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Mechanism of binding of warfarin enantiomers to recombinant domains of human albumin
ABB Archives of Biochemistry and Biophysics 414 (2003) 83–90 www.elsevier.com/locate/yabbi
Mechanism of binding of warfarin enantiomers to recombinant domains of human albumin S.M. Twine,a,1 M.G. Gore,a P. Morton,b B.C. Fish,b,2 A.G. Lee,a and J.M. Easta,* a
Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, SO16 7PX, UK b Delta Biotechnology Limited, Castle Boulevard, Nottingham, NG7 1FD, UK Received 8 January 2003, and in revised form 20 March 2003
Abstract Domain fragments of human serum albumin corresponding to domains 1 and 2 (D12) and domains 2 and 3 (D23) were expressed in yeast. The kinetics of warfarin binding to these fragments were investigated using stopped-flow fluorescence spectroscopy. Binding can be characterized by a two-step process, a rapid diffusion-controlled step and a slower rate-limiting step in which a stable drug–protein complex is formed. The equilibrium constant for step 1 is greater for both D12 and D23 than for albumin, probably as a result of reduced steric hindrance offered by the domain fragments. Binding step 2, thought to be the result of a conformational change as warfarin is accommodated by the protein, is faster for D12 and D23. Albumin and the domain fragments show an increased preference for the R enantiomer, but the preference is particularly enhanced for domain fragment D12. These preferences can largely be explained by the domains having different rates for step 2 of the binding process. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Albumin; Warfarin; Ligand binding; Fluorescence; Enantiomers; Drug binding; Stopped flow
Introduction As the most abundant human plasma protein, human serum albumin (HSA)3 has numerous functions including regulation of colloid osmotic pressure and transport of various endogenous ligands throughout the circulation [1]. HSA also binds a wide variety of drug molecules and is an important factor in determining drug pharmacokinetics including distribution and elimination [1,2]. A large number of drugs are chiral and in most cases only one enantiomer is responsible for therapeutic activity [3]. Albumin has been shown to discriminate between various pairs of enantiomers [1]. The anticoagulant warfarin and the nonsteroidal antiinflammatory drug ibuprofen are examples of chiral * Corresponding author. Fax: +44-2380-594-459. E-mail address: [email protected] (J.M. East). 1 Present address: National Research Council, Institute of Biological Sciences, 100 Sussex Drive, Ottawa, Ontario, Canada. 2 Present address: Cambridge Antibody Technology Limited, Milstein Building, Granta Park, Cambridge, CB1 6GH, UK. 3 Abbreviation used: HSA, human serum albumin; rHA, recombinant human albumin.
drugs that have been extensively characterized [2,4]. Warfarin has a single chiral center, and the stereoisomers have been reported to bind to albumin with a modest variation in affinity [1]. A recent crystallographic study has revealed some clues about the relative positions of the enantiomers within the binding site [5]. Such studies do not, however, provide information with regard to the mechanism by which albumin discriminates between the enantiomers. HSA contains a single polypeptide chain of 585 amino acid residues with three structurally homologous a-helical domains (I–III). Each of these domains can be further divided into subdomains A and B containing six and four a helices, respectively [6,7]. Numerous studies have shown the presence of two major drug-binding sites on the protein [1] designated sites I and II by Sudlow et al. [8]. Subsequent crystallographic studies have mapped sites I and II to subdomains IIA and IIIA, respectively [6]. Both competition binding methods and crystallographic methods have mapped the warfarin binding site to Sudlow et al.Õs site I [1,2]. The dissociation constant for drug binding can provide information with regard to the chiral selectivity of a
0003-9861/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-9861(03)00173-5
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protein, but it provides no information about the rate of formation and dissociation of the drug–protein complex. These data may be obtained by studying the kinetics of drug binding to albumin. Such studies have been conducted [5,9,10] and the data were consistent with a two-step binding mechanism, where the first step is rapid and diffusion controlled and the second step is rate limiting and probably the result of a conformational change [2]. In this study we investigated the mechanism of binding of warfarin enantiomers to recombinant human albumin (rHA) to determine the step(s) in the binding mechanism responsible for chiral discrimination of warfarin enantiomers. Domain fragments corresponding to domains 1 and 2 (residues 1– 387, designated D12) and to domains 2 and 3 (residues 183–585, designated D23) were used to further dissect the warfarin-binding properties of albumin and the potential role of interdomain interactions in this process.
Materials and methods Essentially fatty acid-free human serum albumin was obtained from Sigma. rHA was obtained from Delta Biotechnology Ltd. (Notthingham, UK). All proteins were delipidated by the method of Chen [11]. Ligands of the highest purity were purchased from Sigma, with the exception of warfarin enantiomers, which were purified by the method of West et al. [12].
