Báo cáo khoa học: Reactions of gold(III) complexes with serum albumin docx

Reactions of gold(III) complexes with serum albumin Giordana Marcon 1 , Luigi Messori 1 , Pierluigi Orioli 1 , Maria Agostina Cinellu 2 and Giovanni Minghetti 2 1 Department of Chemistry, University of Florence, Italy; 2 Department of Chemistry, University of Sassari, Italy The reactions of a few representative gold(III) complexes – [Au(ethylenediamine) 2 ]Cl 3 , [Au(diethylentriamine)Cl]Cl 2 , [Au(1,4,8,11-tetraazacyclotetradecane)](ClO 4 ) 2 Cl, [Au(2,2¢, 2¢-terpyridine)Cl]Cl 2 ,[Au(2,2¢-bipyridine)(OH) 2 ][PF 6 ]and the organometallic compound [Au(6-(1,1-dimethylbenzyl)- 2,2¢-bipyridine-H)(OH)][PF 6 ] – with BSA were investigated by the joint use of various spectroscopic methods and separation techniques. Weak metal–protein interactions were revealed for the [Au(ethylenediamine) 2 ] 3+ and [Au(1,4,8,11-tetraazacyclotetradecane)] 3+ species, whereas progressive reduction of the gold(III) centre was observed in the cases of [Au(2,2¢-bipyridine)(OH) 2 ] + and [Au(2,2¢,2¢- terpyridine)Cl] 2+ . In contrast, tight metal–protein adducts areformedwhenBSAisreactedwitheither[Au(diethylen- triamine)Cl] 2+ and [Au(6-(1,1-dimethylbenzyl)-2,2¢-bipyri- dine-H)(OH)] + . Notably, binding of the latter complex to serum albumin results in the appearance of characteristic CD bands in the visible spectrum. It is suggested that adduct formation for both of these gold(III) complexes occurs through coordination at the level of surface histidines. Sta- bility of these gold(III) complexes/serum albumin adducts was tested under physiologically relevant conditions and found to be appreciable. Metal binding to the protein is tight; complete detachment of the metal from the protein has been achieved only after the addition of excess potassium cyanide. The implications of the present results for the pharmacolo- gical activity of these novel cytotoxic agents are discussed. Keywords: gold(III) complexes; serum albumin; spectro- scopic measurements. Following the success of platinum(II) compounds in cancer chemotherapy, several families of nonplatinum metal complexes have been studied intensely as potential cytotoxic and antitumour agents. In particular, in recent years, various gold(III) complexes of sufficient stability in the physiological environment have been prepared and evalu- ated for in vitro anticancer properties. Some of them turned out to exhibit relevant cytotoxic effects in vitro and were the subject of further biochemical and pharmacological inves- tigations [1]. Studies of the interactions of these gold(III) complexes with DNA, the classical target of platinum(II) complexes, pointed out that binding of these compounds to nucleic acids is not as tight as in the case of platinum drugs, suggesting the occurrence of a different mechanism for the observed biological effects [2,3]. Surprisingly, at variance with the reactions with nucleic acids, the reactions of antitumour metal complexes with proteins have been poorly explored until now although they may be of extreme relevance for the biodistribution, the mechanism of action and the toxic effects of several metallodrugs. For example, only a few studies exist on the reactions of the well known anticancer platinum complexes with proteins [4,5]. However, despite the results obtained so far often being incomplete and fragmentary, we believe that the direct damage inflicted on specific proteins by metal complexes, following the formation of strong coordinate bonds, may be of crucial relevance to explain the biological effects of several metallodrugs, either established clinically or experi- mentally. In the present study, we have considered the reactions of a series of representative gold(III) complexes, of different structure and of known biological profile, developed in our laboratory, with bovine serum albumin, selected both as the most abundant plasma protein and as a general model for globular proteins. Serum albumins have many physiological functions. They contribute to colloid osmotic blood pres- sure and are chiefly responsible for the maintenance of blood pH [6]. There is evidence of a significant antioxidant activity of serum albumins. These molecules may represent the major plasma components that protect against oxidative stress [7]. The most outstanding property of albumins is their ability to reversibly bind