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Journal of Inorganic Biochemistry 100 (2006) 1936–1945

InorganicBiochemistry

An in vitro study of interactions between insulin-mimeticzinc(II) complexes and selected plasma components

Eva Anna Enyedy a, Laszlo Horvath a, Krisztina Gajda-Schrantz a,Gabor Galbacs a, Tamas Kiss a,b,*

a Department of Inorganic and Analytical Chemistry, University of Szeged, P.O. Box 440, H-6701 Szeged, Hungaryb Bioinorganic Chemistry Research Group of the Hungarian Academy of Sciences, P.O. Box 440, H-6701 Szeged, Hungary

Received 27 June 2006; received in revised form 18 August 2006; accepted 21 August 2006Available online 30 August 2006

Abstract

The speciations of some potent insulin-mimetic zinc(II) complexes of bidentate ligands: maltol and 1,2-dimethyl-3-hydroxypyridinonewith (O,O) and picolinic acid with (N,O) coordination modes, were studied via solution equilibrium investigations of the ternary complexformation in the presence of small relevant bioligands of the blood serum such as cysteine, histidine and citric acid. Results show thatformation of the ternary complexes, especially with cysteine, is favoured at physiological pH range in almost all systems studied. Besidesthese low molecular mass binders, serum proteins among others albumin and transferrin can bind zinc(II) or its complexes. Accordingly,the distribution of zinc(II) between the small and high molecular mass fractions of the serum was also studied by ultrafiltration. Mod-elling calculations relating to the distribution of zinc(II), using the stability constants of the ternary complexes studied and those of theserum proteins reported in the literature, confirmed the ultrafiltration results, namely, the primary role of albumin in zinc(II) bindingamong the low and high molecular mass components of the serum.� 2006 Elsevier Inc. All rights reserved.

Keywords: Insulin-mimetic zinc(II) complexes; Solution equilibrium study; Ternary complexes; Ultrafiltration

1. Introduction

Diabetes mellitus is a worldwide disease that affectsmillions of patients. The development of orally active com-pounds in place of insulin injections, and therefore theinsulin-mimetic effect of vanadium ions and their com-plexes, have been investigated widely since their influenceon the blood glucose level was first confirmed [1–3]. Itwas proved that many oxovanadium(IV) complexes withvarious coordination modes can normalize the blood glu-cose level with high efficiency, even at low concentrations,with low toxicity [4–6]. However, there is a certain aversion

0162-0134/$ - see front matter � 2006 Elsevier Inc. All rights reserved.

doi:10.1016/j.jinorgbio.2006.08.003

* Corresponding author. Address: Department of Inorganic and Ana-lytical Chemistry, University of Szeged, P.O. Box 440, H-6701 Szeged,Hungary. Tel.: +36 62 454337; fax: +36 62 420505.

E-mail address: tkiss@chem.u-szeged.hu (T. Kiss).

to the use of vanadium in medical treatment and interesthas turned to the development of insulin-mimetic com-plexes of essential metal ions, such as zinc(II) [2,7–12].

Zinc is an important trace element in all living systemsand plays a crucial role in many proteins and metalloen-zymes; it is known to be important in the production andmetabolism of insulin [13]. Zinc(II) is directly involved ininsulin synthesis, secretion and signalling. It was recentlyreported that zinc(II) supplementation is effective in pre-venting type 1 and type 2 diabetes in several rodent modelsand it is possible that zinc(II) can protect against progres-sive b-cell injury in type 2 diabetes [13]. Since the firstreport of ZnCl2 exerting an effect on the glucose level in1980 [7], numerous complexes of zinc(II) have been pre-pared and their insulin-mimetic activities have been inves-tigated both in vivo and in vitro especially in the past tenyears [2,8–12,14–16]. These zinc(II) complexes promote

E.A. Enyedy et al. / Journal of Inorganic Biochemistry 100 (2006) 1936–1945 1937

glucose uptake into the adipocytes and stimulate lipogene-sis, which is similar to the action of insulin, via effects onthe tyrosine kinase of the insulin receptors, phosphatidylinositol-3-kinase, glucose transporters, and the activationof phosphodiesterase [12].

Through complex formation, the insulin-mimetic effectof the metal ion can be regulated and enhanced. Potentligands form a neutral bis complex with higher lipophilic-ity, which ensures better bioavailability through a moreefficient membrane transport. Zinc(II) complexes with(O4), (N2O2), (N2S2), (O2S2), and (S4) coordinationmodes have been reported to display considerable insulin-mimetic effects in in vitro experiments on enzyme inhibi-tions and blood glucose-lowering effects [2,8–12,14,15]. Itshould be mentioned, however, that the efficiency of thezinc(II) complexes in insulin mimetic potential never sur-passed the corresponding VO(IV) species. The effectiveligands include maltol (mal), picolinic acid (pic), 6-meth-ylpicolinic acid (6Me-pic) and different pyrone or pyridi-none derivatives, as in the case of vanadium(IV) [2]. Thebiological relevance of the complexes of zinc(II) formedwith these ligands has been proved [2,8–12,14–16]. Thesolid state structures of the applied bis complexes havebeen revealed [9–11], but, the solution speciations of thesepotential drugs remain relatively unexplored [17–19]. Theoriginal form of the candidate bis complexes most proba-bly changes in the extracellular or intracellular biologicalfluids, and the carrier ligand may be partly or completelydisplaced by suitable endogenous zinc(II) binders. Conse-quently, a solution study of ternary complex formationbetween insulin-mimetic zinc(II) complexes and relevantbioligands may well contribute to a better understandingof the transport processes of zinc(II) complexes underphysiological conditions. Both low molecular mass(LMM) molecules, such as inorganic anions, amino acids,dicarboxilyc acids and hydroxy (oligo)carboxylic acids,and high molecular mass (HMM) proteins may participatein the serum transport processes.

