Báo cáo Y học: Effect of ibuprofen and warfarin on the allosteric properties of haem– human serum albumin A spectroscopic study potx

Effect of ibuprofen and warfarin on the allosteric properties of haem–human serum albumin A spectroscopic study Simona Baroni 1 , Marco Mattu 2 , Alessandro Vannini 2 , Rita Cipollone 2 , Silvio Aime 1 , Paolo Ascenzi 2 and Mauro Fasano 3 1 Department of Chemistry ‘IFM’, University of Torino, Italy; 2 Department of Biology, University ‘Roma Tre’, Rome, Italy; 3 Department of Structural and Functional Biology, University of Insubria, Italy Haem binding to human serum albumin (HSA) endows the protein with peculiar spectroscopic properties. Here, the effect of ibuprofen and warfarin on the spectroscopic properties of ferric haem–human serum albumin (ferric HSA–haem) and of ferrous nitrosylated haem –human serum albumin (ferrous HSA–haem-NO) is reported. Ferric HSA–haem is hexa-coordinated, the haem-iron atom being bonded to His105 and Tyr148. Upon drug binding to the warfarin primary site, the displacement of water molecu- les 2 buried in close proximity to the haem binding pocket 2 induces perturbation of the electronic absorbance properties of the chromophore without affecting the coordination number or the spin state of the haem-iron, and the quenching of the 1 H-NMR relaxivity. Values of K d for ibuprofen and warfarin binding to the warfarin primary site of ferric HSA– haem, corresponding to the ibuprofen secondary cleft, are 5.4 ^ 1.1  10 24 M and 2.1 ^ 0.4  10 25 M, respectively. The affinity of ibuprofen and warfarin for the warfarin primary cleft of ferric HSA– haem is lower than that reported for drug binding to haem- free HSA. Accordingly, the K d value for haem binding to HSA increases from 1.3 ^ 0.2  10 28 M in the absence of drugs to 1.5 ^ 0.2  10 27 M in the presence of ibuprofen and warfarin. Ferrous HSA – haem-NO is a five-coordinated haem-iron system. Drug binding to the warfarin primary site of ferrous HSA–haem-NO induces the transition towards the six-coordinated haem-iron species, the haem-iron atom being bonded to His105. Remarkably, the ibuprofen primary cleft appears to be functionally and spectroscopically uncoupled from the haem site of HSA. Present results represent a clear-cut evidence for the drug-induced shift of allosteric equilibrium(a) of HSA. Keywords: allostery; haem–human serum albumin; human serum albumin; ibuprofen; warfarin. Human serum albumin (HSA), the most prominent protein in plasma, is best known for its exceptional ligand binding capacity, the most strongly bound compounds being hydrophobic organic anions of medium size, long-chain fatty acids, haem and bilirubin. Smaller and less hydrophobic compounds (e.g. tryptophan) are held less strongly, but their binding can still be highly specific. For many compounds, HSA provides a depot so they will be available in quantities well beyond their solubility in plasma. Moreover, HSA abundance (concentration of 45 mg : mL 21 in the serum of human adults) makes it an important determinant of the pharmacokinetic behaviour of many drugs. In other cases, HSA holds some ligands in a strained orientation, allowing their metabolic modification, and renders potential toxins harmless by transporting them to disposal sites. HSA also accounts for most of the antioxidant capacity of human serum, either directly or by binding and carrying radical scavengers, or by sequestering transition metal ions with pro-oxidant activity. Finally, HSA acts as a nitric oxide depot and carrier, leading to covalent modification(s) of (macro)molecules [1–8]. The amino acid sequence of HSA shows the occurrence of three homologous domains, probably arising from divergent evolution of a degenerated ancestral gene followed by a fusion event. However, the three domains deduced from the primary structure do not correspond to domains found in the three-dimensional model. Rather, terminal regions of sequential domains contribute to the