a large variety of endogenous and exogenous ligands. It is worthwhile remembering that serum albumins have often been considered as general ligands for fatty acids, which are otherwise insoluble in blood, and exhibit a high affinity for hematin, bilirubin, and small, negatively charged, hydrophobic molecules; more- over albumins bind various metal ions [8]. The reactions of gold(III) complexes with serum albumin were investigated primarily through the analysis of the Correspondence to L. Messori, Department of Chemistry, University of Florence, via della Lastruccia, 3, 50019 Sesto Fiorentino (Florence), Italy. Fax: + 39 055 4573385, Tel.: + 39 055 4573284, E-mail: luigi.messori@unifi.it Abbreviations: en, ethylenediamine (1,2-diaminoethane); dien, diethy- lentriamine; cyclam, 1,4,8,11-tetraazacyclotetradecane; terpy, 2,2¢, 2¢-terpyridine; bipy, 2,2¢-bipyridine; bipy c , 6-(1,1-dimethylbenzyl)- 2,2¢-bipyridine; CDDP, cis-diammine dichloro platinum(II); ESI-MS, electrospray ionization mass spectrometry; LMCT, ligand to metal charge transfer. (Received 11 July 2003, revised 29 September 2003, accepted 2 October 2003) Eur. J. Biochem. 270, 4655–4661 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03862.x characteristic bands of the gold(III) centre in the visible spectrum. Our experiments show that markedly divergent reactivity patterns with serum albumin have clearly emerged for the various gold(III) complexes in relation to their chemical structure and reactivity. The implications of such differences in reactivity are discussed in relation to the pharmacological properties of the individual compounds. Materials and methods Materials [Au(ethylenediamine) 2 ]Cl 3 ([Au(en) 2 ]Cl 3 )wasprepared according to [9]. A gummy yellow precipitate was formed by the addition of a solution of 1,2-ethylendiamine mono- hydrate in ether to a solution of HAuCl 4 in ether; the yellow precipitate was dissolved in water giving an orange solution. A white precipitate of [Au(en) 2 ]Cl 3 formed upon adding ethyl alcohol to the latter solution. [AuCl(diethylentriamine)]Cl 2 ([AuCl(dien)]Cl 2 )waspre- pared according to [10]. A solution of diethylenetriamine/ 3HCl in water was added slowly and with stirring to a solution of HAuCl 4 (20%, w/v) and a yellow precipitate immediately formed. A solution of NaOH was added to the mixture until pH 3 and stirred for 2 h at 0 °C. The yellow precipitate was then filtered and washed with ethanol. [Au(1,4,8,11-tetraazacyclotetradecane)](ClO 4 ) 2 Cl ([Au (cyclam)](ClO 4 ) 2 Cl) was prepared by following the pro- cedure reported by Kimura et al. [11]. Treatment of NaAuCl 4 2H 2 O with equimolar amounts of cyclam in CH 3 CN for 1 h yielded the [Au(cyclam)](ClO 4 ) 2 Cl complex. [Au(2,2¢,2¢-terpyridine)Cl]Cl 2 ([Au(terpy)Cl]Cl 2 )waspre- pared by addition of terpyridine to a HAuCl 4 solution under a 1 : 1 stoichiometry according to [12]. [Au(2,2¢-bipyri- dine](OH) 2 ][PF 6 ] ([Au(bipy])(OH) 2 ][PF 6 ]) was prepared according to [13]. An aqueous suspension of Ag 2 Owas added to a solution of [Au(bipy)Cl 2 ][PF 6 ] in acetone. The mixture was stirred for 24 h at room temperature. AgCl was removed by filtration and the solution evaporated to dryness under reduced pressure. The residue was extracted with acetonitrile and filtered over Celite (Sigma-Aldrich). The pale-yellow filtrate was concentrated to a small volume and diethyl ether was added to give a white precipitate of [Au(bipy)(OH) 2 ][PF 6 ]. An aqueous solution of KOH (33 mg, 0.59 mmol) was added to an aqueous suspension of [Au(bipy c -H)Cl][PF 6 ] (179 mg, 0.27 mmol) [14,15]. The mixture was refluxed for 1 h under stirring and filtered. The volume of the colourless filtrate was reduced on a rotary evaporator until crystal- lization was observed. The white product [Au(bipy c - H)(OH)][PF 6 ] was collected by filtration and dried under vacuum. All the products obtained were checked by elemental analysis; in all cases, the purity of the compounds was higher than 98%. Further evidence for the correct identi- fication of the obtained compounds is provided by electronic spectra and mass spectra (vide infra). BSA was purchased from Fluka BioChemika (product number 05470). The powder, lyophilized and crystallized, was ‡ 98.0% pure (purified by HPCE) and of a molecular mass  66 kDa. All the other reagents, purchased from Sigma-Aldrich, were of analytical