gation, followed by fourfold dilution and adjustment of the pH to 9.0. The diluted supernatant (32 l) was passed through a diethylaminoethylcellulose column, equilibrated with 0.1 M sodium acetate buffer, pH 9.0. After washing with 10 column volumes of equilibration buffer, elution was carried out with a 0–1 M NaCl gradient. Samples were then concentrated by filtration using a stirred cell (Amicon) to a final concentration of 100 mg/ ml and were applied to a 500-ml Sephacryl S200 column equilibrated with phosphate-buffered saline. Protein purity was assessed by SDS–PAGE analysis. Concentrations of the domain fragments were estimated using e280 , calculated on the basis of amino acid composition [18]. Far-UV circular dichroism Far-UV CD measurements were carried out using a Jasco J720 spectropolarimeter, calibrated as per the manufacturerÕs specifications. Spectra were recorded using 1 mg/ml solutions of each protein in buffer (0.1 M sodium phosphate, pH 7.4, containing 0.14 M NaCl) in a cell of 0.01 cm path length. Scans were carried out between 200 and 250 nm with a bandwidth of 1.0 nm and a scan speed of 5 nm/min. Each spectrum was the average of five accumulations. Secondary structure estimation was carried out using Jasco J-700 Secondary Structure Estimation software. Fluorescence assays
Cloning and expression of domain fragments Cloning. D23 was constructed by amplification of the N-terminal 60 amino acids of domain 2 by the polymerase chain reaction with Pvu polymerase (Boehringer) using a cDNA clone of HSA as a template. The PCR product was then cloned into pAYE 467 digested with NcoI/HindIII containing the C-terminal region of domain 2 and the complete domain 3. Clones were confirmed by DNA sequencing. The D23 expression cassette, containing a leader sequence and the alcohol dehydrogenase terminator, was then ligated into the Escherichia coli–yeast shuttle vector pSAC 35 [13,14] digested with NcoI. Recombinant plasmids were then transformed into yeast strains by electroporation [15]. Transformants were screened for the ability to secrete D23 using nutrient agar plates containing anti-HSA antibodies, as described previously [16]. The cloning of D12 was carried out in the same manner. Expression. Standard protocols and media formulations were used. High-cell-density fermentation was carried out in minimal media in 10-l fed-batch culture essentially as described by Collins [17]. Purification. All manipulations were carried out at ambient temperature unless stated otherwise. Fermentation culture supernatants were harvested by centrifu-
Unless otherwise stated the fluorescence assays were performed at 25 °C in 0.1 M sodium phosphate buffer, pH 7.4, containing 0.14 M NaCl, using Hitachi F-4500 or Perkin–Elmer LS-3B spectrofluorimeters. Ligands were added to assay volumes of 3 ml from 3 mM stock solutions in methanol. Concentration of warfarin stock solutions were confirmed using e310 (13,610 M1 cm1 ). Excitation and emission wavelengths were 330 and 375 nm, respectively. Stopped-flow fluorescence spectrofluorimetry Stopped-flow experiments were carried out using an Applied Photophysics SX-18MV (deadtime 1.5 ms) as outlined previously [19]. The temperatures of both drive syringes and the reaction path were controlled at 25 °C by a circulating water bath. The excitation wavelength (330 nm) was selected by a monochromator with a 1-mm slit width and fluorescence emission was detected at right angles to the exciting beam through a 360-nm cutoff filter. Equal volumes (100 ll) of solutions of protein (in 100 mM phosphate or 40 mM tricine buffer at a given pH) and warfarin (in the same buffer) were rapidly mixed. The concentration of protein after mixing was 1 lM for rHA and 0.1 lM for the domain frag-
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ments. The lowest ligand concentration used was a minimum of 10-fold excess over the protein concentration which allowed the observed rate constant kobs , of the reaction to be analyzed as pseudo-first order processes [19] using the relationship Ft ¼ F0 expkt þC;
ð1Þ
where F0 and Ft are the fluorescence intensities at time 0 and time t respectively, k is the apparent first order rate constant for the reaction, and C is a constant. Each trace analyzed was the average of three to nine individual determinations. The values of k2 were determined experimentally using phenylbutazone to displace warfarin from a preformed complex with albumin or albumin domains; 1 lM solutions of protein–warfarin complexes were mixed 1:1 v/v with 100–500 lM phenylbutazone. Treatment of kinetic data
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Results Fig. 1A illustrates the SDS–PAGE analysis of rHA and domain fragments after purification. The gel shows that the Mr s of rHA, D12, and D23 are 66,500, 46,000, and 45,500, respectively, as predicted and confirmed by mass spectrometry (data not shown); no other bands were apparent on the gel. HPLC gel filtration experiments showed the proteins to contain approximately 4% of the respective dimers (data not shown). Fig. 1B gives the far-UV CD spectra for HSA, rHA, D12, and D23 and shows that the proteins all exhibit spectral characteristics of a folded protein containing a-helical motifs. Secondary structural analysis suggested that HSA and rHA were 40% a-helical and that D12 and D23 were 34 and 35% a-helical, respectively. The kinetics of the binding of warfarin enantiomers to its high-affinity site can be followed by measuring the change in warfarin fluorescence with time after mixing
It has been proposed that the binding of warfarin to albumin occurs in at least two steps [2]. The proposed binding mechanism is outlined below in Eq. (2),
ð2Þ
where W represents warfarin, k1 is the on rate of the first step, k1 is the off rate of the first step, and k2 and k2 are the forward and reverse rates of the second step (postulated conformational rearrangement), respectively. This model assumes that the first binding step is rapid and diffusion controlled, with half times predicted to be within the microsecond range [2]. This is followed by a second step in which the warfarin-binding site is adapted to bind the warfarin molecules by a postulated local conformational change [10] resulting in the formation of a stable warfarin–albumin complex. It is the second step which is rate limiting and can be monitored by stoppedflow fluorimetry. The kinetic data were analyzed using the following equation, kobs ¼ k2
ðK1 ½WÞ þ k2 ; ðK1 ½W þ 1Þ
ð3Þ
where K1 ¼ k1 =k1 and [W] is the total warfarin concentration in the mixing cell. Plotting kobs against [W] allowed the calculation of K1 and k2 , as described previously [10]. The dissociation constant for the overall binding process was calculated from Eq. (4): Kd ¼ 1=ðK1 :ðk2 =k2 ÞÞ
ð4Þ
Fig. 1. (A) Denaturing nonreducing SDS–PAGE (10%) analysis of rHA, D12, and D23. Lanes 1 and 7, Mr markers; lane 2, D12 (20 lg); lane 3, D23 (20 lg); lane 4, rHA (1 lg); lane 5, rHA (0.5 lg); lane 6, rHA (0.1 lg). D12 and D23 were taken from pooled fractions after chromatography purification of fermentation supernatant. (B) Far-UV circular dichroism spectra of 1 mg/ml solutions of HSA (–––), rHA ( ), D12 (- - -), and D23 (– – –) in 0.1 M sodium phosphate buffer (0.14 M NaCl), pH 7.4.
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with protein [2,10]. The primary warfarin-binding site is located in domain 2 [1,5,6] and since both D12 and D23 contain intact primary warfarin-binding sites it is possible to follow the binding of warfarin to the domain fragments. Fig. 2 shows typical reaction progress curves obtained when the proteins were mixed with 25 lM warfarin in 0.1 M sodium phosphate buffer containing 0.14 M NaCl, pH 7.4, at 20 °C. In each case the reaction results in a rapid increase in warfarin fluorescence. Fig. 2 shows that similar first order traces were obtained for the
binding of warfarin to domain fragments D12 and D23. Rates of racemic warfarin binding to the various protein species were as follows: rHA, 15 s1 ; D12, 198 s1 ; and D23, 320 s1 ; all determined with 25 lM warfarin. Fig. 3 shows the observed rate constant, kobs , for the binding of racemic warfarin to rHA and the domain fragments as a function of warfarin concentration. At low warfarin concentrations the observed rate increases in a linear manner. A similar profile was observed for warfarin binding to either domain fragment. The
Fig. 2. Fluorescence change with time for the binding of rac-warfarin to rHA and domain fragments. (A) rHA (1 lM), kobs ¼ 24:4 s1 ; (B) D12 (0.1 lM), kobs ¼ 198 s1 ; (C) D23 (0.1 lM), kobs ¼ 320 s1 . In each case the racemic warfarin concentration was 25 lM. Solutions were made up in 0.1 M sodium phosphate buffer (0.14 M NaCl), pH 7.4. Excitation wavelength was 330 nm and emission wavelength was selected using a 360-nm cutoff filter.
