A spectroscopic study of the reaction of NAMI, a novel ruthenium(III)anti-neoplastic complex, with bovine serum albumin

Luigi Messori1, Pierluigi Orioli1, Daniela Vullo1, Enzo Alessio2 and Elisabetta Iengo2

1Department of Chemistry, University of Florence, Italy; 2Department of Chemistry, University of Trieste, Italy

The reaction of Na[transRuCl4Me2SO(Im)] (NAMI; where Im is imidazole), a novel anti-neoplastic

ruthenium(III) complex, with BSA, was studied in detail by various physico-chemical techniques. It is shown

that NAMI, following chloride hydrolysis, binds bovine serum albumin tightly; spectrophotometric and atomic

absorption data point out that up to five ruthenium ions are bound per albumin molecule when BSA is incubated

for 24 h with an eightfold excess of NAMI. CD and electronic absorption results show that the various ruthenium

centers bound to albumin exhibit well distinct spectroscopic features. The first ruthenium equivalent produces a

characteristic positive CD band at 415 nm whereas the following NAMI equivalents produce less specific and

less marked spectral effects. At high NAMI/BSA molar ratios a broad negative CD band develops at 590 nm.

Evidence is provided that the bound ruthenium centers remain in the oxidation state +3. By analogy with the case

of transferrins it is proposed that the BSA-bound ruthenium ions are ligated to surface histidines of the protein;

results from chemical modification experiments with diethylpyrocarbonate seem to favor this view. Spectral

patterns similar to those shown by NAMI are observed when BSA is reacted with two strictly related

ruthenium(III) complexes Na[transRuCl4(Me2SO)2] and H(Im)[transRuCl4(Im)2] (ICR), implying a similar

mechanism of interaction in all cases. It is suggested that the described NAMI-BSA adducts may form in vivo

and may be relevant for the biological properties of this complex; alternatively NAMI/BSA adducts may be tested

as specific carriers of the ruthenium complex to cancer cells. Implications of these findings for the mechanism of

action of NAMI and of related ruthenium(III) complexes are discussed.

Keywords: ruthenium; albumin; cancer; circular dichroism.

Na[transRuCl4Me2SO(Im)] (NAMI; where Im is imidazole),a ruthenium(III) complex (developed in Trieste) showsencouraging anti-tumor and anti-metastatic properties [1±4].The imidazolium salt of NAMI, NAMI-A, which has improvedcharacteristics of stability in the solid state compared to NAMI,is currently being tested in clinical phase I studies as an anti-metastatic drug [5]. The complex is pseudooctahedral with fourequatorial chloride ligands, and DMSO and imidazole as axialligands (Scheme 1) [6]. The behavior of NAMI underphysiological conditions was previously studied in detail;notably the complex slowly looses its chloride ligands andtransforms into the corresponding, more reactive, aquated-species [4,7,8]. The hydrolysis process can be easily monitoredspectrophotometrically [4]. Apparently, the loss of twocoordinated chlorides is the prerequisite for any furtherreactivity [7]. The resulting bis-aquaspecies may bind variousbiomolecular targets and is very likely responsible for thebiological effects of NAMI. Yet, the final targets and themechanisms through which NAMI and its parent ruthenium(III)complexes exert their anti-tumor effects are largely unknown,and controversial opinions still exist on this issue [3,5].

Within this frame it is of interest to consider the interactionsof NAMI with plasma proteins, and in particular with serumalbumin (by far the most abundant protein in the plasma) asthese proteins represent the first possible targets for ruthenium(III) compounds after intravenous administration. Moreover,detailed knowledge of the interactions of anti-tumor ruthenium

Eur. J. Biochem. 267, 1206±1213 (2000) q FEBS 2000

Correspondence to P. Orioli, Department of Chemistry, University of

Florence, via Gino Capponi 7, 50121 Florence, Italy. Fax: + 3955 2757555,

Tel.: + 3955 2757554, E-mail: orioli@risc1.cerm.unifi.it

Abbreviations: Im, imidazole; Ind, indazole; NAMI,

Na[transRuCl4MeSO2(Im)]; ICR, H(Im)[transRuCl4(Im)2]; DEPC,

diethylpyrocarbonate.