formation of inter- domain helices linking domain I to II, and II to III, respectively. On the other hand, each domain is known to consist of two separate sub-domains, connected by a random coil. Therefore, HSA can be considered as an ensemble of four globular domains, namely IA, IB 1 IIA, IIB 1 IIIA, and IIIB, freely linked by extended random coils. It is thus reasonable to hypothesize allosteric conformational tran- sition(s) occurring in HSA upon ligand binding. Note that the flexibility of the HSA structure allows it to adapt readily to ligands and that its three-domain design provides a variety of binding sites. In particular, the conformational adaptability of HSA involves more than the immediate Correspondence to M. Fasano, Department of Structural and Functional Biology, University of Insubria, Via Jean H. Dunant 3, I-21100 Varese, Italy. Fax: 1 39 0332 421500, Tel. : 1 39 0332 421523, E-mail: mauro.fasano@uninsubria.it Note: S. Baroni and M. Mattu contributed equally to this work. (Received 2 July 2001, revised 27 September 2001, accepted 3 October 2001) Abbreviations: HSA, human serum albumin; ferric HSA –haem, ferric haem– human serum albumin; ferrous HSA –haem-NO, ferrous nitrosylated haem–human serum albumin. Eur. J. Biochem. 268, 6214–6220 (2001) q FEBS 2001 vicinity of the binding site(s), affecting both the structure and the ligand binding properties of the whole HSA molecule [1– 11]. The interaction of ligands with HSA occurs mainly in two regions. According to the Sudlow’s nomenclature, bulky heterocyclic anions bind to site I (located in subdomain IIA), whereas site II (located in subdomain IIIA) is preferred by aromatic carboxylates with an extended conformation. Remarkably, ibuprofen, a nonsteroidal anti-inflammatory agent [12], and warfarin, an anticoagulant drug [12], are considered as stereotypical ligands for Sudlow’s site II and Sudlow’s site I, respectively [1,9,11,13,14]. Ibuprofen binds to Sudlow’s site II with K d ¼ 3.7  10 27 M [1,15], whereas warfarin binds to Sudlow’s site I with K d ¼ 3.0  10 26 M [1,16–18]. Secondary binding clefts have been found for ibuprofen and warfarin to be located on domain I. Remarkably, the ibuprofen secondary site corresponds to the warfarin primary cleft (i.e. to Sudlow’s site I) [1,3,11]. Moreover, multiple recognition sites for binding of anaesthetics, fatty acids, and triiodobenzoic acid to HSA have also been identified [2,5,6,14,19]. Among hydrophobic molecules, haem binding to HSA is of peculiar relevance for the haem iron reuptake following hemolytic events [1,20]. The haem binding site has been located primarily at the interface between domains I and II of HSA, while on domains II and III secondary binding clefts have been found [3,7,10]. The binding of this spectroscopically active label to HSA makes it possible to follow a number of events involving the holoprotein by taking advantage of electronic absorption spectroscopy, EPR spectroscopy and 1 H-NMR relaxometry [7,8,10,21,22]. Here, the effect of ibuprofen and warfarin on the electronic absorption spectroscopic and 1 H-NMR-relaxometric proper- ties of ferric haem –human serum albumin (ferric HSA – haem) as well as on the EPR spectroscopic properties of ferrous nitrosylated haem–human serum albumin (ferrous HSA–haem-NO) is reported. MATERIALS AND METHODS HSA, haem chloride, ibuprofen (Fig. 1), warfarin (see Fig. 1), and the NO-donor S-nitroso-N-acetylpenicillamine were from Sigma. Gaseous NO was purchased from Aldrich Chemical Co. All the other products were from Merck AG. All chemicals were of analytical or reagent grade and used without further purification. Ferric HSA–haem was prepared by adding 0.83- M defect of ferric haem, dissolved in 1.0  10 21 M KOH, to an HSA solution, in 1.0  10 21 M phosphate buffer plus 1.0  10 21 M NaCl, pH 7.0 [8,21]. Both in the absence and presence of ibuprofen and warfarin, haem binds to HSA mostly at the high affinity site and virtually no free haem is present