grade. Where not stated otherwise, experiments were performed in phosphate buffer containing 50 m M Na 2 HPO 4 , 100 m M NaCl, pH 7.4. Spectroscopic measurements The interaction of all complexes with BSA was analysed by monitoring the electronic spectra of freshly prepared solutions of each complex after mixing with BSA (in the ratio 1 : 1) in the reference buffer. The concentration of [Au(en) 2 ]Cl 3 , [Au(dien)Cl]Cl 2 and [Au(cyclam)](ClO 4 ) 2 Cl was 1 · 10 )3 M , while [Au(terpy)Cl]Cl 2 was 1 · 10 )4 M , [Au(bipy)(OH) 2 ][PF 6 ] and [Au(bipy c -H)(OH)][PF 6 ] 2.25 · 10 )4 M . Visible absorption spectra were carried out with a PerkinElmer Lambda Bio 20 spectrophotometer. The measurements were done at room temperature (25 °C). Fluorescence spectra were registered with a Jasco FP- 750 spectrofluorimeter working at room temperature with k ex ¼ 295 nm; BSA 5 · 10 )5 M was titrated with [Au(bi- py c -H)(OH)][PF 6 ] at the ratios [Au(bipy c -H)(OH)][PF 6 ]/ BSA r ¼ 0.5–5.0 (where r is moles of drug per mole of BSA). Ultrafiltration experiments The adducts between gold(III) compounds and BSA, prepared as described above, were filtered after 24 h incubation at room temperature, using Centricon YM-10 (Amicon Bioseparations, Millipore Corporation, USA) at 1370 g and the starting volume reduced by half; finally, the absorption spectra of the upper and lower portions of the solution were recorded. Extensive ultrafiltration was applied to the same samples and the absorption spectra were recorded after three cycles of washing with the buffered solution. Additional experiments were conducted by ultracentri- fuging at half volume [Au(dien)Cl]Cl 2 /BSA solutions at molar ratios of 1 : 1, 2 : 1, 4 : 1 and 8 : 1. Complex content in the upper and lower solution was analysed spectro- photometrically. Circular dichroism spectra CD spectra of BSA samples at increasing [Au(bipy c - H)(OH)][PF 6 ]/BSA molar ratios, in phosphate buffer, were recorded on a Jasco J500C dichrograph and analysed through the standard JASCO software. The time dependence of the spectra was analysed over several hours; the final spectra were recorded after 24 h incubation at 25 °C. Reaction with cyanide [Au(dien)Cl]Cl 2 /BSA and [Au(bipy c -H)(OH)][PF 6 ]/BSA adducts were treated with a 10 : 1 stoichiometric excess of cyanide. The UV-Vis spectra were recorded before and immediately after the addition of a concentrated solution of sodium cyanide. Reaction with imidazole The interaction of [Au(bipy c -H)(OH)][PF 6 ]2.5· 10 )4 M and [Au(dien)Cl]Cl 2 1 · 10 )3 M with imidazole (in the ratio 4656 G. Marcon et al.(Eur. J. Biochem. 270) Ó FEBS 2003 1 : 1) was analysed by monitoring the electronic spectra of a freshly prepared solutions in the reference buffer at 25 °C, 5 h long. Results Structure and solution chemistry of the investigated gold(III) complexes In the present study we have considered the following six gold(III) complexes: [Au(en) 2 ]Cl 3 , [Au(dien)Cl]Cl 2 ,[Au(cy- clam)](ClO 4 ) 2 Cl, [Au(terpy)Cl]Cl 2 , [Au(bipy)(OH) 2 ][PF 6 ] and [Au(bipy c -H)(OH)][PF 6 ], recently investigated in our laboratory (Fig. 1). The choice of these gold(III) complexes was dictated by their favourable chemical properties in terms of solubility in water and stability within a physio- logical-like environment; in addition, most of these com- plexes are endowed with relevant cytotoxic properties toward cultured human tumour cell lines, as previously reported. The solution behaviour of these complexes, within a reference physiological buffer, was further assayed by monitoring the characteristic visible bands over several hours. An appreciable stability was revealed for all men- tioned gold(III) complexes in line with previous reports [1,3]. Spectrophotometric studies of the reaction with BSA As all these gold(III) complexes, under physiological conditions, exhibit intense and characteristic charge transfer bands in the visible, their reactions with BSA were monitored directly by visible absorption spectroscopy. BSA was added in 1 : 1 stoichiometric amounts to buffered solutions of each gold(III) complex and the visible spectra of the resulting mixture recorded over several hours at room temperature. The obtained spectrophotometric patterns are showninFig.2. Different behaviours clearly emerge from direct inspec- tion of the spectral profiles. It is apparent that the spectra of either [Au(en) 