In the present work the mixed ligand complex formationbetween zinc(II), mal, pic or 1,2-dimethyl-3-hydroxypyrid-inone (dhp) (ligand A) as potent insulin-mimetics and citricacid (cit), histidine (His) or cysteine (Cys) (ligand B) hasbeen studied by pH-potentiometry and 1H NMR spectrom-etry. These B ligands were chosen as the most probablezinc(II) binding molecules among the low molecular masscomponents of the serum, when the composition of theserum is considered [20]. The complex formation equilibria(stoichiometry and stability constants) for the parent sys-tems are known in the literature under our selected exper-imental conditions in almost all cases [17,22,23]. However,serum proteins can also bind considerable amounts ofzinc(II) or its complexes [24,25]. The hypothesis is widelyheld that circulating zinc is transported mainly by albumin,and that transferrin is the carrier of newly absorbed zinc inthe portal plasma [24]. Metallothioneins, which are ones ofthe strongest biological zinc(II) binders do not occur in sig-nificant concentration in blood serum. Accordingly, their

role in the metal ion transport processes can be neglected,however, not in the storage processes. The zinc-bindingabilities of albumin and transferrin are known [24–31] andconditional stability constants under physiological condi-tions are also available in the literature [27–31]. Ultrafiltra-tion has been employed to separate the zinc(II) bound tothe high molecular mass proteins from the zinc(II) boundto those of the low molecular mass, containing the carrierligand (mal, pic or dhp) and cit, His and Cys, and thedifferent fractions have been analysed by inductively cou-pled plasma atomic emission spectroscopy (ICP-AES).

2. Experimental

2.1. Chemicals

All ligands and proteins used except dhp were commer-cially available products of puriss quality (Aldrich, Sigmaor Fluka). Dhp was prepared from mal by a literatureprocedure [32]. Their purities were checked and the exactconcentrations of the stock solutions prepared were deter-mined by the Gran method [33]. A ZnCl2 stock solutionwas made by dissolution of anhydrous ZnCl2 in a knownamount of HCl, and its concentration was determined bycomplexometry via ethylenediaminetetraacetate (EDTA)complexes, and gravimetrically via the oxinate.

2.2. pH-potentiometric studies

The pH-metric measurements for determination of thestability constants of the proton and zinc(II) complexesof the ligands were carried out at an ionic strength of0.2 M (KCl) and at 25.0 ± 0.1 �C. The titrations were per-formed with a carbonate-free KOH solution of known con-centration (ca. 0.2 M). An Orion 710 A pH-meter equippedwith a Metrohm combined electrode (type 6.0234.100) anda Metrohm 665 Dosimat burette was used for the pH-met-ric measurements. The electrode system was calibratedaccording to Irving et al. [34], and the pH-meter readingscould therefore be converted into hydrogen ion concentra-tions. The water ionization constant, pKw, is 13.76 ± 0.01under the conditions employed. The pH-metric titrationswere performed in the pH range 2.0–11.0. The initial vol-ume of the samples was 10.00 mL. The ligand concentra-tions were varied in the range 2 · 10�3–4 · 10�3 M. Inthe binary systems the metal ion to ligand ratios were1:1, 1:2 and 1:4; in the ternary systems, metal ion:ligandA:ligand B was 1:1:1, 1:2:1, 1:1:2 or 1:2:2. The acceptedlevel of fitting of the titration curves was always<0.01 mL and the uncertainties (3SD values, i.e. 3 timesthe standard deviation) in the last digit(s) of the stabilityconstants are given in parentheses in the Tables. The sam-ples were in all cases completely deoxygenated by bubblingpurified argon for ca. 15 min before the measurementsand argon was also passed over the solutions during thetitrations. pH-potentiometric results were utilized to estab-lish the stoichiometry of the species and to calculate the

1938 E.A. Enyedy et al. / Journal of Inorganic Biochemistry 100 (2006) 1936–1945

stability constants (logb). Calculations were always madeon the experimental results obtained before precipitation,with the computer program PSEQUAD [35].