(Received 4 June 1999, revised 19 November 1999, accepted

22 December 1999)

Scheme 1. NAMI.

q FEBS 2000 Interactions of ruthenium(III) complexes with BSA (Eur. J. Biochem. 267) 1207

complexes with plasma proteins is of concern because bindingto proteins might result into drastic modifications, or even loss,of the biological properties of the starting compounds.Alternatively, a protein bound ruthenium species might stillrepresent an active species provided that some amount ofruthenium is transported through biological fluids and even-tually released at the cellular level [9].

Previous work was devoted to the analysis of the interactionsof ruthenium complexes with plasma proteins [9±12]. Forexample, serum transferrin was shown to bind variousequivalents of H(Ind)[transRuCl4(Ind)2] (where Ind is indazole)and H(Im)[transRuCl4(Im)2] (ICR) at different sites, underphysiological conditions (unpublished results). A crystallo-graphic investigation of ruthenium modified lactoferrin, carriedout by Baker et al. allowed precise identification of the mainbinding sites for ruthenium ions on the lactoferrin molecule[13]; according to this study ruthenium binding was shown tooccur predominantly at the level of solvent exposed histidineresidues.

In the present paper the reaction of NAMI with bovine serumalbumin, under physiological conditions, was analysed in detailthrough various spectroscopic methods. Attempts are made toelucidate the molecular mechanism of binding to BSA and tocharacterize the resulting adducts. A general model is presentedto describe the interactions that take place between ruthenium(III) complexes, with labile ligands, and proteins.

M A T E R I A L S A N D M E T H O D S

Preparation of NAMI and of related ruthenium(III) complexes

NAMI and Na[transRuCl4(Me2SO)2] were prepared accordingto the procedure reported in literature [6]. ICR was a kind giftof B. Keppler (University of Vienna, Austria). The purity ofruthenium(III) complexes was checked through elementalanalysis and 1H NMR spectroscopy. BSA was purchased fromSigma Chemical Company. All the other reagents were ofanalytical grade.

Electronic and 1H NMR spectra

The absorption spectra in the visible range were recorded on aPerkin-Elmer Bio 20 spectrophotometer operating at roomtemperature. The hydrolysis experiments were carried out bymonitoring the electronic spectra of freshly prepared solutionsof the complex, both alone and after interaction with BSA, inphosphate buffer (0.05 m Na2HPO4, pH 7.4, NaCl 0.1 m). 1HNMR spectra were carried out on an MSL 200 Brukerinstrument.

CD spectra

CD spectra of NAMI/BSA samples at increasing NAMI/BSAmolar ratios, in phosphate buffer (0.05 m Na2HPO4, pH 7.4,NaCl 0.1 m), were recorded on a Jasco J500C dichrograph andanalysed through the standard Jasco software package. The timedependence of the spectra was analysed over several hours; thefinal spectra were recorded after 24 h incubation at 25 8C.

Atomic absorption measurements

Flame atomic absorption spectroscopy (FAAS) measurementsof ruthenium were carried out with a FAAS Varian 4775instrument. Calibration curves were built up for each complex.Various ruthenium/BSA samples, prepared in the buffer atincreasing molar ratios, were extensively ultradialysed, after24 h incubation at 25 8C, and analysed for ruthenium content.

Diethylpyrocarbonate (DEPC) modification

DEPC is known to be a specific probe for chemicalmodification of proteins [14]. DEPC preferentially modifieshistidine residues of proteins although other residues may beaffected as well [15]. DEPC modification results into specificchanges of the UV spectrum of the protein that are easilydetected in the UV difference spectra. For the chemicalmodification experiments BSA samples (either native orruthenated) in phosphate buffer (0.05 m Na2HPO4, pH 7.4)were treated with DEPC at a 15 : 1 DPEC/BSA molar ratio, at25 8C. UV spectra were monitored continuously for 30 minafter mixing and the corresponding difference spectra obtainedby spectral subtraction.