2 ]Cl 3 or [Au(cyclam)](ClO 4 ) 2 Cl are not signi- ficantly affected by addition of BSA. These observations suggest that the gold(III) chromophore of these complexes is not – or is only slightly – perturbed by protein addition. Small changes are observed in the main charge transfer band for both [Au(dien)Cl]Cl 2 and [Au(bipy c -H)(OH) ][PF 6 ]. For [Au(dien)Cl]Cl 2 , the changes are complete within about 2 h, while only a few minutes are needed in the case of [Au(bipy c -H)(OH)][PF 6 ]. In contrast, in the cases of [Au(terpy)Cl]Cl 2 and [Au(bipy)(OH) 2 ][PF 6 ], a progressive decrease in intensity of the visible bands is observed until complete disappear- ance. Under the experimental conditions that we have used, the process is complete within 2 h in the case of [Au(terpy)Cl]Cl 2 and within about 6 h in the case of [Au(bipy)(OH) 2 ][PF 6 ]. After ultrafiltration of the adducts between BSA and [Au(terpy)Cl]Cl 2 or [Au(bipy)(OH) 2 ] [PF 6 ], the lower solutions were spectrophotometrically analysed and found to contain the free ligands terpyridine and bipyridine. No gold was detected in these solutions. As these gold(III) complexes are fairly stable, the best explan- ation of the above observation is that gold(III) undergoes reduction and the complexes break down with release of the ligands. In turn, gold may be reduced to gold(I) or even to colloidal gold associated with the protein. Adduct formation as assessed by ultrafiltration experiments Further information on the reactions of gold(III) complexes with BSA was gained by the application of classical biochemical separation techniques. The main goal of these experiments was to provide at least qualitative information on the strength of the interactions between gold(III) complexes and BSA. Buffered solutions of the individual gold(III) complexes and BSA were prepared, at 1 : 1 stoichiometry, and incubated for 12–24 h at room tempera- ture. Ultrafiltration with a Centricon device was carried out to reduce sample volumes from 2 to 1 mL, and the upper and lower solutions analysed spectrophotometrically. We noticed that [Au(en) 2 ]Cl 3 and [Au(cyclam)](ClO 4 ) 2 Clare readily removed from the protein by ultrafiltration, imply- ing that the interaction is relatively weak and most likely electrostatic in nature. Figure 3A shows the results obtained with [Au(cyclam)](ClO 4 ) 2 Cl). In the case of [Au(bipy)(OH) 2 ][PF 6 ] (Fig. 3B), disruption of the gold(III) complex is confirmed by the appearance of the characteristic UV-Vis bands of the free ligand 2,2¢-bipyridine (at 230 and 280 nm) in the lower solution after ultrafiltration. In contrast, both [Au(dien)Cl]Cl 2 and [Au(bipy c - H)(OH)][PF 6 ] are not easily displaced from the protein. For [Au(dien)Cl]Cl 2 , the protein-bound complex after a single ultrafiltration is about 80%, while for [Au(bipy c - Fig. 1. Schematic drawings of some representative gold(III) complexes. [Au(en) 2 ]Cl 3 , [Au(dien)Cl]Cl 2 , [Au(cyclam)](ClO 4 ) 2 Cl, [Au(terpy)Cl] Cl 2 , [Au(bipy)(OH) 2 ][PF 6 ] and [Au(bipy c -H)(OH)][PF 6 ]. Ó FEBS 2003 Interactions of cytotoxic gold(III) complexes with BSA (Eur. J. Biochem. 270) 4657 H)(OH)][PF 6 ] it is more than 96%, suggesting that these complexes are tightly bound to BSA through coordinate bonds. However, [Au(dien)Cl]Cl 2 may be removed by repeated cycles of ultrafiltration while [Au(bipy c - H)(OH)][PF 6 ] is not. Representative results of repeated ultrafiltration experiments are shown in Fig. 4. The [Au(dien)Cl]Cl 2 /BSA system The appreciable stability of the [Au(dien)Cl]Cl 2 /BSA adducts prompted us to analyse this system in more detail. Specifically, we tested whether protein binding is reversible and whether multiple binding sites are available for [Au(dien)Cl]Cl 2 on the protein surface. To address these issues, [Au(dien)Cl]Cl 2 /BSA solutions were prepared at molarratiosof1:1,2:1,4:1and8:1;thegoldcontent in the upper and lower solutions was analysed spectropho- tometrically after extensive ultracentrifugation. From ana- lysis of the experimental results, it is apparent that the relative percentage of bound gold decreases as the [Au(dien)Cl]Cl 2 /BSA ratio increases (Table 1). When BSA is exposed to an 8 : 1 [Au(dien)Cl]Cl 2 molar excess, about 2.4 gold atoms are found associated with each protein molecule after extensive washing. Overall, these findings suggest that multiple binding sites for [Au(dien)Cl]Cl 2 are present on BSA, of progressively lower affinity. CD spectrum of the [Au(bipy c -H)(OH)][PF 6 ]/BSA adduct Further information on the spectral features of BSA-bound gold(III) centres was obtained by CD spectroscopy, a particularly well-suited technique to analyse the specific environment of protein-bound metal centres [16]. Asampleof[Au(bipy c -H)(OH)][PF 6 ]/BSA was prepared at a 1 : 1 molar ratio, and analysed by CD, immediately after mixing, at 25 °C (Fig. 5). Notably this adduct is characterized by an intense CD negative band in the visible spectrum, at k ¼ 410 nm, diagnostic of the fact that the gold(III) species is bound to a chiral matrix such as the protein. With [Au(dien)Cl]Cl 2 , only minor modifications were observed in the CD spectra of 10 )4 M BSA when the gold(III) complex was added in the ratios 1 : 1, 2 : 1, 4 : 1 and 8 : 1; however, no clear characteristic CD band appeared in the visible spectrum (data not shown). Gold removal from BSA by potassium cyanide To further assess the stability of the adducts, either [Au(dien)Cl]Cl 2 /BSA and [Au(bipy c -H)(OH)][PF 6 ]/BSA were treated with a 10 : 1 stoichiometric excess of cyanide. It is well known that excess cyanide leads to the formation of a very stable tetracyanoaurate complex and we therefore wanted to check whether such a strong ligand is able to remove gold(III) from the protein, both kinetically and thermodynamically. Indeed, treatment with cyanide results in quick disappearance of the peculiar visible bands of the gold(III) centres in both adducts implying that the bound gold is accessible and that the kinetics of release are fast. In contrast, treatment of these derivatives with lower amounts of cyanide did not result in complete detachment of gold from the protein. Fig. 2. Time-dependent spectral profiles of gold(III) compounds/BSA adducts. Visible absorption spectra of buffered solutions con- taining gold(III) complexes and BSA in a 1 : 1 ratio. Spectra correspond to [Au(en) 2 ]Cl 3 1 · 10 )3 M (A), [Au(dien)Cl]Cl 2 1 · 10 )3 M (B), [Au(cyclam)](ClO 4 ) 2 Cl1· 10 )3 M (C), [Au(terpy)Cl]Cl 2 1 · 10 )4 M (D), [Au(bipy) (OH) 2 ][PF 6 ]2.25· 10 )4 M (E) and [Au(bipy c -H)(OH)][PF 6 ]2.25· 10 )4 M (F), before (a) and after the addition of BSA. The further evolution of the various systems over time is reported until the spectral changes reach completion. The buffer (pH 7.4) con- tains 50 m M Na 2 HPO 4 and 100 m M NaCl. 4658 G. Marcon et al.(Eur. J. Biochem. 270) Ó FEBS 2003 Surface histidines as the probable binding site for gold(III) complexes: the reaction with imidazole Imidazoles of surface histidines are good candidates as donors for the gold(III) centre. To elucidate this issue we carried out the reaction of [Au(dien)Cl]Cl 2 and [Au(bipy c - H)(OH)][PF 6 ] with imidazole, within the same buffer, and analysed the modifications of the visible spectra of the gold(III) chromophore. Interestingly, spectral changes similar to those observed upon reaction of the same complex with albumin were detected. This observation, although not conclusive, favours the view that histidines are the probable binding sites for the gold(III) containing fragments. Fluorescence studies Fluorescence measurements give information about the molecular environment in the vicinity of the chromophore molecules. The intensity of intrinsic fluorescence of two tryptophan residues (Trp213 and Trp314) and a shift in wavelength of their emission maxima were chosen as indicators of protein conformational changes in serum albumin. Notably, the addition of [Au(bipy c -H)(OH)][PF 6 ]to BSA-buffered solutions results in a net decrease of fluores- cence intensity; indeed, progressive fluorescence quenching is observed as the [Au(bipy c -H)(OH)][PF 6 ]/BSA molar ratio increases from 0.5 to 5 (Fig. 6). At higher ratios, saturation is reached and the final fluorescence spectrum is assigned to the protein-bound form of [Au(bipy c -H)(OH)][PF 6 ]. Whereas the residual fluorescence intensity is only  5% of the original value, the position of the maximum moved toward red wavelengths (a Dk ¼+13 nm has been deter- mined for r ¼ 5). The shift in the position of the emission maximum corresponds to the changes of the polarity around the chromophore molecule. The slight red-shift observed indi- cates that tryptophan residues were placed in a more polar environment and were more exposed