in solution (see [1,8,21], and present study). Values of the apparent dissociation equilibrium constant (K d ) for haem binding to the HSA primary site are 1.3 ^ 0.2  10 28 M in the absence of drugs, and 1.5 ^ 0.2  10 27 M in the presence of ibuprofen and warfarin (5.0  10 22 M) (see [1,22,23] and the present study). Ferrous HSA – haem-NO was obtained under anaerobic conditions: (a) by sequential addition of a 10- M excess of sodium dithionite or sodium ascorbate and a 4- M excess of KNO 2 to ferric HSA–haem, in 1.0  10 21 M phosphate buffer plus 1.0  10 21 M NaCl, pH 7.0; (b) by blowing purified NO over the ferric HSA– haem solution (1.0  10 21 M phosphate buffer plus 1.0  10 21 M NaCl, pH 7.0), in the absence and presence of a 5- M excess of sodium dithionite or sodium ascorbate; and (c) by sequential addition of a 10- M excess of dithiothreitol and a 4-M excess of S-nitroso-N-acetylpenicillamine (which releases NO) to ferric HSA –haem, in 1.0  10 21 M phosphate buffer plus 1.0  10 21 M NaCl, pH 7.0 [8,21]. HSA solutions were prepared by dissolving the protein in 1.0  10 21 M phosphate buffer plus 1.0  10 21 M NaCl, at pH 7.0 and 25.0 8C. Haem solutions were prepared in 1.0  10 21 M KOH. Ibuprofen solutions were prepared by dissolving the drug in 1.0  10 21 M phosphate buffer plus 1.0  10 21 M NaCl, at pH 7.0 and 25.0 8C. Warfarin solutions were prepared by stirring the drug in 1.0  10 21 M phosphate buffer plus 1.0  10 21 M NaCl at pH 12.0 until it dissolved, then adjusting to pH 7.0 with HCl (at 25.0 8C). Binding of ibuprofen and warfarin to ferric HSA –haem was followed by electronic absorption spectroscopy at pH 7.0, in 1.0  10 21 M phosphate buffer plus 1.0  10 21 M NaCl, and 25.0 8C. Electronic absorption spectra of ferric HSA–haem (5.0  10 26 M to 2.0  10 24 M), in the absence and presence of ibuprofen and warfarin (1.0  10 24 M to 5.0  10 22 M), were collected between 350 nm and 700 nm. The electronic absorption spectra were recorded in 1-mm to 1-cm path length cuvettes. The ibuprofen- and warfarin-induced electronic absorption spectroscopic transition of ferric HSA–haem was complete within the time to achieve the sample preparation (, 10 min). Test measurements performed after 2 h excluded slow kinetic effects. Fig. 1. Chemical structures of ibuprofen and warfarin. q FEBS 2001 Drug binding to HSA–haem (Eur. J. Biochem. 268) 6215 Binding of ibuprofen and warfarin to ferric HSA –haem was also followed by 1 H-NMR relaxometry at pH 7.0 (1.0  10 21 M phosphate buffer plus 1.0  10 21 M NaCl) and 25.0 8C. 1 H-NMR relaxometry of ferric HSA–haem (1.0  10 23 M) in the absence and presence of ibuprofen and warfarin (1.0  10 24 M to 1.0  10 21 M) was investi- gated on a Stelar SpinMaster Spectrometer (Stelar S.n.c., Mede, PV, Italy). Water proton relaxation rate (R 1 ) measurements were obtained at 0.47 T (i.e. at 20 MHz proton Larmor frequency) by means of the Inversion- Recovery technique (16 experiments, four scans). Magneti- zation values were obtained by averaging the first 128 data points of the Free Induction Decay. A phase cycle (1x, –x, –x, 1x) was applied on the 908 observation pulse to cut off the y-scale receiver offset. A typical 908 pulse width was 3.5 ms. The t -values were increased linearly from a starting value corresponding to one-seventh of the estimated null-point (0.693/R 1 ), so that the null-point occurs on the middle of the inversion-recovery curve (seventh experiment). In the 16th experiment the Free Induction Decay is acquired after a single 908 pulse, to obtain the M 1 value [24]. The reproducibility in R 1 measurements was ^ 0.5%. The temperature was controlled by a Stelar VTC- 91 airflow heater (Stelar S.n.c.), equipped with a copper- constantan thermocouple; the actual temperature in the probe head was measured with a Fluke 52k/j digital thermometer (Fluke AG, Zu ¨ rich, Switzerland), with an uncertainty of ^ 0.3 8C. Values of the paramagnetic contribution to the overall water