Fig. 3. Effect of varying the ligand concentration on the observed rate constants (kobs ) for the binding of warfarin to HSA (d), rHA (s), D12 (.), and D23 (r) for (A) rac-warfarin, (B) R-warfarin, and (C) Swarfarin. Kinetic parameters derived are shown in Table 1. Protein concentrations were 1 lM for HSA and rHA and 0.1 lM for D12 and D23. Warfarin concentrations were varied between 10 and 200 lM for HSA and rHA, between 2 and 100 lM for D12, and between 2 and 40 lM for D23. All solutions were in 0.1 M phosphate buffer, pH 7.4 (0.14 M NaCl). Excitation wavelength was 330 nm, and emission wavelength was selected using a 360-nm cutoff filter.
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Table 1 Kinetic parameters for the binding of rac-warfarin to HSA, rHA, D12, and D23 Protein
K1 (05 M)
k2 ðs1 Þ
k2 ðs1 Þ
Kd ðlMÞ
HSA rHA D12 D23
0:11 0:03 0:11 0:01 3:00 0:8 2:1 0:7
77 4 76 5 130 7 260 10
3:0 0:9 2:9 1:1 73 5 79 4
3:5 0:3 3:4 0:4 1:9 0:3 1:5 0:2
Determined from the data in Fig. 3 as described under Materials and methods. Each experiment was repeated three times.
Table 2 Kinetic parameters for the binding of the enantiomers of warfarin to HSA, rHA, D12, and D23 Protein
Enantiomer
K1 ( 105 M)
k2 ðs1 Þ
k2 ðs1 Þ
Kd ðlMÞ
HSA rHA D12 D23 HSA rHA D12 D23
R R R R S S S S
0:17 0:02 0:17 0:04 2:2 0:4 2:5 0:4 0:12 0:04 0:13 0:01 3:4 0:7 1:6 0:3
70 4 70 4 180 10 270 10 55 5 57 3 73 5 260 20
2:2 0:2 2:2 0:2 27 3 120 10 3:0 0:3 3:2 0:9 70 4 91 9
1:9 0:5 1:9 0:3 0:7 0:02 1:7 0:2 4:6 0:8 4:4 0:6 2:9 0:4 2:2 0:2
Determined from the data in Fig. 3 as described under Materials and methods. Each experiment was repeated three times.
maximal observed rates for the binding of racemic warfarin were 80 s1 for HSA/rHA, 198 s1 for D12, and 350 s1 for D23. Table 1 shows the kinetic parameters derived from Fig. 3 for the binding of racemic warfarin to the proteins. The kinetic parameters for the binding of racemic warfarin to the domain fragments are notably different from those of the full-length protein. Considering warfarin binding (Table 1), for D12 in comparison to rHA, k2 is approximately 1.6 times greater, k2 is 24-fold greater, and K1 is 27 times greater. For D23, k2 is 3-fold greater than that observed with warfarin binding to the full-length proteins, k2 is 26-fold greater, and K1 is 19-fold greater. There is no notable difference between the binding kinetics of warfarin enantiomers to rHA and those of the warfarin enantiomers to HSA. These results are shown in Table 2. The maximum observed rates for the binding of the R enantiomer were 72, 207, and 390 s1 for rHA, D12, and D23, respectively. In the case of the S enantiomer the maximal observed rates were 60, 143, and 351 s1 for rHA, D12, and D23, respectively. The kinetic parameters for the binding of the enantiomers of warfarin to HSA, rHA, and the domain fragments are shown in Table 2. Values for k2 were calculated from the y intercept of plots shown in Fig. 3. These values were confirmed experimentally using phenylbutazone to displace warfarin from its binding site on each of the proteins. Fig. 4 illustrates typical reaction progress curves for the measurement of k2 using phenylbutazone to displace racemic warfarin from a complex with the site I of rHA. For rHA (Fig. 4A and inset table) and D23 (data not shown) the observed dissociation rate did not change markedly with increasing phenylbutazone concentra-
tion. The values of k2 obtained from these data (2:2 s1 for rHA and 78 s1 for D23) were in reasonable agreement with those obtained from the plots kobs against ligand concentration, shown in Table 2. Fig. 4B shows the displacement of warfarin from D12 by phenylbutazone. The rate of displacement increases with phenylbutazone concentration, as shown in Fig. 4C.
Discussion Fragments of rHA containing domains 1 and 2 and domains 2 and 3 have been prepared. Initial structural analysis of the proteins using far-UV CD, illustrated in Fig. 1B, show the domain fragments to contain 34 and 35% a helix for D12 and D23, respectively. This compares with rHA and HSA which were estimated to be approximately 40% a-helical. The extensive disulfide cross-linking of the molecule [20–22] provides sufficient constraints on the folding of the polypeptide chain to allow the fragments to retain a predominantly a-helical conformation. Thus when the domains 1 and 2 or the domains 2 and 3 are expressed, they form a structure similar to that adopted by the complete protein. From the X-ray crystal structure it can be estimated that both domain fragments contain identical proportions of a helix and the same proportions as the full-length protein. Differences in the observed secondary structure of the domains relative to that of the full-length protein must be attributed to the contribution of domain interactions. The binding of racemic warfarin to HSA has been well characterized in a number of studies [1,8] and warfarin is known to bind specifically within site I. Domain fragments D12 and D23 contain site I, and as
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Fig. 4. Determination of k2 by displacement of warfarin by phenylbutazone. (A) rHA (1 lM)–warfarin (1 lM) in the presence of 100 lM phenylbutazone. The curve fitted to k2 ¼ 2:18 s1 , t1=2 ¼ 0:2 s. Inset: observed k2 values with increasing phenylbutazone, indicating that k2 is independent of phenylbutazone concentration. This was also observed for the displacement of warfarin from D23 (data not shown). (B) D12 (1 lM)–warfarin (1 lM) mix with (i) 100 lM phenylbutazone, curve fitted to k2 ¼ 45:5 s1 , t1=2 ¼ 0:018 s, (ii) 150 lM phenylbutazone, curve fitted to k2 ¼ 60:0 s1 , t1=2 ¼ 0:01 s; and (iii) 300 lM phenylbutazone, curve fitted to k2 ¼ 110:0 s1 , t1=2 ¼ 0:006 s. D12 and warfarin concentrations were 2 lM. (C) Variation of k2 with phenylbutazone concentration for the displacement of warfarin from D12. All solutions were made up in 0.1 M sodium phosphate buffer (0.14 M NaCl), pH 7.4. Excitation wavelength was 330 nm and emission wavelength was selected using a 360-nm cutoff filter.