2.3. NMR measurements

1H NMR spectra were recorded on a Bruker DRX-500instrument at 25 �C. Chemical shifts are reported in ppm(d), in D2O solutions, with 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) as internal reference. The samples wereprepared in water containing 10% D2O to provide theNMR lock signal. Spectra of 0.004 M solutions of theligands were measured at various pH values (pH = 5–10).

2.4. Membrane ultrafiltration and ICP-AES measurements

The samples were separated by ultrafiltration through10 kDa membrane filters (Microcon YM-10 centrifugalfilter unit, Amicon, Millipore) according to the standardprocedure. All 0.5 mL samples contained 630 lM humanserum albumin (HSA), or 200 lM apo-transferrin (apo-Tf), or 630 lM HSA and 37 lM apo-Tf with the complex[Zn(II)A2] at 200 lM in 0.1 M 4-(2-hydroxyethyl)pipera-zine-1-ethanesulfonic acid (HEPES) buffer at pH = 7.4and 0.025 M NaHCO3; 99 lM cit, 77 lM His, 33 lMCys. These concentrations are the relevant total serum con-centrations for both the low molecular mass and the highmolecular mass components [20]. Control measurementswere carried out with samples under the same conditions,but containing only the proteins without the zinc(II) com-plex, or only the zinc(II) complex without the proteins.

Maltol 1,2-dimethyl-3-hydr

Citric acid H

HO C C

CH2OH

O

O

HO

CH2O

OH

O

O

OH

CH3 N

O

CH3

H2N

CH

C

HO

N

Scheme 1. Formulae of

After the separation, low and high molecular mass frac-tions were obtained. The high molecular mass fractionwas washed with 0.20 mL buffer solution and filtered threetimes. The filtrate contained the low molecular mass spe-cies. Two parallel measurements were made.

The Zn contents of the samples were determined byICP-AES. Prior to the analysis, the samples were digestedin an acid mixture containing 0.5 mL of concentratedHNO3 and 0.5 mL of 30% H2O2 (Suprapur, Merck). Theresulting solutions were transferred to 5 mL volumetricflasks and filled up to the mark with Millipore MilliQquality deionized water. The analysis was performed atthe Zn I emission wavelength of 213.86 nm, using aJobin-Yvon 24 all-argon spectrometer equipped with aTeflon V-groove nebulizer and a Gilson Minipuls IIIperistaltic pump. Two determinations on each solutionwere carried out, using a 2-point background correction,a 4-point direct calibration and Gaussian spectral driftcompensation. Standard solutions for calibration wereprepared from a 1 g/L commercial zinc(II) stock solution(Merck) through dilution with Millipore MilliQ qualitydeionised water.

3. Results and discussion

3.1. Binary systems

The formulae of the ligands in their neutral forms are tobe found in Scheme 1. The stoichiometries of the complexesand the stability constants with some derived and stepwiseequilibrium constants are collected in Table 1.

oxypyridinone Picolinic acid

istidine Cysteine

OH

CH3

N

O OH

CH2

O

NH

NH2

CHC

CH2

HO

O

HS

the ligands studied.

Table 1Proton (logK) and zinc(II) stability constants (logb) of the ligands studied, together with some derived and stepwise stability constants for the complexesformed in the proton-ligand and Zn(II)-ligand systems at 25 �C and I = 0.20 M KCla

malb dhpb picb Citc Hisd Cyse

logK (HA) 8.44 9.77 5.19 5.58(3) 9.13 10.16logK (H2A) 3.67 �1 4.27(3) 6.06 8.10logK (H3A) 2.91(9) 1.68 1.86

logb (ZnAH) 8.39(3) 11.37 14.76logb (ZnA) 5.57 7.24 5.19 4.64(3) 6.31 8.2logb (ZnA2H2) 29.93logb (ZnA2H) 17.55 24.43logb (ZnA2) 10.29 13.55 9.52 6.96(9) 11.84 18.05logb (ZnA3) 12.71 15.2 13.07logb (ZnA2H�1) �0.1 2.3 0.7logb (Zn2A2H�2) �2.82(3)logb (Zn2A3) 29.2logb (Zn3A4H) 49.01logb (Zn3A4) 42.11logK (ZnA) 5.57 7.24 5.19 4.64 6.31 8.2logK (ZnA2) 4.72 6.31 4.33 2.32 5.53 9.85logK (ZnA3) 2.42 1.65 3.55

log(K(ZnA)/K(ZnA2)) 0.85 0.93 0.86 2.32 0.78 �1.65log(K(ZnA2)/K(ZnA3)) 2.30 4.66 0.78

a Standard deviations in the last digit (3SD) are shown in parenthesis if the values were determined in the present work. ‘‘A’’ generally denotes thecompletely deprotonated form of the ligands in this table.

b Ref. [17].c Fitting parameter: 3.81 · 10�3 mL (the average difference between the experimental and the calculated titration curves expressed in the volume of the

titrant), number of titration points: 282, pH range: 2.0–9.2.d Ref. [22].e Ref. [23].