to the solvent. It is possible that [Au(bipy c -H)(OH)][PF 6 ]stickstoBSAmole- cules and consequently rearranges the tryptophan micro- environment. Fig. 4. The exhaustive ultrafiltration experiments of two representative gold(III) compounds/BSA adducts. Visible absorption spectra of the adduct before (a) and after exhaustive ultrafiltration: the spectra of the lower (l) and the upper (u) solutions are shown. These data refer to the [Au(dien)Cl]Cl 2 /BSA (A) and [Au(bipy c -H)(OH)][PF 6 ]/BSA (B) adducts (1 : 1). Fig. 3. The ultrafiltration experiments at half volume of two represen- tative gold(III) compounds/BSA adducts. Visible absorption spectra of the lower (l) and upper (u) solution obtained after ultrafiltration (reducing the volume to half). These data refer to the [Au(cy- clam)](ClO 4 ) 2 Cl/BSA (A) and [Au(bipy)(OH) 2 ][PF 6 ]/BSA (B) adducts (1 : 1). Table 1. Percentages of [Au(dien)Cl]Cl 2 in the upper and lower solutions after ultracentrifugation. Percentage of complex found in the upper and lower fractions after ultracentrifugation of solutions containing the [Au(dien)Cl]Cl 2 /BSAsystemintheratios1:1,2:1,4:1and8:1. Fraction 1 : 1 2 : 1 4 : 1 8 : 1 Upper solution 70.3 60.0 46.3 31.8 Lower solution 29.7 40.0 53.7 68.2 Ó FEBS 2003 Interactions of cytotoxic gold(III) complexes with BSA (Eur. J. Biochem. 270) 4659 Biological properties of the adduct [Au(bipy c -H)(OH)][PF 6 ]/BSA 1 : 1 It is still a matter of debate whether protein adducts of cytotoxic metallodrugs retain, at least in part, the anti- tumour properties of the free metal complex. In order to address this point the biological activity of the adduct [Au(bipy c -H)(OH)][PF 6 ]/BSA 1 : 1 was tested toward some representative human tumour cell lines. We observed that the adduct retained to a good extent the cytotoxic activity of the free metal complex; probably the protein behaves as a ÔreservoirÕ of the free gold(III) compound (Table 2). Discussion The reactions of anticancer metal complexes with proteins have been scarcely investigated until now. We believe that this issue is of particular relevance in view of the established reactivity of metal complexes with model proteins, and deserves, in any case, greater attention. In fact, metal–protein interactions may play key roles in the biodistribution, in the mechanism of action and in the toxic effects of antitumour metal complexes. Moreover, this subject is becoming more important because the paradigm that DNA is a primary target for antitumour metallodrugs is rapidly declining, and seems to be no longer valid, at least for some families of nonplatinum anticancer metal complexes. Obviously, this observation has prompted new interest in the search of novel proteins as possible targets for such metallodrugs. Even in the case of cisplatin, the knowledge of the interactions with proteins is limited to a few studies only, from which, notwithstanding, it emerges that the largest portion of administered platinum is associated with pro- teins. Cole reported that cisplatin binds in vitro almost irreversibly to BSA [17]; due to the apparent irreversibility (both in vivo and in vitro) of the protein/195mPt–cisplatin complex, it is unlikely that the protein-bound fraction of the administered free drug will serve as a therapeutically useful drug reservoir [18]. Other studies have been reported on the interactions of some well known anticancer ruthenium(III) complexes and of auranofin with plasma proteins [19–21]. Very scarce information exists on the reaction of gold(III) complexes with proteins. In fact gold(III) complexes gen- erally behave as strong oxidizing agents; hence it is commonly believed that they are quickly reduced to gold(I) compounds or to colloidal gold by low molecular mass biomolecules and by protein side chains. Thus, in the present paper, we have tried to detail the reactions of a series of emerging antitumour gold(III) complexes of appreciable redox stability with serum albu- min, used as a general model for globular proteins. In the compounds investigated the oxidizing properties of the gold(III) centre are drastically decreased by the presence of strong multidentate ligands in such a way that interaction studies are feasible. However, the stronger oxidizing agents in our series ([Au(terpy)Cl]Cl 2 and [Au(bipy)(OH) 2 ][PF 6 ]) are still able to slowly oxidize the protein side chains. At