expected these domain fragments bind racemic and R and S enantiomers of warfarin (Tables 1 and 2). The binding of warfarin enantiomers to HSA has been studied using a number of methods, yielding different results. Two warfarin-binding sites have been reported by groups using circular dichroism or HSA immobilized on a Sepharose column to monitor warfarin binding [4]. Other research groups [23–26] have also utilized immobilized HSA and have shown that the S enantiomer of warfarin has marginally higher affinity for HSA than the R enantiomer [25]. This finding was in agreement with others studies using HSA in solution [21]. Some studies report no stereoselectivity in the binding of warfarin enantiomers [27–29], while others have again suggested two binding sites for warfarin, with only the secondary sites demonstrating stereose-
lectivity [30]. Discrepancies in the reported stereospecificity of albumin for warfarin may be attributed to differences in experimental conditions. The albumin molecule is highly sensitive to factors such as pH [1], temperature, fatty acid content [31], and chloride ion concentration [31,32]. HSA undergoes conformational changes between pH 7.4 and 9.0 (the N–B transition), which is known to have marked affects upon the binding characteristics of warfarin [31]. Chloride ions have been shown to displace warfarin from HSA, with variable effects on the binding of warfarin enantiomers [32]. Studies conducted at extremes of pH [24], without removal of bound fatty acids [21] and with variations in chloride ion concentration, are therefore, difficult to compare. In the studies reported here conditions have been maintained at pH 7.4, by 100 mM sodium phosphate buffer with 0.14 M NaCl, initially to reflect the ligand binding properties in vivo. We have shown R-warfarin to bind to HSA and rHA with higher affinity than Swarfarin (Table 2). Although D21 and D23 show higher affinity for binding of both enantiomers than rHA and HSA, the stereoselectivity is comparable to that of the full-length protein. Plots of kobs against ligand concentration, shown in Fig. 3, show an increase in the rate of warfarin binding until a plateau is reached, where kobs becomes independent of warfarin concentration. This is observed in the binding of warfarin to the domain fragments and to the full-length protein, indicating that warfarin binds to both the full-length protein and the domain fragments by a similar mechanism. This is consistent with the findings of Bos et al. [2] who used HSA domain fragments produced by proteolysis. The rate of dissociation of warfarin from the binding site, k2 , was estimated from the y intercept of the plots shown in Fig. 3 and confirmed by displacement studies. Phenylbutazone is known to bind within site I with high affinity (Kd ¼ 1:4 lM) [1] and is nonfluorescent. When mixed with warfarin–albumin complexes, the phenylbutazone readily displaces warfarin, and this process can be monitored by the decrease in warfarin fluorescence. The experimental values of k2 were found to be in reasonable agreement with the calculated values (Tables 1 and 2) and, in the case of rHA and D23, k2 was found to be independent of phenylbutazone concentration (Fig. 4A, inset table). The rate of displacement of warfarin from D12 was, however, found to be dependent upon the concentration of phenylbutazone. This suggests that the phenylbutazone is not simply displacing the warfarin from site I, but that a more complex process is involved. From Fig. 3 the differences in kobs in the binding of racemic warfarin to albumin and the domain fragments is apparent. The kobs for the binding of warfarin to the domain fragments is markedly greater than that seen
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with the full-length protein. The differences in kinetics are reflected in k2 , k2 , and K1 (Table 1). K1, the equilibrium constant for the first step in the binding of warfarin, is 27- and 19-fold greater for the binding of racemic warfarin to D12 and D23, respectively, in comparison to the full-length protein. This could result from changes in k1 or k1 . This first binding step is diffusion controlled and even at low ligand concentrations occurs within the mixing time of the stopped-flow apparatus, so differences in k1 and k1 cannot be determined experimentally. Wilting et al. [9], determined K1 to be 1:2 105 M1 and estimated k1 to be in the region of 3 108 M1 s1 for HSA. Factors influencing K1 include the size of the proteins, which will influence the rate of diffusion, and the charge distribution, which could result in a greater or lesser degree of electrostatic repulsion. The encounter frequency will be determined by the radius of the respective particles, the diffusion constants, and the charges on the molecules. The differences in the kinetic constants of the second step in the warfarin binding process, k2 and k2 , likely result from differences in the overall structure of the domain fragments. While the circular dichroism data have shown D12 and D23 to be predominantly a-helical, both domain fragments have structures that differ from that of the full-length protein, due to the absence of a third domain. Both D12 and D23 have notably faster k2 and k2 rates for the binding of racemic R- and S-warfarin, in comparison to those observed for binding to the full-length protein. In particular, the rate of the second step, k2 , is markedly faster for the binding of racemic, Rand S-warfarin to D23. Absence of a domain may reduce the steric hindrance encountered by warfarin when entering the binding site. Interdomain interactions have long been known to play a role in ligand binding to HSA [1]. Indeed, the albumin molecule appears conformationally labile, with structural changes resulting from alterations in pH, fatty acid binding, and salt concentration. It would not seem surprising that removal of a domain would markedly affect the conformation and flexibility of the molecule. More interestingly, the removal of domain 1 in D23 appears to have a more dramatic impact upon k2 than does the removal of domain 3 in D12. These differences are not reflected in k2 , which show little difference between D12 and D23 in the case of racemic warfarin. With respect to the binding of the warfarin enantiomers to rHA, the R enantiomer is bound with a 2-fold higher affinity than the S enantiomer. Kinetically, the differences in binding affinity lay primarily in the second binding step. The values of K1 for the binding of R- and S-warfarin to rHA are similar. The differences in k2 , however, are more marked. The binding of the R enantiomer occurs with a k2 that is 1.3-fold faster than that of the S enantiomer. The k2 value, which is 1.5-fold slower in the case of R-warfarin, suggests that a tighter