R E S U L T S

Hydrolysis of NAMI in the presence of BSA

The hydrolysis of NAMI in the presence of increasing amountsof BSA, at 25 8C, in phosphate buffer (0.05 m Na2HPO4,pH 7.4, NaCl 0.1 m), was analysed spectrophotometrically; forcomparison purposes the hydrolysis of NAMI alone wasfollowed under the same solution conditions (Fig. 1). Frominspection of the spectral profiles it is apparent that thepresence of BSA does not modify appreciably the hydrolysispattern previously reported for NAMI alone [4]. In all caseshydrolysis proceeds through two well distinct steps: firstly, thedecrease of the characteristic band at 400 nm (band a) and theconcomitant increase of a band of similar intensity at 346 nm(band b) is observed; secondly, the progressive decrease of thelatter band takes place. These spectral changes werepreviously interpreted in terms of sequential replacement oftwo ruthenium-coordinated chloride groups by water molecules[4]. More precisely, the band at 400 nm has been assigned tothe [transRuCl4(Im)Me2SO]2, and the band at 346 nm to the[transRu(OH)Cl3(Im)Me2SO]2 species [4]. Notably, only thefinal steps of NAMI hydrolysis are affected by the presence ofalbumin: the final species obtained in the presence of albuminis spectrally distinct from that observed in the case of NAMIalone. In addition, the presence of albumin, while notmodifying the pattern of the first step of NAMI hydrolysis,leads to a significant increase in the rate of this process (the rateof hydrolysis of the first chloride increases by a factor of < 2for a NAMI/BSA ratio of 5 : 1); the increases of the hydrolysisrate of the first chloride were found to correlate roughly withthe BSA/NAMI molar ratio.

NAMI is known to be easily reduced in vivo by mildreductants and to transform into the corresponding ruthenium(II) species [5]; the effects of the addition of a slight excess ofsodium ascorbate on the visible spectrum of NAMI wereanalysed. It is found that ascorbate reduces quickly, andquantitatively, ruthenium(III) to ruthenium(II) [5]; reductionresults into virtual disappearance of the intense charge transferband of NAMI at 400 nm as shown in Fig. 2. The fact that thelatter band, characteristic of ruthenium(III), is still observedwhen NAMI is reacted with albumin, allows us to state that thereaction with albumin does not cause reduction of ruthenium(III) to ruthenium(II).

Characterization of the adducts prepared at increasingNAMI/BSA ratios

In order to better characterize the adducts that are formed uponreacting NAMI with bovine serum albumin, NAMI was

1208 L. Messori et al. (Eur. J. Biochem. 267) q FEBS 2000

incubated with BSA, at different molar ratios, for 24 h at 25 8C.Then, samples were extensively ultradialysed against purebuffer and the resulting products analysed through electronicabsorption spectroscopy and atomic absorption spectroscopy.The electronic absorption spectra of samples prepared atincreasing NAMI/BSA molar ratios (1 : 1, 2 : 1, 4 : 1 and8 : 1, respectively) are shown in Fig. 3. The spectra arecharacterized by intense transitions in the visible range thatare assigned as LMCT transitions of the ruthenium(III) centers.Remarkably, the spectral profiles of these adducts showimportant variations in dependence of the NAMI/BSA ratio,suggesting the presence of multiple binding sites for NAMI onserum albumin. For example, the 1 : 1 and 2 : 1 NAMI/BSA

adducts show a main transition centered around 330 nm; at highNAMI to BSA ratios (4 : 1 and 8 : 1), new intense transitionsappear at 460 and 610 nm.

The NAMI/BSA adducts were analysed for rutheniumcontent by flame atomic absorption spectroscopy. The amountof ruthenium present in the samples, after 24 h incubation withNAMI and extensive ultradialysis against the buffer, wasmeasured through FAAS. For comparison purposes, similarexperiments were carried out with some strictly relatedruthenium(III) complexes, i.e. Na[transRuCl4 (Me2SO)2], andICR (Scheme 2). The FAAS results point out that, in all cases, alarge percentage of added ruthenium binds to the protein(Table 1). Notably, at the highest ruthenium/BSA ratiosemployed in this study (i.e. 8 : 1), BSA is found to bind < 5ruthenium equivalents.