variance with this, the complexes with less pronounced oxidizing properties do not give rise to significant redox chemistry but tend to form adducts with BSA that appear to be of different strength. The tight adducts that formed with either [Au(dien)Cl]Cl 2 or [Au(bipy c -H)(OH)][PF 6 ]were further investigated. Compared to the cisplatin–BSA adduct, the adduct between the organometallic gold(III) Fig. 5. Circular dichroism spectra of the [Au(bipy c -H)(OH)][PF 6 ]/BSA adduct. Circular dichroism spectra of BSA and of the [Au(bipy c - H)(OH)][PF 6 ]/BSA adduct in the 1 : 1 ratio. The spectrum of the adduct was recorded immediately after mixing and after 3 h. BSA concentration was 2 · 10 )4 M . Fig. 6. Titration of BSA with [Au(bipy c -H)(OH)][PF 6 ] studied by fluorescence. Fluorescence spectra of 5 · 10 )5 M BSA upon addition of increasing amounts of [Au(bipy c -H)(OH)][PF 6 ], in the reference buffer are shown. In the course of the experiment, r varies from 0.5 to 5.0. Table 2. Cytotoxic activity of [Au(bipy c -H)(OH)][PF 6 ] and of its adduct with BSA. Inhibitory effects of [Au(bipy c -H)(OH)][PF 6 ], the adduct Au(bipy C -H)(OH)][PF 6 ]/BSA and cisplatin on the growth of some cisplatin-sensitive (A2780/S) and -resistant (A2780/R, SKOV3) human tumour cell lines. ED 50 is defined as the concentration of drug required to inhibit cell growth by 50% compared to control. Cell line ED 50 (l M ) [Au(bipy C - H)(OH)][PF 6 ] [Au(bipy C - H)(OH)][PF 6 ]/BSA cisplatin A2780/S 2.3 7.4 2.1 A2780/R 12.0 50 27.7 SKOV3 11.3 45 32.1 4660 G. Marcon et al.(Eur. J. Biochem. 270) Ó FEBS 2003 compound and BSA, once formed, is stable and retains its cytotoxic activity; in other words it seems to be a good candidate for further pharmacological evaluation. Notably, the main features of the gold(III) centre are conserved after association with BSA. The adducts are relatively stable and may be destroyed only by the addition of strong ligands for gold(III) such as cyanide. This behaviour is interpreted in terms of either weak electrostatic interactions or direct metal coordination to surface residues of the protein. The ability of selected complexes to tag either cysteine or histidine residues may result in specific damaging of crucial proteins, which could account for the pharmacological and toxic effects. Some reports exist in the literature indicating that histidine residues are preferred binding sites for ruthenium(III) on the protein surface [22,23]. The antiarthritic gold(I) drug Auranofin is known to bind specifically Cys34 of human serum albumin [24]. In the light of the above examples it might well be that selective modification of surface protein residues by gold(III) complexes constitutes the molecular basis for their biological effects. Concluding remarks In this study we have investigated the reactions of six representative gold(III) complexes with bovine serum albu- min used as a general model for plasma proteins. Different patterns of reactivity emerge for the various compounds in relation to the specific chemical properties of the gold(III) complexes. In some cases tight adducts are formed in which the bound gold(III) centres are probably coordinated to surface histidines of the protein. It is hypothesized that the ability of selected gold(III) complexes to tag either cysteine or histidine residues may result in specific damaging of crucial intracellular proteins thus accounting for the relevant cytotoxic effects of these compounds. Acknowledgements The Cassa di Risparmio di Firenze and MIUR are gratefully acknowledged for a generous grant. We thank Dr Costanza Landi and Alessandro Vaccini for helping us in the experimental work. References 1. Messori, L., Abbate, F., Marcon, G., Orioli, P., Fontani, M., Mini,E.,Mazzei,T.,Carotti,S.,O’Connell,T.&Zanello,P. (2000) Gold (III) complexes as potential antitumor agents: solu- tion chemistry and citotoxic properties of some selected gold (III) compounds. J. Med. Chem. 43, 3541–3548. 2. Messori, L., Marcon, G., Tempi, C. & Orioli, P., (2001) Inter- actions of selected gold (III) complexes with calf thymus DNA. Biochem. Biophys. Res. Commun. 281, 352–360. 3. Marcon, G., Carotti, S., Coronnello, M., Messori, L., Mini, E., Orioli, P., Mazzei, T., Cinellu, M.A. & Minghetti, G. (2002) Gold (III) complexes with bipyridyl ligands. solution chemistry, cytotoxicity and DNA binding properties. J. Med. Chem. 45, 1672–1677. 