized in Table 2.

for square planar copper complexes with four equatorial N nuclei but are very similar to those of Cu(II)-phthalocyanine (g|| ) 2.160 and |A||| ) 616 MHz).13 The g|| value is sufficiently low and |A||| sufficiently large to exclude pyramidalization at Cu(II) by the carboxylate oxygen of Asp1. The EPR and optical data correlate with a square planar environment of copper. This conclusion is in agreement with a recent X-ray study on the serum albumin mimic pseudopeptide glycylglycylhistamine.22 A similar conclusion is arrived at by considering correlations between g|| and A|| (known as P-B diagrams).23 These correlations arise from a variety of factors which have been summarized for natural and artificial copper proteins. It has been well established, particularly for CuN4 systems, that tetrahedral distortions of a planar CuX4 moiety markedly reduce |A||| while simultaneously increasing g||. The tetrahedral distortion arising from the dependence of g|| on the dihedral angle led to the introduction of the quotient g||/|A||| as a convenient measure of the degree of tetrahedral distortion.24 The EPR data for the [CuHSA] complex give a value of 103 cm which suggests a square planar environment around the copper center with practically negligible tetrahedral distortion. This is in agreement with an MO study of the binding site fragment modeled within the ZINDO10 method, and the optimized geometry is shown in Figure 5. Superhyperfine Splitting and Bonding Characteristics. In estimating the covalency of the metal-ligand bond, the coppernitrogen bond length is taken to be the same as that in copper complex with glycylglycylhistamine (2.0 Å).22 The ligand coefficient can then be computed using the following equation:25

AN⊥ ) (1/2R′2)

[16π3 βg β φ (2s)(γ ) + 2

2

N N

14 βg β 〈1/r3〉2p(1 - γ2) (2) 5 N N

]

where γ2 ) 1/3 for assumed sp2 hybridization.26 The dipolar correction was made for AN⊥. Using the values of Hurd and Coodin,27 5.6 au and 3.6 au, for φ 2(2s) and 〈1/r3〉2p, respectively, eq 2 gives R′2 ) 0.32, which together with the normalization condition assuming the overlap integral S ) 0.092 gives R2 ) 0.87. Thus, there is a significant covalent character for the in-

Figure 5. Optimized geometry for [Cu(II)-HSA] complex fraction involving R-NH2 terminal of Asp1, two deprotonated peptide nitrogens (NH of Ala2, and NH of His3), and imidazole nitrogen of His3 bound to copper atom. Unrestricted Hartree-Fock (UHF) ZINDO/1 calculations were performed in order to obtain this geometry.

Figure 6. First-derivative EPR spectrum (X-band, 9.46 GHz) of a frozen solution of a copper(II)-glycine (1:2) complex in PBS at pH 7.3: E, experiment; S, computer simulation using parameters summarized in Table 2.

plane bonds. We note that independent evaluation of bonding parameters of shf splitting can be found in ref 28. [Cu(gly)2]-DSA Interaction. The low-temperature X-band EPR spectrum of DSA in the presence of CuCl2 (1:1) at physiological pH yields overlapping EPR peaks, indicating considerable heterogeneity in the copper(II) binding sites on DSA (spectrum not shown, see also ref 5d). The use of [Cu(His)2] failed in the elucidation of the high-affinity binding site and demonstrated that DSA cannot compete with histidine. Thus, as a next step, a weaker chelator, glycine, was used, which is still a good biomimetic complex. The difference between the low-temperature EPR spectra of [Cu(gly)2] (Figure 6, Table 2) and [Cu(gly)2]-DSA (1:1) (Figure 3B, Table 2) at physiological pH provides evidence for copper(II) bound to a single site of DSA. The difference in low-temperature EPR spectrum of [Cu(gly)2] and [Cu(gly)2]-DSA (1:1) (Figure 3, Table 2) at physiological pH provides evidence for copper(II) bound to a single site of DSA, the high-affinity binding site. There are no