Circular dichroism spectra of NAMI/BSA samples

Titration of BSA with NAMI: CD spectra. More detailedinformation on the spectral features of BSA-bound rutheniumcenters was obtained by CD spectroscopy. CD is a particularlysuitable technique for analysing the specific environment ofprotein-bound metal centers [16]. The CD titration of BSA withincreasing amounts of NAMI is shown in Fig. 4. Four sampleswere prepared at NAMI/BSA ratios of 1 : 1, 2 : 1, 4 : 1 and8 : 1, respectively, and analysed through CD after 24 hincubation at 25 8C. At low ruthenium/BSA ratios (1 : 1;2 : 1) a well defined CD spectrum in the visible, with adominant positive transition at 410 nm, is observed. Thisfinding suggests that some preformed site exists on BSAcapable of binding the first NAMI equivalent with high affinity.The following NAMI equivalents bind albumin more loosely,probably to surface residues of the protein, producing broaderand less characteristic CD features. At high ruthenium/BSAratios (4 : 1 or 8 : 1) the CD spectrum is dominated by a broadnegative band centered at 590 nm (Fig. 4) whereas the bandaround 410 nm disappears.

Remarkably, the visible CD spectra of these adducts developfully after several hours of incubation of BSA with NAMI atroom temperature; indeed the CD spectra recorded on the samesamples after only 1 h incubation showed very weak Cottoneffects (data not shown) in agreement with the view thatformation of the final adducts takes hours.Fig. 1. (A) Hydrolysis of NAMI in the presence of albumin: first phase

(NAMI/BSA ratio 5 : 1), and (B) hydrolysis of NAMI alone. (A) The

electronic spectra were measured every 5 min after mixing (buffer

phosphate 0.1 m, NaCl 0.1 m, pH 7.4) at 25 8C. BSA concentration:

2 � 1025 m; NAMI concentration: 1 � 1024 m. (B) Electronic spectra of

NAMI 1 � 1024 m in physiological buffer (phosphate 0.1 m, NaCl 0.1 m,

pH 7.4) measured every 5 min.

Table 1. Ruthenium content of different NAMI/BSA, ICR/BSA and

Na[transRuCl4(Me2SO)2]/BSA samples as determined by flame atomic

absorption spectroscopy. ri indicates the initial ruthenium/BSA stoichio-

metry; rb is the amount of ruthenium found associated to the protein after

extensive ultradialysis against the buffer (phosphate 0.05 M, NaCl 0.1 M,

pH 7.4). BSA concentration for all the experiments was 1 mm; in all cases

ruthenium concentration is expressed as moles of ruthenium per moles of

BSA.

ri

rb

(ruthenium/BSA) NAMI ICR Na[transRuCl4(MeSO2)2]

1 : 1 0.91 �^ 0.04 0.72 �^ 0.03 0.70 �^ 0.02

2 : 1 1.80 �^ 0.05 1.41 �^ 0.04 1.53 �^ 0.06

4 : 1 2.92 �^ 0.17 2.80 �^ 0.12 3.21 �^ 0.16

8 : 1 4.80 �^ 0.24 5.52 �^ 0.16 5.10 �^ 0.15

q FEBS 2000 Interactions of ruthenium(III) complexes with BSA (Eur. J. Biochem. 267) 1209

Electronic and CD spectra of BSA adducts with `reduced'NAMI. As shown above, the ruthenium(III) center in NAMImay be easily and promptly reduced to ruthenium(II) byaddition of sodium ascorbate without disruption of thecomplex. Thus, for comparison purposes, absorption and CDspectra were carried out on BSA samples incubated for 24 h, at

room temperature, with increasing amounts of `reduced'NAMI. Reduction of NAMI was accomplished by addition ofa slight excess of sodium ascorbate and checked spectro-photometrically. Then `reduced' NAMI was added to BSAsolutions to reach final stoichiometries of 1 : 1, 2 : 1, 4 : 1 and8.1. Absorption and CD spectra of the resulting adducts are

Fig. 2 Reduction of NAMI with ascorbate.