E.A. Enyedy et al. / Journal of Inorganic Biochemistry 100 (2006) 1936–1945 1939

The speciation and stability constants of the insulin-mimetic zinc(II) complexes were earlier studied in ourlaboratory and the results have been published [17]. Maland dhp coordinate to zinc(II) through O donor atomswith (O,O) coordination mode and pic via N and O atoms.All three bidentate ligands form mono, bis and tris com-plexes, and in the pH range of the formation of tris com-plexes mixed hydroxo species can also be found.

Cit, His and Cys were chosen as ligands B: their concen-trations in the serum [20] and their complex formationproperties with zinc(II) at physiological pH [21–23] suggestthat they are the most potent binders among the lowmolecular mass serum components. Computational model-ling has led other authors to a similar conclusion [36].

Complexation of His and Cys under our conditions hasalso been reported [22,23]. Mono and bis complexes andprotonated complexes of the two amino acids have beendescribed, and the formation of some polynuclear specieswas proved in the case of Cys. Because of the lack of acrystal field effect, zinc(II) readily forms complexes withoctahedral or tetrahedral geometry and, since it is a border-line metal ion, different types of donor atoms can be foundin the coordination sphere. Both His and Cys are typicaltridentate and ambidentate ligands: His can coordinatevia the carboxylate group, the amino group or the imidaz-ole group, while for Cys, besides the carboxylate O andamino N, the thiolate S can also be involved in chelationin the zinc(II) complexes formed. At lower pH values, the

coordination is generally rather glycine-like (COO�,NH2), and in the higher pH range the contribution of theside-chain imidazole-N donor to the coordination is moreimportant in the case of His. This ligand coordinates to zin-c(II) in a tridentate mode in [Zn(His)2], and the geometryof the complex is irregular octahedral, with an approxi-mately tetrahedral distortion of the four amino-N atomsand the two carboxylate O atoms are situated further away[22,37]. Cys coordinates via O and S donor atoms at lowerpH, and through N and S donors at pH > 6, but the tri-dentate (COO�, NH2, S�) coordination mode is also prob-able in the octahedral complexes. However, in the biscomplex the geometry is planar with (S,N) coordination,resulting in an unusual stability sequence, since the log(K(ZnA)/K(ZnA2)) is negative [23,37]. All the dissociationconstants of the ligands and the stability constants of theparent complexes were redetermined in the present work,and values identical with the literature data (within theexperimental error) were obtained.

No stability constants for the zinc(II) complexes of citare available in the literature for our experimental condi-tions. The stability data determined in the present workfor these parent complexes are listed in Table 1. The speci-ation model which gave the best fit with the experimentaltitration data was the same as it was published earlier[38]. Cit contains three dissociable protons, belonging tothe carboxylic groups, and forms mono ([Zn(cit)H], and[Zn(cit)]�) and bis ([Zn(cit)2]4�) complexes in the acidic

Zn

O

O

O,N

(Im)N

O,NZn

S-

O

O,N

-S

H2NZn

H2O

H2N

O,N

O

COO-

1940 E.A. Enyedy et al. / Journal of Inorganic Biochemistry 100 (2006) 1936–1945

pH range. In the bis complex, the ligand most probablycoordinates in a tridentate way [39]. Above pH 7, a verystable dinuclear alcoholate-bridged complex is the predom-inant species [38].

[ZnA(His)] [ZnA2(His)] [ZnA(Cys)2]

NH2 NH2N (Im)

Scheme 2. Binding modes of the ternary complexes [ZnA(His)], [ZnA2

(His)] and [ZnA(Cys)2].

0.0

0.2

0.4

0.6

0.8

1.0

3 4 5 6 7 8 9 10pH

Mol

ar f

ract

ion

of Z

n(II

)

Zn(II)

[Zn(cit)]-

[Zn(cit)H] [Zn(mal)(cit)H]-

[Zn(mal)(cit)]2-

[Zn(cit)2]4-

[Zn(mal)2]

[Zn(mal)]+

[Zn(mal)2(cit)]3-

[Zn(mal)(cit)H-1]3-

[Zn(mal)3]-

[Zn2(cit)2H-2]4-

0.0

0.2

0.4

0.6

0.8

1.0

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it

[Zn(cit)]-H(cit)2-

(cit)3-H2(cit)-

H3(cit)

[Zn(cit)H] [Zn(mal)(cit)H]-[Zn(mal)(cit)]2-

[Zn(cit)2]4- [Zn(mal)2(cit)]3-

[Zn(mal)(cit)H-1]3-

[Zn2(cit)2H-2]4-

Fig. 1. Concentration distribution curves of zinc(II) (a) and cit (b) for thecomplexes formed in the Zn(II)–mal–cit system at a 1:2:2 metal ion to malto cit ratio, cZn(II) = 0.002 M.

3.2. Ternary systems

The equilibrium models and calculated stability con-stants for the mixed-ligand complexes are listed in Table 2.