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final ligand protein complex is formed. Based on these data, it can be concluded that it is the second step in the binding of warfarin to rHA in which the chiral discrimination occurs. It has been postulated by other authors that the second step in warfarin binding involves a conformational change in the protein as it adapts to accommodate the ligand [10]. The nature of this conformational change may differ depending upon the enantiomer occupying the binding site, leading to the difference in k2 values. As shown in Table 2, D12 displays a greater degree of stereoselectivity toward the warfarin enantiomers than does the full-length protein. The differences in the binding kinetics of R- and S-warfarin to D12 are similar to those observed with the binding of warfarin enantiomers to rHA. The K1 values differ slightly, but the most distinct differences are observed in the k2 values. The k2 for R-warfarin is 2.5 times more rapid than the binding of the S enantiomer. The k2 value, however, is 2.6-fold slower in the case of the R enantiomer. In fact, the S enantiomer dissociates at almost the same rate at which it associates. These data suggest that the R enantiomer binds more favorably than does the S enantiomer, forming a more stable ligand–protein complex, which dissociates less readily. The degree of stereoselectivity of D23 toward the enantiomers of warfarin is lower than that exhibited by D12 or rHA. As observed with the binding of racemic warfarin to D23, the overall binding process for both warfarin enantiomers is more rapid in comparison to the kinetic values seen for enantiomer binding to rHA. Little difference is observed in k2 for R- and S-warfarin binding to D23. However, small differences lie in the k2 and K1 values for the binding of R- and S-warfarin. These data suggest that domain 1 is of key importance in determining the stereoselectivity of binding site I toward the enantiomers of warfarin. The structure of albumin with the enantiomers of warfarin bound has been resolved [5]. From this it can be seen that the warfarin-binding pocket is formed by all six helices of subdomain IIA. The R-(+) and S-()) enantiomers were observed to bind in the pocket in a similar orientation. The main difference noted in the conformation of bound enantiomers was in the acetonyl group, which branches from the chiral carbon. This is located near the mouth of the pocket. In this position the chiral groups within the albumin are not so constrained by the binding pocket, which would explain why the differences in the binding of the stereoisomers of warfarin are relatively small. The structure of rHA containing bound R- and Swarfarin was determined in the presence of myristate [33]. Fatty acids are known to enhance the affinity of HSA for warfarin and a number of other site I ligands [31,34] and could potentially influence the binding of the individual enantiomers. Fatty acid binding causes
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a substantial change in protein conformation involving rotations of domains I and III relative to domain II [33]. The principle driving force behind this conformational change is the binding of a fatty acid to FA2 (the second fatty acid binding site) that spans the interface between subdomains IA and IIA. This interface lies close to the warfarin-binding site. Overall, the binding of fatty acid results in the removal of Tyr 150 from the warfarinbinding pocket and rotation of the aliphatic portion of Arg 257, which are thought to render the warfarinbinding pocket more hydrophobic. It has been proposed that this results in the increased binding affinity of warfarin observed in the presence of fatty acids, but the presence of myristic acid bound to the rHA may subtly alter the binding of the warfarin enantiomers.
Conclusion We have produced domain fragments of recombinant human albumin that possess a functional warfarinbinding site that retains the ability to discriminate between pairs of warfarin enantiomers. We have also demonstrated that it is the second step in the postulated warfarin-binding mechanism that is responsible for the chiral discrimination. These studies complement other binding and structural studies in unraveling the complex ligand binding behaviour of human serum albumin.
Acknowledgments S.M.T. was funded by a CASE studentship from the BBSRC. The authors thank Andrew Turner for his technical support.
References [1] T.J. Peters, All About Albumin, Academic Press, London, 1996. [2] O.J.M. Bos, M.J.E. Fischer, J. Wilting, L.H.M. Janssen, Biochem. Pharmacol. 38 (1989) 1979–1984. [3] W. Konig, in: I.W. Wainer (Ed.), Drug Stereochemistry, Marcel Dekker Inc., New York, 1999, pp. 113–145. [4] T.C. Pinkerton, K.A. Koeplinger, Anal. Chem. 62 (1990) 2114– 2122.
[5] I. Peptitpas, A.A. Bhattacharya, S.M. Twine, M. East, S. Curry, J. Biol. Chem. 276 (2001) 22804–22809. [6] X. He, D.C. Carter, Nature 358 (1992) 209–215. [7] U. Kragh-Hansen, Danish Med. Bull. 37 (1990) 57–84. [8] G. Sudlow, D.J. Birkett, D.N. Wade, Mol. Pharmacol. 11 (1975) 824–832. [9] J. Wilting, W.F. van der Giesen, L.H.M. Janssen, Biochem. Pharmacol. 20 (1981) 1025–1031. [10] J.M.H. Kremer, G. Bakker, J. Wilting, Bichim. Biophys. Acta 708 (1982) 239–242. [11] Chen, J. Biol. Chem. 242 (1967) 173–181. [12] B.D. West, S. Preis, C.H. Schroeder, K.P. Link, J. Am. Chem. Soc. 83 (1961) 2676–2679. [13] S.A. Chinery, E. Hinchliffe, 14th International Conference on Yeast Genetics and Molecular Biology S (1988), p. 123. [14] S.A. Chinery, E. Hinchliffe, Curr. Genet. 16 (1989) 21–25. [15] J.D. Beggs, Nature 275 (1978) 104–109. [16] D. Sleep, G.P. Belfield, D.J. Ballance, J. Steven, S. Jones, L.R. Evans, P.D. Moir, A.R. Goodey, Biotechnology 9 (1991) 183–187. [17] S.A. Collins, in: T.J.R. Harris (Ed.), Protein Production in Biotechnology, Elsevier Applied Science, Amsterdam, 1990, pp. 61–77. [18] S.C. Gill, P.H. von Hippel, Anal. Biochem. 182 (1989) 319– 326. [19] M.G. Gore, in: M.G. Gore (Ed.), Spectrophotometry & Spectrofluorimetry, A Practical Approach, Oxford Univeristy Press, Oxford, 2000, pp. 241–264. [20] M.J. Geisow, G.H. Beaven, Biochem. J. 163 (1977) 477–484. [21] E.M. Sellers, J. Koch-Weser, Pharmacol. Res. Commun. 7 (1975) 331–336. [22] J.R. Brown, Fed. Proc. 34 (1975) 591. [23] C. Lagercrantz, T. Larsson, I. Denfors, Comp. Biochem. Physiol. 69C (1981) 375–378. [24] I. Fitos, J. Visy, A. Simoncsits, Biochem. Pharmacol. 46 (1993) 1159–1163. [25] B. Loun, D.S. Hage, Anal. Chem. 68 (1996) 1218–1225. [26] C. Lagercrantz, T. Larsson, H. Karlsson, Anal. Biochem. 99 (1979) 352–364. [27] R.A. OÕReilly, J. Clin. Invest. 46 (1967) 829–837. [28] R.A. OÕReilly, Ann. N. Y. Acad. Sci. (1967) 293–308. [29] N.A. Brown, E. Jahnchen, W.E. Muller, U. Wollert, Mol. Pharmacol. 13 (1977) 70–79. [30] K. Veronich, G. White, A. Kapoor, J. Pharm. Sci. 68 (1979) 1515– 1518. [31] G. Wilding, R.C. Feldhoff, E.S. Vesell, Biochem. Pharmacol. 26 (1977) 1143–1146. [32] J. Wilting, W.F. van der Giesen, L.H.M. Janssenn, M.M. Weideman, M. Otagiri, J. Biol. Chem. 255 (1980) 3032–3037. [33] A.A. Bhattacharya, T. Grune, S. Curry, J. Mol. Biol. 303 (2000) 721–732. [34] S. Curry, H. Mandelkow, P. Brick, N. Franks, Nat. Struct. Biol. 5 (1998) 827–835.