Electronic spectra of NAMI (0.8 � 1024 m)

freshly dissolved in the physiological buffer

before (a) and after addition of a slight

stoichiometric excess (1.2 : 1) of sodium

ascorbate (b).

Scheme 2. ICR (A) and Na[transRuCl4(Me2SO)2] (B).

Fig. 3 Electronic spectra of ultradialysed

NAMI/BSA samples. Samples were prepared at

the following NAMI/BSA ratios: (a) 1 : 1;

(b) 2 : 1; (c) 4 : 1 and (d) 8 : 1. Buffer:

phosphate 0.05 m, NaCl 0.1 m, pH 7.4;

incubation time: 24 h at 25 8C; BSA

concentration: 1 � 1023 m.

1210 L. Messori et al. (Eur. J. Biochem. 267) q FEBS 2000

shown in Fig. 5. It is evident that preliminary reduction ofNAMI to the corresponding ruthenium(II) species results into amarked modification of the spectral pattern of the absorptionand CD titrations as compared to NAMI. Again, BSA is able tobind several ruthenium(II) equivalents. The various equivalentsof reduced NAMI, bound to BSA, give rise to well distinct

spectral features implying that also in this case binding to BSAis sequential and that the ruthenium binding sites arestructurally different from one another. More precisely, theCD spectra of the adducts at low ruthenium/BSA ratios arecharacterized by a main band at 410 nm (with a shoulder at460 nm) that moves to 450 nm for higher ruthenium/BSAratios. The electronic spectra are dominated by a maintransition at 515 nm. The fact that the spectral pattern observedupon addition of `reduced' NAMI is different from thatproduced by NAMI further supports the view that in the lattercase ruthenium remains in the +3 oxidation state. Notably, thebroad negative CD feature at 600 nm is no more observed in thecase of `reduced' NAMI suggesting that this latter LMCTtransition is characteristic of a ruthenium(III) center ligated toimidazole rings of surface histidines.

CD spectra of BSA adducts prepared with increasing amountsof either Na[transRuCl4(Me2SO)2] or H(Im)[transRuCl4(Im)2].In order to obtain more detailed information on the binding ofruthenium(III) complexes to serum albumin, BSA was treatedwith increasing amounts of either Na[transRuCl4(Me2SO)2][6], or ICR [17] and the resulting adducts analysed throughabsorption and CD spectroscopies. The CD titration profiles areshown in Fig. 6. It is evident that, in both cases, BSA bindsseveral ruthenium(III) equivalents (up to five ruthenium ionsper BSA molecule, on average); again, the BSA-bound

Fig. 5 CD and electronic spectra of `reduced'

NAMI/BSA samples following ultradialysis.

(A) CD spectra of reduced NAMI/BSA adducts at

the following ratios: (a) 1 : 1; (b) 2 : 1; (c) 4 : 1

and (d) 8 : 1, recorded after 24 h incubation.

Protein concentration 1 � 1023 m; buffer:

phosphate 0.05 m, NaCl 0.1 m, pH 7.4.

(B) Absorption electronic spectra of the same

samples.

Fig. 4 CD spectra of NAMI/BSA samples. Samples were prepared at the

following NAMI/BSA molar ratios: (a) 1 : 1; (b) 2 : 1; (c) 4 : 1 and (d)

8 : 1, and were measured after 24 h incubation in buffer (phosphate 0.05 m,

NaCl 0.1 m, pH 7.4, 25 8C). Protein concentration: 1 � 1023 m.

q FEBS 2000 Interactions of ruthenium(III) complexes with BSA (Eur. J. Biochem. 267) 1211

ruthenium centers exhibit spectrally distinct features. Overall,the spectral pattern obtained for either Na[transRuCl4(Me2SO)2] or ICR is similar (but not identical) to that observedin the case of NAMI, supporting the view that rutheniumremains in the +3 oxidation state and that roughly the samebinding sites are sequentially occupied by these ruthenium(III)complexes. The above reported atomic absorption data(Table 1) show that for both Na[transRuCl4(Me2SO)2] andH(Im)[transRuCl4(Im)2] amounts of ruthenium comparable tothose found in the case of NAMI remain associated to BSAafter extensive ultradialysis.