solvent relaxation rate (R 1p ) were determined by subtracting from the observed relaxation rate (R obs 1 ) the blank relaxation rate value (R dia 1 ) measured for solutions containing HSA at the same concentration without the paramagnetic prosthetic group. Test measurements performed after 2 h excluded slow kinetic effects [7]. Haem binding to HSA in the absence and presence of ibuprofen and warfarin was followed by electronic absorption spectroscopy (between 350 nm and 450 nm) at pH 7.0 (1.0  10 21 M phosphate buffer plus 1.0  10 21 M NaCl) and 25.0 8C [22]. The HSA concentration ranged between 3.0  10 28 M and 2.0  10 26 M, the haem concentration was 1.0  10 27 M, and the ibuprofen and warfarin concentrations were 5.0  10 22 M. The electronic absorption spectra were recorded in a 10-cm path length cuvette. The haem-induced electronic absorption spectro- scopic transition of HSA was complete within the time to achieve the sample preparation (, 10 min). Test measurements performed after 2 h excluded slow kinetic effects. Binding of ibuprofen and warfarin to ferrous HSA– haem- NO was followed by X-band EPR spectroscopy at pH 7.0 in 1.0  10 21 M phosphate buffer plus 1.0  10 21 M NaCl, and 2173 8C. X-band EPR spectra of ferrous HSA–haem- NO (3.0  10 24 M) in the absence and presence of ibuprofen and warfarin (5.0  10 22 M) were collected on a Bruker ESP 300 spectrometer, operating at 9.42 GHz microwave frequency, 100 kHz field modulation, 20 mW microwave power, and 0.10 mT modulation amplitude. The ibuprofen- and warfarin-induced EPR-spectroscopic tran- sition of ferrous HSA –haem-NO was complete within the time to achieve the sample preparation (, 10 min). Test measurements performed after 2 h excluded slow kinetic effects [8]. RESULTS AND DISCUSSION Fig. 2 shows the electronic absorption spectra of ferric HSA–haem in the absence and presence of ibuprofen and warfarin at pH 7.0 and 25.0 8C. Electronic absorbance spectroscopy and 1 H-NMR relaxometry indicate that the haem iron atom of ferric HSA–haem is hexa-coordinated, possibly being bonded to His105 and Tyr148 as suggested by docking simulations [7]. Upon drug binding, neither a change in the haem-iron atom coordination number, nor in the spin state of the metal centre, is observed. Spectra shown in Fig. 2 are indicative of a high-spin state of the haem-iron. Actually, even the minor low-spin component [22] seems to diminish in the presence of either drug. In fact, the Soret band is blue-shifted, the charge transfer band is red-shifted and the a band is decreased in intensity with respect to the b band. The spectral features shown in Fig. 2 are indicative of a drug-dependent conformational transition(s) that does not affect the inner coordination sphere of the haem iron atom. Fig. 2. Effect of ibuprofen and warfarin on the electronic absorption spectroscopic properties of ferric HSA–haem. Elec- tronic absorption spectra of ferric HSA–haem were obtained in the absence (spectrum a) and in the presence of 5.0  10 22 M ibuprofen (spectrum b, continuous line) and 5.0  10 22 M warfarin (spectrum b, filled circles) at pH 7.0 and 25.0 8C. The electronic absorption spectra of ferric HSA –haem in the presence of ibuprofen and warfarin are superimposable. The ferric HSA–haem concentration was 8.4  10 26 M. The electronic absorption spectra were recorded in a 1-cm path length cuvette. 6216 S. Baroni et al. (Eur. J. Biochem. 