Mechanism of binding of warfarin enantiomers to recombinant domains of human albumin S.M. Twine,a,1 M.G. Gore,a P. Morton,b B.C. Fish,b,2 A.G. Lee,a and J.M. Easta,* a
Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, SO16 7PX, UK b Delta Biotechnology Limited, Castle Boulevard, Nottingham, NG7 1FD, UK Received 8 January 2003, and in revised form 20 March 2003
Abstract Domain fragments of human serum albumin corresponding to domains 1 and 2 (D12) and domains 2 and 3 (D23) were expressed in yeast. The kinetics of warfarin binding to these fragments were investigated using stopped-flow fluorescence spectroscopy. Binding can be characterized by a two-step process, a rapid diffusion-controlled step and a slower rate-limiting step in which a stable drug–protein complex is formed. The equilibrium constant for step 1 is greater for both D12 and D23 than for albumin, probably as a result of reduced steric hindrance offered by the domain fragments. Binding step 2, thought to be the result of a conformational change as warfarin is accommodated by the protein, is faster for D12 and D23. Albumin and the domain fragments show an increased preference for the R enantiomer, but the preference is particularly enhanced for domain fragment D12. These preferences can largely be explained by the domains having different rates for step 2 of the binding process. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Albumin; Warfarin; Ligand binding; Fluorescence; Enantiomers; Drug binding; Stopped flow
Introduction As the most abundant human plasma protein, human serum albumin (HSA)3 has numerous functions including regulation of colloid osmotic pressure and transport of various endogenous ligands throughout the circulation [1]. HSA also binds a wide variety of drug molecules and is an important factor in determining drug pharmacokinetics including distribution and elimination [1,2]. A large number of drugs are chiral and in most cases only one enantiomer is responsible for therapeutic activity [3]. Albumin has been shown to discriminate between various pairs of enantiomers [1]. The anticoagulant warfarin and the nonsteroidal antiinflammatory drug ibuprofen are examples of chiral * Corresponding author. Fax: +44-2380-594-459. E-mail address: [email protected] (J.M. East). 1 Present address: National Research Council, Institute of Biological Sciences, 100 Sussex Drive, Ottawa, Ontario, Canada. 2 Present address: Cambridge Antibody Technology Limited, Milstein Building, Granta Park, Cambridge, CB1 6GH, UK. 3 Abbreviation used: HSA, human serum albumin; rHA, recombinant human albumin.
drugs that have been extensively characterized [2,4]. Warfarin has a single chiral center, and the stereoisomers have been reported to bind to albumin with a modest variation in affinity [1]. A recent crystallographic study has revealed some clues about the relative positions of the enantiomers within the binding site [5]. Such studies do not, however, provide information with regard to the mechanism by which albumin discriminates between the enantiomers. HSA contains a single polypeptide chain of 585 amino acid residues with three structurally homologous a-helical domains (I–III). Each of these domains can be further divided into subdomains A and B containing six and four a helices, respectively [6,7]. Numerous studies have shown the presence of two major drug-binding sites on the protein [1] designated sites I and II by Sudlow et al. [8]. Subsequent crystallographic studies have mapped sites I and II to subdomains IIA and IIIA, respectively [6]. Both competition binding methods and crystallographic methods have mapped the warfarin binding site to Sudlow et al.Õs site I [1,2]. The dissociation constant for drug binding can provide information with regard to the chiral selectivity of a
0003-9861/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-9861(03)00173-5
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protein, but it provides no information about the rate of formation and dissociation of the drug–protein complex. These data may be obtained by studying the kinetics of drug binding to albumin. Such studies have been conducted [5,9,10] and the data were consistent with a two-step binding mechanism, where the first step is rapid and diffusion controlled and the second step is rate limiting and probably the result of a conformational change [2]. In this study we investigated the mechanism of binding of warfarin enantiomers to recombinant human albumin (rHA) to determine the step(s) in the binding mechanism responsible for chiral discrimination of warfarin enantiomers. Domain fragments corresponding to domains 1 and 2 (residues 1– 387, designated D12) and to domains 2 and 3 (residues 183–585, designated D23) were used to further dissect the warfarin-binding properties of albumin and the potential role of interdomain interactions in this process.
Materials and methods Essentially fatty acid-free human serum albumin was obtained from Sigma. rHA was obtained from Delta Biotechnology Ltd. (Notthingham, UK). All proteins were delipidated by the method of Chen [11]. Ligands of the highest purity were purchased from Sigma, with the exception of warfarin enantiomers, which were purified by the method of West et al. [12].