4. Allardyce, C.S., Dyson, P.J., Coffey, J. & Johnson, N. (2002) Determination of drug binding sites to proteins by electrospray ionisation mass spectrometry: the interaction of cisplatin with transferrin. Rapid Commun. Mass Spectrom. 16, 933–935. 5. LeRoy, A.F. & Thompson, W.C. (1989) Binding kinetics of tet- rachloro-1,2-diaminocyclohexaneplatinum (IV) (tetraplatin) and cis-diamminedichloroplatinum (II) at 37 degrees C with human plasma proteins and with bovine serum albumin. Does aquation precede protein binding? Natl. Cancer Inst 81, 427–436. 6. He, X.M. & Carter, D.C. (1992) Atomic structure and chemistry of human serum albumin. Nature 358, 209–215. 7. Bourdon, E., Loreau, N. & Blache, D. (1999) Glucose and free radicals impair the antioxidant properties of serum albumin. FASEB J. 13, 233–244. 8. Bal, W., Christodoulou, J., Sadler, P.J. & Tucker, A. (1998) Multi- metal binding site of serum albumin. J. Inorg. Biochem. 70, 33–39. 9. Block, B.P. & Bailar, J.C. (1951) The reaction of gold (III) with some bidentate coordinating groups. J. Am. Chem. Soc. 73, 4722–4725. 10. Nardin,G.,Randaccio,L.,Annibale,G.,Natile,G.&Pitteri,B. (1979) Comparison of structure and reactivity of bis (2-amino- ethyl)amine- and bis(2-aminoethyl)amido-chlorogold (III) complexes. J. Chem. Soc., Dalton Trans. 220–223. 11. Kimura, E., Kurogi, Y. & Takahashi, T. (1991) The first gold (III) macrocyclic polyamine complexes and application to selective gold (III) uptake. Inorg. Chem. 30, 4117–4121. 12. Hollis, L.S. & Lippard, S.J. (1983) Aqueous chemistry of (2,2¢,2¢- terpyridine) gold (III). Preparation and structures of [Au (terpy) Cl]Cl 2 3H 2 O and the mixed valence Au(I)-Au(III) salt [Au (terpy)Cl] 2 [AuCl 2 ] 3 [AuCl 4 ]. J. Am. Chem. Soc. 105, 4293–4299. 13. Cinellu, M.A., Minghetti, G., Pinna, M.V., Stoccoro, S., Zucca, A. & Manassero, M. (2000) Gold (III) derivatives with anionic oxygen ligands: mononuclear hydroxo, alkoxo and acetato com- plexes. Crystal structure of [Au(bipy)(OMe) 2 ][PF 6 ]. J. Chem. Soc., Dalton Trans. 1261–1265. 14. Cinellu, M.A., Zucca, A., Stoccoro, S., Minghetti, G., Manassero, M. & Sansoni, M. (1996) Synthesis and characterization of gold (III) adducts and cyclometallated derivatives with 6-benzyl- and 6-alkyl-2,2¢-bipyridines. J. Chem. Soc., Dalton Trans. 4217–4225. 15. Cinellu, M.A., Minghetti, G., Pinna, M.V., Stoccoro, S., Zucca, A. & Manassero, M. (1999) Replacement of the chloride ligand in [Au(C,N,N)Cl][PF 6 ] cyclometallated complexes by C, N, O and S donor anionic ligands. J. Chem. Soc., Dalton Trans. 2823–2831. 16. Rodger, A. & Norde ´ n, B., (1997) Circular Dichroism and Linear Dichroism. Oxford University Press, Oxford, UK. 17. Cole, W.C. & Wolf, W. (1980) Preparation and metabolism of a cisplatin/serum protein complex. Chem. Biol. Interact 30, 223–235. 18. Ivanov, A.I., Christodoulou, J., Parkinson, J.A., Barnham, K.J., Tucker, A., Woodrow, J. & Sadler, P.J. (1998) Cisplatin binding sites on human albumin. J. Biol. Chem. 273, 14721–14730. 19. Messori,L.,Orioli,P.,Vullo,D.,Alessio,E.&Iengo,E.(2000)A spectroscopic study of the reaction of NAMI, a novel ruthenium (III) anti-neoplastic complex, with bovine serum albumin. Eur J. Biochem. 267, 1206–1213. 20. Kratz, F., Hartmann, M., Keppler, B. & Messori, L., (1994) The binding properties of two antitumor ruthenium (III) complexes to apotransferrin. J. Biol. Chem. 269, 2581–2588. 21. Roberts, J.R., Xiao, J., Schliesman, B., Parsons, D.J. & Shaw, C.F. III (1996) Kinetics and mechanism of the reaction between serum albumin and auranofin (and its isopropyl analogue) in vitro. Inorg. Chem. 35, 424–433. 22. Clarke, M.S. & Stubbs, H., (1987) Metal Ions in Biological Sys- tems (Sigel, H., ed), Vol. 33, pp. 728–755. Marcel Dekker, New York, USA. 23. Keppler, B.K. (1993) Metal Complexes in Cancer Chemotherapy. VCH Publishers, Weinheim, Germany. 24. Shaw, C.F. III. (1999) Gold-based therapeutic agents. Chem. Rev. 99, 2589–2600. Ó FEBS 2003 Interactions of cytotoxic gold(III) complexes with BSA (Eur. J. Biochem. 270) 4661 . more- over albumins bind various metal ions [8]. The reactions of gold(III) complexes with serum albumin were investigated primarily through the analysis of the Correspondence. the reactions of a series of representative gold(III) complexes, of different structure and of known biological profile, developed in our laboratory, with