As the data in Table 2 show, the species [ZnABH],[ZnAB] and [ZnABH�1] are formed in all systems. Takinginto account the logK values of the ligands and thelog KZnABH

ZnAB values, in the ternary complexes [ZnABH] thesituation of the proton cannot be assigned to a given ligandexcept to mal, since that has only one dissociable proton,which belongs unquestionably to ligand B. In the complex[ZnAB], ligand B presumably coordinates in a tridentateway and the sixth coordination position is occupied by awater molecule (see Scheme 2). Dissociation of this watermolecule takes place in the pH range �8–9, resulting inthe formation of the complexes [ZnABH�1]; a more correctformula for this complex is therefore [ZnAB(OH)], as isshown in the representative concentration distributioncurves for the Zn(II)–mal–cit system in Fig. 1. Besidesthe bis mixed-ligand complexes, formation of a [ZnA2B]species could be detected in almost all systems. In this com-plex, the coordination number of the metal ion is probablysix, and ligand B coordinates in a bidentate way to the biscomplex of the insulin mimetic carrier ligand (a possiblebinding mode of this complex with His as ligand B is alsoshown in Scheme 2). On the other hand, simultaneouscoordination of the negatively charged bidentate ligand Awith two ligands B to a zinc(II) centre could be found onlyin the case of Cys, most probably because of the low coor-dination number in the complex [Zn(Cys)2]2�, which canaccept further coordinating donors through change of thegeometry of the complex from tetrahedral to octahedral(see also Scheme 2).

Unfortunately, because of the fast proton and ligandexchange processes, 1H NMR measurements on the ternarysystems could not give too much useful information about

Table 2Stability constants (logb) for the mixed-ligand complexes formed in the Zn(II)-ligand A–ligand B ternary systems at 25 �C; I = 0.20 M KCla

A B [ZnABH] [ZnAB] [ZnABH�1] [ZnA2B] [ZnAB2] Fitting (DmL)b No. of points pH range

mal Cit 14.80(15) 9.28(6) 0.52(6) 12.67(15) – 3.58 · 10�3 402 2–10His 17.66(12) 12.00(3) 2.33(9) 15.11(12) – 6.20 · 10�3 421 2–9.3Cys 20.48(15) 14.55(6) 5.07(12) 18.21(12) 20.89(9) 5.80 · 10�3 344 2–9.9

dhp Cit 15.2(6) 10.69(3) 1.75(6) 15.82(9) – 2.53 · 10�3 467 2–10.5His 19.19(9) 13.56(3) 2.89(6) 16.32(12) – 4.63 · 10�3 515 2–11Cys 22.05(9) 15.93(6) 5.24(9) – 20.49(12) 4.21 · 10�3 417 2–10.8

pic Cit 13.72(6) 9.26(3) �0.08(24) 12.58(24) – 3.40 · 10�3 497 1.9–10.2His 16.5(3) 11.01(9) 1.20(12) 14.02(24) – 4.50 · 10�3 495 1.9–10.4Cys 19.78(27) 13.69(12) 4.50(9) 16.6(3) 20.31(15) 5.60 · 10�3 521 2–10.5

a Standard deviations in the last digit(s) (3SD) are shown in parenthesis determined in the present work.b The average difference between the experimental and the calculated titration curves, expressed in the volume of the titrant.

Table 3D logbZnAB values and some molar fractions calculated for the bis mixed-ligand complexes formed in the Zn(II)-ligand A–ligand B systems withsome stepwise stability constants

A B D logbZnABa [ZnAB]

%bR[Zn-A]%b

logK

(ZnA+B)logK

(ZnB +A)

mal Cit 0.35 50 28 3.71c 4.64f

His 0.63 72 7 6.43d 5.69f

Cys 0.08 41 2 8.98e 6.35f

dhp Cit 0.13 40 36 3.45c 6.05g

His 0.56 79 10 6.32d 7.25g

Cys �0.17 37 8 8.69e 7.73g

pic Cit 0.72 39 34 4.07c 4.62h

His 0.03 30 49 5.82d 4.70h

Cys �0.40 15 31 8.50e 5.49h

a D log bZnAB ¼ log bexpZnAB � 1=2ðlog bZnA2

þ log bZnB2þ log 4Þ.

b At pH = 7.4; 1:2:2 Zn(II):ligand A:ligand B ratio, cZn(II) = 0.002 M.c Stepwise stability constant (logK(ZnL + L)) for the parent complexes:

2.32 L = Cit.d 5.53 L = His.e 9.85 L = Cys.f 4.72 L = mal.g 6.31 L = dhp.h 4.33 L = pic (see text).

E.A. Enyedy et al. / Journal of Inorganic Biochemistry 100 (2006) 1936–1945 1941

the binding modes of the complexes. The spectra recordedat different pH values in the Zn(II)–mal–cit ternary systemshowed that the free and the complexed ligand could not bedistinguished. The characteristic d values of both ligandsare upfield shifted with the increase of pH. These changesare compared with those of the binary systems and withthe free ligands in Fig. 2.