gation, followed by fourfold dilution and adjustment of the pH to 9.0. The diluted supernatant (32 l) was passed through a diethylaminoethylcellulose column, equilibrated with 0.1 M sodium acetate buffer, pH 9.0. After washing with 10 column volumes of equilibration buffer, elution was carried out with a 0–1 M NaCl gradient. Samples were then concentrated by filtration using a stirred cell (Amicon) to a final concentration of 100 mg/ ml and were applied to a 500-ml Sephacryl S200 column equilibrated with phosphate-buffered saline. Protein purity was assessed by SDS–PAGE analysis. Concentrations of the domain fragments were estimated using e280 , calculated on the basis of amino acid composition [18]. Far-UV circular dichroism Far-UV CD measurements were carried out using a Jasco J720 spectropolarimeter, calibrated as per the manufacturerÕs specifications. Spectra were recorded using 1 mg/ml solutions of each protein in buffer (0.1 M sodium phosphate, pH 7.4, containing 0.14 M NaCl) in a cell of 0.01 cm path length. Scans were carried out between 200 and 250 nm with a bandwidth of 1.0 nm and a scan speed of 5 nm/min. Each spectrum was the average of five accumulations. Secondary structure estimation was carried out using Jasco J-700 Secondary Structure Estimation software. Fluorescence assays
Cloning and expression of domain fragments Cloning. D23 was constructed by amplification of the N-terminal 60 amino acids of domain 2 by the polymerase chain reaction with Pvu polymerase (Boehringer) using a cDNA clone of HSA as a template. The PCR product was then cloned into pAYE 467 digested with NcoI/HindIII containing the C-terminal region of domain 2 and the complete domain 3. Clones were confirmed by DNA sequencing. The D23 expression cassette, containing a leader sequence and the alcohol dehydrogenase terminator, was then ligated into the Escherichia coli–yeast shuttle vector pSAC 35 [13,14] digested with NcoI. Recombinant plasmids were then transformed into yeast strains by electroporation [15]. Transformants were screened for the ability to secrete D23 using nutrient agar plates containing anti-HSA antibodies, as described previously [16]. The cloning of D12 was carried out in the same manner. Expression. Standard protocols and media formulations were used. High-cell-density fermentation was carried out in minimal media in 10-l fed-batch culture essentially as described by Collins [17]. Purification. All manipulations were carried out at ambient temperature unless stated otherwise. Fermentation culture supernatants were harvested by centrifu-
Unless otherwise stated the fluorescence assays were performed at 25 °C in 0.1 M sodium phosphate buffer, pH 7.4, containing 0.14 M NaCl, using Hitachi F-4500 or Perkin–Elmer LS-3B spectrofluorimeters. Ligands were added to assay volumes of 3 ml from 3 mM stock solutions in methanol. Concentration of warfarin stock solutions were confirmed using e310 (13,610 M1 cm1 ). Excitation and emission wavelengths were 330 and 375 nm, respectively. Stopped-flow fluorescence spectrofluorimetry Stopped-flow experiments were carried out using an Applied Photophysics SX-18MV (deadtime 1.5 ms) as outlined previously [19]. The temperatures of both drive syringes and the reaction path were controlled at 25 °C by a circulating water bath. The excitation wavelength (330 nm) was selected by a monochromator with a 1-mm slit width and fluorescence emission was detected at right angles to the exciting beam through a 360-nm cutoff filter. Equal volumes (100 ll) of solutions of protein (in 100 mM phosphate or 40 mM tricine buffer at a given pH) and warfarin (in the same buffer) were rapidly mixed. The concentration of protein after mixing was 1 lM for rHA and 0.1 lM for the domain frag-
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ments. The lowest ligand concentration used was a minimum of 10-fold excess over the protein concentration which allowed the observed rate constant kobs , of the reaction to be analyzed as pseudo-first order processes [19] using the relationship Ft ¼ F0 expkt þC;
ð1Þ
where F0 and Ft are the fluorescence intensities at time 0 and time t respectively, k is the apparent first order rate constant for the reaction, and C is a constant. Each trace analyzed was the average of three to nine individual determinations. The values of k2 were determined experimentally using phenylbutazone to displace warfarin from a preformed complex with albumin or albumin domains; 1 lM solutions of protein–warfarin complexes were mixed 1:1 v/v with 100–500 lM phenylbutazone. Treatment of kinetic data
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Results Fig. 1A illustrates the SDS–PAGE analysis of rHA and domain fragments after purification. The gel shows that the Mr s of rHA, D12, and D23 are 66,500, 46,000, and 45,500, respectively, as predicted and confirmed by mass spectrometry (data not shown); no other bands were apparent on the gel. HPLC gel filtration experiments showed the proteins to contain approximately 4% of the respective dimers (data not shown). Fig. 1B gives the far-UV CD spectra for HSA, rHA, D12, and D23 and shows that the proteins all exhibit spectral characteristics of a folded protein containing a-helical motifs. Secondary structural analysis suggested that HSA and rHA were 40% a-helical and that D12 and D23 were 34 and 35% a-helical, respectively. The kinetics of the binding of warfarin enantiomers to its high-affinity site can be followed by measuring the change in warfarin fluorescence with time after mixing
It has been proposed that the binding of warfarin to albumin occurs in at least two steps [2]. The proposed binding mechanism is outlined below in Eq. (2),
ð2Þ
where W represents warfarin, k1 is the on rate of the first step, k1 is the off rate of the first step, and k2 and k2 are the forward and reverse rates of the second step (postulated conformational rearrangement), respectively. This model assumes that the first binding step is rapid and diffusion controlled, with half times predicted to be within the microsecond range [2]. This is followed by a second step in which the warfarin-binding site is adapted to bind the warfarin molecules by a postulated local conformational change [10] resulting in the formation of a stable warfarin–albumin complex. It is the second step which is rate limiting and can be monitored by stoppedflow fluorimetry. The kinetic data were analyzed using the following equation, kobs ¼ k2
ðK1 ½WÞ þ k2 ; ðK1 ½W þ 1Þ
ð3Þ
where K1 ¼ k1 =k1 and [W] is the total warfarin concentration in the mixing cell. Plotting kobs against [W] allowed the calculation of K1 and k2 , as described previously [10]. The dissociation constant for the overall binding process was calculated from Eq. (4): Kd ¼ 1=ðK1 :ðk2 =k2 ÞÞ
ð4Þ
Fig. 1. (A) Denaturing nonreducing SDS–PAGE (10%) analysis of rHA, D12, and D23. Lanes 1 and 7, Mr markers; lane 2, D12 (20 lg); lane 3, D23 (20 lg); lane 4, rHA (1 lg); lane 5, rHA (0.5 lg); lane 6, rHA (0.1 lg). D12 and D23 were taken from pooled fractions after chromatography purification of fermentation supernatant. (B) Far-UV circular dichroism spectra of 1 mg/ml solutions of HSA (–––), rHA ( ), D12 (- - -), and D23 (– – –) in 0.1 M sodium phosphate buffer (0.14 M NaCl), pH 7.4.
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with protein [2,10]. The primary warfarin-binding site is located in domain 2 [1,5,6] and since both D12 and D23 contain intact primary warfarin-binding sites it is possible to follow the binding of warfarin to the domain fragments. Fig. 2 shows typical reaction progress curves obtained when the proteins were mixed with 25 lM warfarin in 0.1 M sodium phosphate buffer containing 0.14 M NaCl, pH 7.4, at 20 °C. In each case the reaction results in a rapid increase in warfarin fluorescence. Fig. 2 shows that similar first order traces were obtained for the
binding of warfarin to domain fragments D12 and D23. Rates of racemic warfarin binding to the various protein species were as follows: rHA, 15 s1 ; D12, 198 s1 ; and D23, 320 s1 ; all determined with 25 lM warfarin. Fig. 3 shows the observed rate constant, kobs , for the binding of racemic warfarin to rHA and the domain fragments as a function of warfarin concentration. At low warfarin concentrations the observed rate increases in a linear manner. A similar profile was observed for warfarin binding to either domain fragment. The
Fig. 2. Fluorescence change with time for the binding of rac-warfarin to rHA and domain fragments. (A) rHA (1 lM), kobs ¼ 24:4 s1 ; (B) D12 (0.1 lM), kobs ¼ 198 s1 ; (C) D23 (0.1 lM), kobs ¼ 320 s1 . In each case the racemic warfarin concentration was 25 lM. Solutions were made up in 0.1 M sodium phosphate buffer (0.14 M NaCl), pH 7.4. Excitation wavelength was 330 nm and emission wavelength was selected using a 360-nm cutoff filter.