268) q FEBS 2001 Fig. 3 shows the binding isotherms of ibuprofen and warfarin to ferric HSA–haem, at pH 7.0 and 25.0 8C. Data obtained by different techniques (i.e. electronic absorption spectroscopy and 1 H-NMR relaxometry) are in good agreement. By applying the minimum model accounting for multiple binding sites per monomeric protein, a relationship between the apparent equilibrium dissociation constant (K d ) for ibuprofen and warfarin binding to ferric HSA–haem and the molar fraction of the ligand-bound ferric HSA–haem (a) may be expressed according to Eqn (1) [25]: a ¼ð½L T 2 ½L b Þ n /K d 1 ð½L T 2 ½L b Þ n fg ð1Þ where n is the Hill coefficient, and [L] is the ligand (i.e. drug, HSA, HSA : ibuprofen, or HSA : warfarin) concen- tration in the forms indicated by subscripts T (total) and b (bound), respectively. [L] b was calculated according to Eqn (2) [25]: ½L b ¼ K d 1 n : ½Q T 1 ½L T 2 p ðK d 1 n : ½Q T 1 ½L T Þ 2 ÈÈ 24K d : n : ½Q T : ½L T gg / 2 ð2Þ where [Q] T is the total ferric HSA–haem or haem concentration. The analysis of data given in Fig. 3 according to Eqn (1) allowed the determination of values of K d (¼ 5.4 ^ 1.1  10 24 M) and n (¼ 1.9 ^ 0.1) for ibuprofen binding to ferric HSA –haem, and of K d (¼ 2.1 ^ 0.4  10 25 M) and n (¼ 2.7 ^ 0.1) for ferric HSA–haem : warfarin complex formation at pH 7.0 and 25.0 8C. The K d value for ibuprofen binding to ferric HSA –haem is higher than those reported for drug binding to the ibuprofen primary site (K d ¼ 3.7  10 27 M, at pH 7.4 and 37.0 8C) [1,15] and to the ibuprofen secondary cleft (K d < 4  10 25 M at pH 7.4 and 37.0 8C) [1,15] of haem-free HSA. Also the K d value for warfarin binding to ferric HSA–haem is higher than that reported for drug binding to the warfarin primary site (K d ¼ 3.0  10 26 M at pH 7.4 and 25.0 8C) [1,16 – 18] of haem-free HSA. Fig. 4 shows the binding isotherms of ferric haem to HSA in the absence and presence of ibuprofen and warfarin, as obtained by electronic absorption spectroscopy at pH 7.0 and 25.0 8C. Data analysis according to Eqn (1) allowed the determination of values of the apparent dissociation equilibrium constant (K d ) and of the Hill coefficient (n ) for haem binding to HSA, in the absence and presence of ibuprofen and warfarin. The K d value for ferric haem binding to HSA, in the absence of drugs, is 1.3 ^ 0.2  10 28 M at pH 7.0 and 25.0 8C. Remarkably, the K d value for ferric haem binding to HSA determined here (see Fig. 4) is in excellent agreement with that reported in the literature (K d ¼ 1  10 28 M at pH 7.0 and 24.0 8C) Fig. 3. Ibuprofen and warfarin binding to ferric HSA– haem. Electronic absorption spectroscopic and 1 H-NMR relaxometric binding isotherms of ibuprofen and warfarin to ferric HSA–haem were obtained at pH 7.0 and 25.0 8C. Circles and squares indicate data obtained by electronic absorption spectroscopy and 1 H-NMR relaxometry, respectively. The continuous lines were obtained by using Eqn (1). Best fitting parameters are K d ¼ 5.4 ^ 1.1  10 24 M and n ¼ 1.9 ^ 0.1 for ibuprofen binding, and K d ¼ 2.1 ^ 0.4  10 25 M and n ¼ 2.7 ^ 0.1 for warfarin binding. The ferric HSA–haem concentration was 1.5  10 24 M and 1.0  10 23 M for electronic absorption spectroscopic and 1 H-NMR relaxometric experiments, respectively. The electronic absorption spectra were recorded in a 1-mm path length cuvette. Fig. 4. Haem binding to HSA. Electronic absorption spectroscopic binding isotherms of haem to HSAwere obtained in the absence (A) and presence of 5.0  10 22 M ibuprofen (K) and 5.0  10 22 M warfarin (W) at pH 7.0 and 25.0 8C. The continuous lines were obtained by using Eqn (1). Best fitting parameters for ferric HSA–haem formation are K d ¼ 1.3 ^ 0.2  10 28 M and n ¼ 1.0 ^ 0.1, in the absence of drugs, and K d ¼ 1.5 ^ 0.2  10 27 M and n ¼ 1.0 ^ 0.1, in the presence of ibuprofen and warfarin. The haem concentration was 1.0  10 27 M. The electronic absorption spectra were recorded in a 10-cm path length cuvette. For further details, see text. q FEBS 2001 Drug binding to HSA–haem (Eur. J. Biochem. 