Treatment with DEPC

DEPC is commonly used for modification of histidine residuesin proteins; in fact DEPC preferentially binds exposed histidineresidues and induces characteristic changes of the UV spectrumof the protein [14,15]. We thought that DEPC modificationmight represent an useful tool to help determining if ruthenium

binding occurs at the level of histidine residues in BSA.Ruthenated and native BSA samples were treated with the sameamount of DEPC and the changes of the UV spectrum analyzedwith time. The resulting UV difference spectra are shown inFig. 7. From inspection of these results it is evident that thekinetics of growth of the characteristic difference band at240 nm is markedly different in the two cases, although thefinal UV difference spectra are almost identical. Native BSAresults to be more reactive than the corresponding ruthenatedsample towards DEPC; for example after 2 min incubation theintensity of the UV difference band in the ruthenated sample isonly 20% of the intensity of the same band in native BSA. Thisfinding suggests that exposed histidine residues of native BSA,that react quickly with DEPC, are partially inactivated inNAMI-treated BSA samples in agreement with the view thathistidines are primary target sites for ruthenium(III) complexes.The fact that the final difference UV spectra are nonethelessalmost identical may be explained by reminding that BSApossesses several histidine residues and that DEPC, beyond

Fig. 6 CD spectra of ICR/BSA (A) and

Na[transRuCl4(Me2SO)2]/BSA, and (B)

adducts at increasing ruthenium/BSA ratios.

Ruthenium/BSA ratios are: (a) 1 : 1; (b) 2 : 1;

(c) 4 : 1 and (d) 8 : 1. Spectra were recorded

after 24 h incubation at 25 8C. BSA

concentration: 1 � 1023 M. Buffer: phosphate

0.05 m, NaCl 0.1 m, pH 7.4.

1212 L. Messori et al. (Eur. J. Biochem. 267) q FEBS 2000

histidines, can also react with lysine, tyrosine and cysteineresidues [15].

D I S C U S S I O N

Formation and characterization of NAMI/BSA adducts

The reaction of NAMI with bovine serum albumin has beeninvestigated in detail. Spectrophotometric data point out thatserum albumin does not affect substantially the characteristicprofile of NAMI hydrolysis in buffer solution; however, thepresence of BSA renders the hydrolysis rate of the firstruthenium(III)-coordinated chloride group significantly faster.Also, it is evident that BSA does not reduce NAMI to thecorresponding ruthenium(II) complex.

Following chloride hydrolysis, NAMI is able to bind BSAextensively as demonstrated by ultradialysis experiments andFAAS data. A number of NAMI/BSA adducts were obtainedupon treating BSA with increasing amounts of NAMI; forinstance, when working at ruthenium/BSA ratios as high as8 : 1, BSA is able to bind tightly at least five rutheniumequivalents, as indicated by FAAS data. The spectroscopicfeatures of the individual BSA-bound ruthenium centers are fardifferent from one another implying that the respective binding

sites on the protein are distinct. In particular, the first rutheniumequivalent gives rise to peculiar CD features that are suggestiveof the presence of a preformed, high-affinity ruthenium bindingsite. Less characteristic features are observed for the other BSAbound ruthenium(III) centers. Notably at high NAMI/BSAratios the CD spectrum of the first equivalent fades awayprobably as a consequence of the summation of the negativeCD contributions from the other BSA bound ruthenium centers.

Nature and location of BSA-bound ruthenium centers

The spectroscopic results obtained on a number of NAMI/BSAadducts, prepared at increasing ruthenium/BSA stoichiometries,as well as on the corresponding adducts with ICR andNa[transRuCl4(Me2SO)2], allow us to draw some insight onthe nature and the location of the BSA-bound rutheniumcenters. In all cases a similar, although not identical, CDspectral pattern is observed, suggesting that ruthenium bindsessentially the same protein sites in a similar sequence. The factthat NAMI, ICR and Na[transRuCl4(Me2SO)2] exhibit distinct,although very similar, spectral features favors the idea that theaxial ligands are, at least partially, conserved. Failure to observequick reduction of ruthenium(III) upon interaction with BSAsupport the view that BSA-bound ruthenium centers remain inthe +3 oxidation state; in favor of this view is also the fact thatthe adducts prepared with `reduced' NAMI exhibit largelydifferent spectral properties.