As shown in Fig. 1b cit is mainly in its non-coordinated,deprotonated form in the pH range of formation of the ter-nary complexes, e.g. 80% of cit is non-coordinated atpH = 8 in the ternary systems, which is why the averagesignals of the CH2 protons in cit do not differ from thosein the ligand itself (see Fig. 2). On the other hand, a signif-icant difference can be observed for the chemical shifts ofthe mal protons (especially C(5)H and C(6)H) above pH7 in the ternary system as compared with the parent com-plexes. The coordination of cit in the ternary complexesmost probably results in this electron density shift for theseprotons through the p-electron system of the pyrone ring.This confirms formation of ternary complexes but doesnot give any information about the binding modes of thecomplexes.

The relative stability of mixed-ligand complexes can beevaluated in several ways [40,41]. In many cases, the rela-tive stability of the [ZnAB] complexes is expressed in termsof D logbZnAB for the equilibrium [ZnA2] + [ZnB2] ¡

2[ZnAB], and its value can be calculated via Eq. (1).

D log bZnAB ¼ log bexpZnAB � 1=2ðlog bZnA2

þ log bZnB2þ log 4Þ

ð1ÞD logbZnAB values calculated from the experimentally ob-tained stability constants of the relevant binary and ternarycomplexes are listed in Table 3.

-CH3 (mal)

-C(6)H (mal)

-C(5)H (mal)

-CH2 (cit)

-CH2 (cit)

2.20

2.30

2.40

2.50

2.60

2.70

4.5 5.5 6.5 7.5 8.5 9.5 10.5

6.35

6.45

7.80

7.90

8.00

2.20

2.30

2.40

2.50

2.60

2.70

4.5 5.5 6.5 7.5 8.5 9.5 10.5

6.35

6.45

7.80

7.90

8.00

pH

δ (p

pm)

Fig. 2. Chemical shifts as a function of pH in the systems maltol (r),Zn(II) – mal (h), Zn(II) – mal – cit (m), cit (s), Zn(II) – cit (�).

From these data and the molar fractions of the com-plexes [ZnAB] at physiological pH, where these are thepredominant the ternary complexes (Table 3), it may beconcluded that the formation of such types of species isfavoured. However, for the Zn(II)–mal–Cys system theD logbZnAB values are surprisingly small. The reason is thatD logbZnAB can be calculated only when the parent and theternary complexes have the same geometry and most prob-ably this is not the case because of the tetrahedral bis com-plex of Cys and the probably octahedral ternary complexesformed with mal. Some stepwise stability constants relatingto the equilibria [ZnA] + B ¡ [ZnAB] and [ZnB] + A ¡

[ZnAB] are also listed in Table 3 and from a comparisonof these constants with the corresponding values for thebinary complexes, it can be seen that the formation ofthe [ZnAB] complexes is favoured, but a similar contradic-tion is observed in the equilibrium constants calculated forthe corresponding Cys complexes, because of the very highstability of [Zn(Cys)2]2�. For a comparison of the extentsof ternary complex formation with the different ligandsB, the summed concentration distribution curves (whenthe concentrations of the species formed with mal (A), orligand B (B) or bound in the ternary complexes (A–B)are summed) are more expressive, as demonstrated inFig. 3 in the case of maltol.

These figures show that formation of the ternary com-plexes starts at the lowest pH with cit since there is lessextensive proton competition among the donor groups ascompared with His or Cys due to the lower pK values ofcit. Above pH 6–7, mixed ligand complexes are present inhigh proportions in all systems, especially in the case ofHis, owing to the most favoured formation of the species[ZnAB] (see D log bZnAB value) and, with increasing pH,

pH

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ion

of Z

n(II

)

Zn(II)Σ[Zn-B]

Σ[Zn-A-B]

Σ[Zn-A]

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)

Zn(II)

Σ[Zn-B]

Σ[Zn-A-B]

Σ[Zn-A]

0.0

0.2

0.4

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0.8

1.0

3 4 5 6 7 8 9 10

3 4 5 6 7 8 9 10

pH

Mol

ar f

ract

ion

of Z

n(II

)

Zn(II)

Σ[Zn-B]

Σ[Zn-A-B]

Σ[Zn-A]

Fig. 3. Summed concentration distribution curves for the complexesformed in the Zn(II)-ligand A = mal-ligand B: (a) cit, (b) His, (c) Cyssystems at 1:2:2 metal ion to ligand A to ligand B ratio, cZn(II) = 0.002 M.

0.0

0.2

0.4

0.6

0.8

1.0

3 4 5 6 7 8 9 10pH

Mol

ar f

ract

ion

of Z

n(II

)

Zn(II)

Σ[Z n-cit]

Σ[Zn-pic-cit]

Σ[Zn-pic]

Σ[Zn-Cys]

Σ[Zn-pic-Cys]

Σ[Zn-pic-His]

Fig. 4. Predominance curves for the hypothetical Zn(II)–pic–cit–His–Cyssystem at a ratio of 1:2:2:2:2, cZn(II) = 0.002 M.

1942 E.A. Enyedy et al. / Journal of Inorganic Biochemistry 100 (2006) 1936–1945

formation of the tris ternary species becomes moreimportant.