268) 6217 [23]. In the presence of saturating amounts of ibuprofen and warfarin (5.0  10 22 M) the affinity of haem for HSA decreases by about one order of magnitude, the drug- independent K d value being 1.5 ^ 0.2  10 27 M at pH 7.0 and 25.0 8C. In the absence and presence of drugs, the value of n for haem binding to HSA is 1.0 ^ 0.1 at pH 7.0 and 25.0 8C (see Fig. 4). Data reported in Figs 3 and 4 indicate that haem binding to HSA inhibits ibuprofen and warfarin association to the warfarin primary cleft (i.e. Sudlow’s site I), corresponding to the ibuprofen secondary site [1,3,11]. Then, ibuprofen and warfarin impair ferric HSA– haem formation. Remark- ably, the ibuprofen primary cleft (i.e. Sudlow’s site II) appears to be functionally and spectroscopically uncoupled to the haem site of HSA. Ferric HSA–haem has been widely investigated by 1 H-NMR relaxometry [7]. The high value of the paramagnetic contribution to the water relaxation rate (R 1p )of hexacoordinated ferric HSA– haem (¼ 4.8 m M 21 : s 21 at 20 MHz, pH 7.2 and 25 8C) has been ascribed to the occurrence of slowly exchanging water molecules in the surroundings of the paramagnetic ferric haem center [7]. In the presence of saturating amounts of ibuprofen and warfarin, the R 1p value of hexacoordinated ferric HSA– haem decreases to 0.4 m M 21 : s 21 at 20 MHz, pH 7.0 and 25 8C (data not shown). The decrease of the R 1p value upon drug binding may reflect a conformational transition(s) towards a ferric HSA – haem state where slowly exchanging water molecules are far apart from the paramagnetic centre. On the other hand, the lifetime of the ferric haem centre hydration shell could be shortened to approach the diffusion mean time [26–28]. Fig. 5 shows a ribbon diagram of HSA (PDB code 1E78) [5]. Remarkably, a cavity hosting three water molecules can be located at the interface between the haem cleft and the warfarin primary site (i.e. Sudlow’s site I). These water Fig. 5. Sudlow’s site I and haem cleft location in HSA. Buried water molecules (blue spheres) are located at the interface between the warfarin primary site (i.e. Sudlow’s site I; highlighted in green) and the haem cleft (traced in red). The HSA backbone is rendered as a ribbon model. HSA atomic coordinates were recovered from the Protein Data Bank (PDB ID: 1E78) [19]. For details, see [5,7,10,14] and text. Fig. 6. Effect of ibuprofen and warfarin on the EPR spectroscopic properties of ferrous HSA– haem-NO. X-band EPR spectra of ferrous HSA–haem-NO were obtained in the absence (spectrum a) and in the presence of 5.0  10 22 M ibuprofen (spectrum b, continuous line) and 5.0  10 22 M warfarin (spectrum b, filled circles) at pH 7.0 and 2173 8C. The X-band EPR spectra of ferrous HSA–haem-NO in the presence of ibuprofen and warfarin are superimposable. The ferrous HSA–haem-NO concentration was 3.0  10 24 M. Table 1. X-band EPR parameters and the haem-iron coordination state of HSA–haem-NO. Values listed are for 14 N systems. Experimental conditions were pH 7.0 and 2173 8C. US, unresolved signal. Conditions A 3 (mT) g 1 g 2 g 3 Coordination state Stripped a 1.65 2.095 2.060 2.010 Five Bezafibrate b US 2.064 1.983 2.005 Six Clofibrate b US 2.064 1.983 2.005 Six Ibuprofen a US 2.064 1.983 2.005 Six Warfarin a US 2.064 1.983 2.005 Six a Present study. b From [8]. 6218 S. Baroni et al. (Eur. J. Biochem. 268) q FEBS 2001 molecules are able to exchange with the bulk solvent typically on the submicrosecond timescale, which is sufficient to promote relaxation of the observed water protons [27]. Therefore, upon drug binding, these water molecules are displaced or, alternatively, their residence lifetime is reduced. In either case, a quenching of the paramagnetic contribution to the observed relaxation rate is expected [7,24,26]. Fig. 6 shows the X-band EPR spectra of ferrous HSA– haem-NO in the absence and presence of ibuprofen and warfarin. Ferrous HSA–haem-NO samples obtained by different methods give identical X-band EPR spectra. In the absence of any allosteric effector, the X-band EPR spectrum of ferrous HSA–haem-NO displays a three-line splitting (A 3 ¼ 1.65 mT) in the high magnetic field region (g 3 ¼ 2.010) (see Fig. 6, spectrum a, and Table 1). This X-band EPR spectrum has been associated with the five- coordinate haem-iron state of ferrous HSA– haem-NO [8], in agreement with data reported for several ferrous nitrosylated haemoglobin systems [8,29–34]. Addition of