5596 J. Phys. Chem. B, Vol. 103, No. 26, 1999 features attributable to any residual [Cu(gly)2], also further supported by a marked shift in λmax in the optical spectrum (see Table 2). The EPR spectrum of [Cu(II)-DSA] exhibits rhombicity with g1,2,3 ) 2.034, 2.059, and 2.268 and |A3| ) 563 MHz. The considerable increase in g3 compared to HSA would suggest a marked distortion about the copper center, with the g3/|A3| ratio being 121 cm. Detailed inspection of the shf structure reveals quintets on g1 and g2 with coupling constant of 43 MHz (Figure 3, inset II). We note that in this case the low-frequency EPR spectrum (S-band) did not enhance the resolution. The quintet indicates the presence of two equivalent nitrogens29 bound to the copper, which indicates that His3 is absent. This absence might produce a certain degree of distortion in the square planar geometry of the copper surroundings. X-ray crystallography indicates that the N-terminus of albumin is flexible and therefore free to move in space.1b Thus, residues of non-nitrogen origin localized in the main body of albumin may be involved in the copper binding site. Binding in this manner would explain why DSA exhibits protection from the Sanger reaction seen by Peters.30 We note that the same absence of His3 was discovered in proalbumin Nagasaki-3 genetic variants of HSA containing basic propeptide that is not removed during posttranscriptional processing because of a mutation.31 Conclusion A new approach in metal-albumin binding studies applied to the elucidation of the high-affinity binding site for copper in human serum albumin (HSA) and dog serum albumin (DSA) is presented. To avoid nonspecific metal binding to apoenzyme, the presentation of the metal to apoenzyme is in the form of well-characterized histidine or glycine chelates. This method minimizes binding to lower affinity sites to the point of their being practically unobservable, while still allowing binding to high-affinity sites. Detailed analysis of the X-band and S-band EPR and optical spectra of the [Cu(II)-HSA] complex revealed that copper forms a complex with HSA involving R-NH2 terminal and two deprotonated peptide nitrogens (NH of Ala2, and NH of His3) as well as imidazole nitrogen of the His3. The spin Hamiltonian parameters and a visible absorption maximum at 535 nm are consistent with a square planar arrangement of four nitrogens around copper atom. The EPR study of the [Cu(II)-DSA] complex confirmed the presence of two bonded nitrogens which indicate the absence of strategically located His3. Acknowledgment. We thank Professor R. Linn Belford (University of Illinois) for the EPR simulation program (QPOW). Sponsorship of this work by the British Council, Bratislava, The Royal Society, and the Grant Agency for Science (No. 1/733/95) is gratefully acknowledged. M.V. thanks the Royal Society for a postdoctoral fellowship. E. J. L. McInnes is an EPSRC postdoctoral fellow. The National EPSRC MultiFrequency EPR Centre located at the Chemistry Department, University of Manchester is supported by the EPSRC. References and Notes (1) (a) Carter, D. C.; Ho, J. X. AdV. Protein Chem. 1994, 45, 153. (b) He, M. X.; Carter, D. C. Nature 1992, 358, 209. (c) Carter, D. C.; He, X. Science 1990, 249, 302. (d) Peters, T, Jr. AdV. Protein Chem. 1985, 37, 161. (e) Ni Dhubhghaill, O. M.; Sadler, P. J.; Tucker, A. J. Am. Chem. Soc. 1992, 114, 1118. (f) Christodoulou, J.; Sadler, P. J.; Tucker, A. FEBS Lett. 1995, 376, 1. (2) (a) Lippard, S. J.; Berg, J. M. Principles of Bioinorganic Chemistry; University Science Book: Mill Valley, CA, 1994; p 16. (b) Lippard, S. J. Metals in Medicine. In Bioinorganic Chemistry; Bertini, I., Gray, H. B.,

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