A recent crystallographic study of ICR-treated lactoferrinsuggested that solvent exposed imidazole rings of histidinerepresent preferential binding site for ruthenium(III) complexeson the protein surface [13]; several other reports exist in theliterature indicating that histidine residues are preferred bindingsites for ruthenium(III) on the protein surface (e.g. pentam-mineruthenium(III) complex) [18,19]. The results of ourinvestigation are roughly consistent with the hypothesis thatruthenium binding takes place at the level of histidines. Indeed,the band observed in the absorption spectra of NAMI/BSAadducts is centered at 330 nm as in the case of theruthenium(III) imidazole chromophore [20]; in addition thehere reported DEPC modification experiments point out thatruthenated BSA samples are less reactive than native BSAsamples toward diethylpyrocarbonate modification, suggestingthat histidine residues are less accessible after ruthenation.

Implications of protein binding for the activity of NAMI andrelated compounds

Our results point out that NAMI binds serum albuminextensively. Since experimental anti-tumor ruthenium(III)complexes are usually administered intravenously, extensivebinding in vivo of these drugs to plasma proteins may beanticipated, with important consequences on the biodistributionand bioavailability of these compounds. It is probable thatbinding to serum albumin may result into inactivation ofruthenium(III) complexes so that only the free fraction of thecomplex is responsible for the observed biological effects [21].Alternatively the NAMI/BSA adducts might retain, at leastpartially, the biological activity of the original complexes andbehave as carriers of ruthenium inside cells through permeationand/or endocytosis mechanisms. To elucidate this issueexperiments are presently being carried out to compare thepharmacological properties of NAMI/BSA adducts with thoseof free NAMI; similar experiments were previously performedin vitro with transferrin adducts of either H(Im)[trans-RuCl4(Im)2] or H(Ind)[transRuCl4(Ind)2].

Fig. 7. UV Difference spectra of BSA (A) and NAMI/BSA (8 : 1

ruthenium/BSA ratio) (B) samples following modification with DEPC.

20 mm protein samples in buffer: phosphate 0.05 m, pH 7.4 were treated

with DEPC to a final 0.3 mm concentration. The difference spectra were

obtained at time intervals of 1 minute (a±m) and 5 min (n).

q FEBS 2000 Interactions of ruthenium(III) complexes with BSA (Eur. J. Biochem. 267) 1213

Also, it is of interest to consider the potential ofruthenium(III) complexes to bind and modify proteins, as itemerges from the present results and from previous literaturedata. For instance, binding of ruthenium to proteins might causesevere inhibition of some fundamental enzyme activity, thusexplaining the important biological effects produced byanti-tumor ruthenium(III) complexes in vivo and in vitro.Alternatively, direct protein damage might represent thebiological basis for the systemic toxic effects of ruthenium(III)complexes, that are, nevertheless, far more limited than thoseproduced by cisplatin.

Recently, serious doubts were raised on the hypothesis thatthe biological effects of experimental ruthenium(III) anti-cancer drugs are mediated by a direct DNA damage, as in thecase of the classical platinum complexes; modification andinactivation of some yet unidentified target proteins might wellconstitute the molecular basis for the mechanism of anti-tumorruthenium(III) compounds [22].

Concluding remarks.

Overall, the above reported results show that: (a) NAMI bindsBSA up to a final stoichiometry of 5 ruthenium moles per BSAmole; (b) structurally different BSA sites are sequentiallyoccupied by ruthenium(III) ions; (c) nonlabile axial ligands areprobably retained upon binding; (d) the oxidation state ofbound ruthenium is +3; and (e) probable protein ligands forNAMI are solvent exposed His residues.

A C K N O W L E D G E M E N T S

The Ministry of University and Scientific and Technologic Research is

acknowledged for financial support in the frame of the project

`Pharmacological and diagnostic aspects of metal complexes'. The Cassa

di Risparmio di Firenze is gratefully acknowledged for a generous grant.

We thank Prof. B. K. Keppler for the gift of ICR samples.

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