The predominance curves calculated for a hypotheticalsystem Zn(II)–pic–cit–His–Cys (Fig. 4), show that the ter-nary complexes of cit are present at high concentration inthe acidic pH range, due to the fact that the coordinationof cit via O donor atoms to the (N,O)-coordinated pic com-plex is favoured, but at basic pH zinc(II) is bound mainlyby Cys in both the ternary and binary complexes.

These results are in agreement with the general findingsthat the possibility of the mixed binding of different typesof donor atoms, (O,N) or (N,N) of His and (O,S) or

(S,N) of Cys, leads to more considerable ternary complexformation [41].

3.3. Model calculations and ultrafiltration study on the

binding of zinc(II) in a serum model

Besides the low molecular mass compounds, the threemetal-binding proteins albumin, a-macroglobulin andtransferrin have been proved to have the ability to complexzinc(II) in human serum [25,29,42]. In most papers, HSA isregarded as the primary zinc(II)-binder protein, and therole of transferrin in zinc(II) transport is questioned fromtime to time [24,25,43]. In this paper, the zinc(II) bindingproperties of HSA and apo-Tf are considered.

HSA is a single chain of 585 amino acids with a molec-ular weight of �67 kD [26]. It is a carrier molecule for fattyacids, a free radical scavenger, and an osmotic regulator,and it is implicated in the transport and storage of metalions such as copper(II) and nickel(II). It contains threestructurally homologous domains, which are mainly heli-cal, and each domain consists of two subdomains. Themajor zinc(II)-binding site is situated between domains Iand II, which is a secondary binding site for copper(II)and nickel(II). The proposed zinc(II)-binding site has a dis-torted trigonal bipyramid geometry: two His N atoms, anAsn carboxylate and an Asp carboxylate are coordinated,and a water ligand is at the fifth place. The binding con-stants of HSA for zinc(II) under physiological conditionsare given in the literature as logK* � 7.1–7.9 [27,28]. Trans-ferrin contains 679 amino acids and has a molecular weightof �79 kDa. This protein is divided into two lobes (N andC), which are linked by short spacers. Each lobe consists oftwo domains with an iron-binding site. At the metal-bind-ing sites two Tyr, one Asp and one His can be found in adistorted octahedral arrangement. For effective binding,two further O atoms are required, donated by a carbonatemolecule. The HCO�3 content of the serum at 0.025 M con-centration is important from this point of view. The biolog-ical function of transferrin is related to its high affinity foriron (logK* � 22), since this protein is responsible for the

Table 4Calculated and measured percentage of zinc(II) bound to different fractions of the ultrafiltered samples containing Zn(II)–ligand A–ligand B–serumproteins under serum conditions

mal (A) dhp (A) pic (A)

Calc. (%) Meas. (%) Calc. (%) Meas. (%) Calc. (%) Meas. (%)

Low molecular mass 2.2 3 3.3 5 14.0 10In ternary complexes

R [ZnA(cit)] 0.1 0.1 0.8R [ZnA(His)] 0.5 0.9 1.0R [ZnA(Cys)] 0.8 0.9 1.0

In binary complexes

R [Zn(Cys)] 0.4 0.4 0.3In insulin mimetics

R [ZnA] 0.4 1.0 10.9

High molecular mass 97.8 97 96.6 95 85.9 90Bound to the proteins

R [Zn-albumin] 87.0 85.9 76.1R [Zn-apo-Tf] 10.8 10.7 9.8

0

20

40

60

80

100

a c

Zn

%

mal dhp pic

ba cba cb

Fig. 5. Measured (stripe bars) and predicted (black bars) percentagedistributions of zinc(II) bound to the high molecular mass fraction inthe mal, dhp and pic containing systems at pH = 7.40, cZn(II) = 200 lM,cligand A = 400 lM, ccit = 99 lM, cHis = 77 lM, cCys = 33 lM, cNaHCO3 =0.025 M, cHEPES = 0.10 M and cHSA = 630 lM (a), capo-Tf = 200 lM (b),cHSA = 630 lM, capo-Tf = 37 lM (c).

E.A. Enyedy et al. / Journal of Inorganic Biochemistry 100 (2006) 1936–1945 1943

transport of iron from the sites of absorption and storageto utilization [30]. For studies of the metal-binding abilityof transferrin its iron-free form apo-Tf is usually used.For the zinc(II)-binding ability of apo-Tf at pH = 7.4,log K�1 ¼ 7:8 and log K�2 ¼ 6:4ðat 0:015 M HCO�3 Þ [29]are generally accepted in the literature for the binding ofthe first and the second metal ions, but some lowerconditional constants can also be found e.g. (log K�1 ¼5:9ðat 0:02 M HCO�3 Þ [24]). However, both proteins canbind the zinc(II) ion strongly under the circumstances ofthe serum. Since HSA is typically present in the serum at630 lM and transferrin at 37 lM, HSA is suggested to bethe main transporter molecule of this metal ion [29,42].