either ibuprofen or warfarin to ferrous HSA–haem-NO induces the transition towards a species characterized by an X-band EPR spectrum with a rhombic shape and a weak superhyperfine pattern in the g z region (see Fig. 6, spectrum b, and Table 1). Such behaviour is similar to that observed in the presence of bezafibrate and clofibrate, which has been attributed to the shift of the conformational equilibrium towards the six-coordinated haem-iron state of ferrous HSA–haem-NO [8]. The His242 residue has been postulated to be a likely candidate for the sixth axial ligand of the haem iron in ferrous HSA–haem-NO in the presence of bezafibrate and clofibrate [8]. However, more recent results are consistent with the suggestion that His105 might be responsible for the sixth axial bonding of the haem iron [7,10,11]. Remarkably, His242 has been shown to be hydrogen-bonded to warfarin [14]. As the binding of warfarin to Sudlow’s site I does not affect the coordination state of haem iron (see Fig. 2), His242 does not appear to be a likely candidate for the axial haem bonding. CONCLUSIONS The effect of ibuprofen and warfarin on the electronic absorption spectroscopic, 1 H-NMR relaxometric and X-band EPR spectroscopic properties of ferric HSA–haem and ferrous HSA–haem-NO is in keeping with the allosteric conformational transition(s) induced by these therapeutic drugs in HSA. Note that the Sudlow’s site I and the Sudlow’s site II ligands induce the N (normal) to B (basic) conformer transition in HSA. Accordingly, the affinity of therapeutic drugs for the B-form of HSA is higher than that observed for the N-form [1,3,4,14,35,36]. Haem inhibits drug binding, possibly stabilizing the N-species of HSA (present study). Accordingly, binding of ibuprofen and warfarin to Sudlow’s site I impairs ferric HSA – haem formation (present study). Finally, drug-dependent spectro- scopic properties of ferric HSA–haem and ferrous HSA– haem-NO may be helpful in investigating ligand binding and allosteric properties of HSA, as already reported for haemoglobin (see [37,38]). Therefore, the therapeutic use of warfarin and ibuprofen may affect haem transfer to hemopexin and consequently its plasma level. In parallel, haem may affect pharmacokinetics of drugs carried out by HSA. ACKNOWLEDGEMENTS The authors thank M. Coletta and A. Desideri for helpful discussions and F. Tiberi for technical assistance. This work was partially supported by grants from the Ministry for University, Scientific Research and Technology of Italy (MURST ‘Fondi per lo Sviluppo, Universita ` Roma Tre 2001’ to P. A., and MURST ‘Projects of Relevant National Interest’ to S. A.), as well as from the National Research Council of Italy (CNR, Target oriented project ‘Biotecnologie’ to P. A. and M. F.). S. 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(1981) Molecular aspects of ligand binding to serum albumin. Pharmacol. Rev. 33, 17–53. 37. Ascenzi, P., Bertollini, A., Coletta, M. & Lucacchini, A. (1999) Stabilization of the T-state of ferrous human adult haemoglobin by chlorpromazine and trifluoperazine. Biotechnol. Appl. Biochem. 30, 185– 187. 38. Ascenzi, P., Colasanti, M., Fasano, M. & Bertollini, A. (1999) Stabilization of the T-state of human hemoglobin by proflavine, an antiseptic drug. Biochem. Mol. Biol. Int. 47, 991–995. 6220 S. Baroni et al. (Eur. J. Biochem. 268) q FEBS 2001 . Effect of ibuprofen and warfarin on the allosteric properties of haem human serum albumin A spectroscopic study Simona Baroni 1 , Marco Mattu 2 , Alessandro Vannini 2 , Rita Cipollone 2 ,. not appear to be a likely candidate for the axial haem bonding. CONCLUSIONS The effect of ibuprofen and warfarin on the electronic absorption spectroscopic, 1 H-NMR relaxometric and X-band EPR spectroscopic. extended conformation. Remarkably, ibuprofen, a nonsteroidal anti-inflammatory agent [12], and warfarin, an anticoagulant drug [12], are considered as stereotypical ligands for Sudlow’s site II and Sudlow’s