During the application of an insulin-mimetic zinc(II)complex, a crucial question, with regard to its efficiency,is the form in which it can be found in the serum, andthe extent to which it can retain its original form. Amongthe few in vivo studies relating to insulin-mimetic zinc(II)complexes, Sakurai et al. studied the zinc(II) level in theblood of normal rats, followed by use of the [Zn(mal)2]and [Zn(6-Me-pic)2] complexes in 1–30 mg/kg body weightdoses, dependent on the type of administration [16]. It wasfound that the Zn(II) concentration in the blood was usu-ally increased up to �170–220 lM within 30–90 min,dependent on whether it was administered intravenously,intraduodenally or orally. Sakurai et al. studied the effi-ciency of some insulin-mimetic zinc(II) complexes in vitro,via the inhibitory activities at 200–300 lM [12]. These arethe reasons why a zinc(II) complex concentration of200 lM was chosen for the modelling and for the ultrafil-tration studies in the present work.

In view of the stability constants to be found in theliterature for the two proteins (for HSA, a mean valueof logK* = 7.3 [28], and for apo-Tf, log K�1 ¼ 7:8 andlog K�2 ¼ 6:4 [29]) and for the ternary complexes of thelow molecular mass zinc(II)-binders of the serum formedwith the different insulin-mimetic complexes determinedhere, model calculations could be performed under serum

conditions to estimate the biodistribution of zinc(II) inthe serum. The results of these computational modellingsare listed in Table 4.

These calculations reveal that zinc(II) is bound mainlyto the high molecular mass fractions of the serum, particu-larly to albumin.

The low and high molecular mass fractions of samplescontaining the insulin-mimetic zinc(II) complexes, the pro-teins (HSA and apo-Tf) and the bioligands (Cys, His andcit) and NaHCO3 (0.025 M) at physiological pH were sep-arated by ultrafiltration, followed by the determination ofthe zinc(II) contents of the different fractions by ICP-AES. The experimental results, together with the relevantcalculated values are given in Fig. 5. It can be concludedthat the measured percentage distribution of zinc(II) is ingood agreement (within the experimental error) with thepredicted values obtained by computational modelling. Inall cases, zinc(II) is found mostly in the high molecular

1944 E.A. Enyedy et al. / Journal of Inorganic Biochemistry 100 (2006) 1936–1945

mass fraction. The results show that mal and dhp have sim-ilar abilities to keep the metal ion bound to the low molec-ular mass fraction, but in the case of pic the zinc content ofthe high molecular mass fraction is definitely lower,because of the stronger zinc(II)-binding ability of thisligand as compared with the other two studied.

4. Conclusions

Literature biological studies revealed that about twothirds of the zinc(II) in the human plasma is bound to albu-min and one third to a-macroglobulin [42]. Our modellingcalculations and ultrafiltration studies confirmed the pri-mary role of albumin in zinc(II) binding. In contrast withthe suggestions of various reports [25], our measurementsdid not prove that transferrin is an efficient zinc(II) binderin serum. Moreover, a small proportion (�3–5% of zinc(II))is present in low molecular mass forms, as was predicted inRef. [29]. The calculations and ultrafiltration measurementssuggest that the presence of the carrier ligands of the insu-lin-mimetic complex have practically no effect on the biodis-tribution of zinc(II) in the serum, and thus on the transportof the bioactive metal ion to the cells, where it can exert itsglucose-lowering effect. The results, however, allow the for-mation of ternary complexes between the carrier complexesand the proteins, and in this way the zinc(II) carrier ligandsmay have further roles than merely facilitating the absorp-tion of the zinc(II) complex in the gastro-intestinal tract.The measurements do not allow to make any concreteassumption for the possible binding of the carrier ligandin this ternary complex. We have only very vague idea basedon chemical evidences. Namely, the interaction can beeither primarily by displacement of some weaker bindingdonors of the protein presumably side chain COO�

function(s), or only much weaker secondary interactions,hydrogen bonding or hydrophobic interactions with the‘semi-aromatic’ rings of some of the carrier ligands. Studiesfocusing on the interactions between the zinc(II) complexesand the serum proteins are in progress in our laboratory. Itis also worth mentioning that the binding abilities of theVO(IV) and the corresponding zinc(II) complexes to serumproteins show somewhat opposite feature. Namely, VO(IV)complexes seem to have higher affinity to transferrin [5],while zinc(II) complexes to albumin and this might havesome differences in the way how they can reach the cell toexert the biological effect.

5. Abbreviations

apo-Tf apo-transferrin

cit citric acid cys cysteine dhp 1,2-dimethyl-3-hydroxypyridinone DSS 2,2-dimethyl-2-silapentane-5-sulfonate EDTA ethylenediaminetetraacetate HEPES 4-(2-hydroxyethyl)piperazine-

1-ethanesulfonic acid

His

histidine HMM high molecular mass HSA human serum albumin ICP-AES inductively coupled plasma atomic

emission spectroscopy

LMM low molecular mass mal maltol pic picolinic acid

Acknowledgements

This work was supported by Hungarian Research Foun-dation (OTKA grants T49417, NI61786 and PD050011).

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