Fig. 3. Effect of varying the ligand concentration on the observed rate constants (kobs ) for the binding of warfarin to HSA (d), rHA (s), D12 (.), and D23 (r) for (A) rac-warfarin, (B) R-warfarin, and (C) Swarfarin. Kinetic parameters derived are shown in Table 1. Protein concentrations were 1 lM for HSA and rHA and 0.1 lM for D12 and D23. Warfarin concentrations were varied between 10 and 200 lM for HSA and rHA, between 2 and 100 lM for D12, and between 2 and 40 lM for D23. All solutions were in 0.1 M phosphate buffer, pH 7.4 (0.14 M NaCl). Excitation wavelength was 330 nm, and emission wavelength was selected using a 360-nm cutoff filter.
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Table 1 Kinetic parameters for the binding of rac-warfarin to HSA, rHA, D12, and D23 Protein
K1 (05 M)
k2 ðs1 Þ
k2 ðs1 Þ
Kd ðlMÞ
HSA rHA D12 D23
0:11 0:03 0:11 0:01 3:00 0:8 2:1 0:7
77 4 76 5 130 7 260 10
3:0 0:9 2:9 1:1 73 5 79 4
3:5 0:3 3:4 0:4 1:9 0:3 1:5 0:2
Determined from the data in Fig. 3 as described under Materials and methods. Each experiment was repeated three times.
Table 2 Kinetic parameters for the binding of the enantiomers of warfarin to HSA, rHA, D12, and D23 Protein
Enantiomer
K1 ( 105 M)
k2 ðs1 Þ
k2 ðs1 Þ
Kd ðlMÞ
HSA rHA D12 D23 HSA rHA D12 D23
R R R R S S S S
0:17 0:02 0:17 0:04 2:2 0:4 2:5 0:4 0:12 0:04 0:13 0:01 3:4 0:7 1:6 0:3
70 4 70 4 180 10 270 10 55 5 57 3 73 5 260 20
2:2 0:2 2:2 0:2 27 3 120 10 3:0 0:3 3:2 0:9 70 4 91 9
1:9 0:5 1:9 0:3 0:7 0:02 1:7 0:2 4:6 0:8 4:4 0:6 2:9 0:4 2:2 0:2
Determined from the data in Fig. 3 as described under Materials and methods. Each experiment was repeated three times.
maximal observed rates for the binding of racemic warfarin were 80 s1 for HSA/rHA, 198 s1 for D12, and 350 s1 for D23. Table 1 shows the kinetic parameters derived from Fig. 3 for the binding of racemic warfarin to the proteins. The kinetic parameters for the binding of racemic warfarin to the domain fragments are notably different from those of the full-length protein. Considering warfarin binding (Table 1), for D12 in comparison to rHA, k2 is approximately 1.6 times greater, k2 is 24-fold greater, and K1 is 27 times greater. For D23, k2 is 3-fold greater than that observed with warfarin binding to the full-length proteins, k2 is 26-fold greater, and K1 is 19-fold greater. There is no notable difference between the binding kinetics of warfarin enantiomers to rHA and those of the warfarin enantiomers to HSA. These results are shown in Table 2. The maximum observed rates for the binding of the R enantiomer were 72, 207, and 390 s1 for rHA, D12, and D23, respectively. In the case of the S enantiomer the maximal observed rates were 60, 143, and 351 s1 for rHA, D12, and D23, respectively. The kinetic parameters for the binding of the enantiomers of warfarin to HSA, rHA, and the domain fragments are shown in Table 2. Values for k2 were calculated from the y intercept of plots shown in Fig. 3. These values were confirmed experimentally using phenylbutazone to displace warfarin from its binding site on each of the proteins. Fig. 4 illustrates typical reaction progress curves for the measurement of k2 using phenylbutazone to displace racemic warfarin from a complex with the site I of rHA. For rHA (Fig. 4A and inset table) and D23 (data not shown) the observed dissociation rate did not change markedly with increasing phenylbutazone concentra-
tion. The values of k2 obtained from these data (2:2 s1 for rHA and 78 s1 for D23) were in reasonable agreement with those obtained from the plots kobs against ligand concentration, shown in Table 2. Fig. 4B shows the displacement of warfarin from D12 by phenylbutazone. The rate of displacement increases with phenylbutazone concentration, as shown in Fig. 4C.
Discussion Fragments of rHA containing domains 1 and 2 and domains 2 and 3 have been prepared. Initial structural analysis of the proteins using far-UV CD, illustrated in Fig. 1B, show the domain fragments to contain 34 and 35% a helix for D12 and D23, respectively. This compares with rHA and HSA which were estimated to be approximately 40% a-helical. The extensive disulfide cross-linking of the molecule [20–22] provides sufficient constraints on the folding of the polypeptide chain to allow the fragments to retain a predominantly a-helical conformation. Thus when the domains 1 and 2 or the domains 2 and 3 are expressed, they form a structure similar to that adopted by the complete protein. From the X-ray crystal structure it can be estimated that both domain fragments contain identical proportions of a helix and the same proportions as the full-length protein. Differences in the observed secondary structure of the domains relative to that of the full-length protein must be attributed to the contribution of domain interactions. The binding of racemic warfarin to HSA has been well characterized in a number of studies [1,8] and warfarin is known to bind specifically within site I. Domain fragments D12 and D23 contain site I, and as
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Fig. 4. Determination of k2 by displacement of warfarin by phenylbutazone. (A) rHA (1 lM)–warfarin (1 lM) in the presence of 100 lM phenylbutazone. The curve fitted to k2 ¼ 2:18 s1 , t1=2 ¼ 0:2 s. Inset: observed k2 values with increasing phenylbutazone, indicating that k2 is independent of phenylbutazone concentration. This was also observed for the displacement of warfarin from D23 (data not shown). (B) D12 (1 lM)–warfarin (1 lM) mix with (i) 100 lM phenylbutazone, curve fitted to k2 ¼ 45:5 s1 , t1=2 ¼ 0:018 s, (ii) 150 lM phenylbutazone, curve fitted to k2 ¼ 60:0 s1 , t1=2 ¼ 0:01 s; and (iii) 300 lM phenylbutazone, curve fitted to k2 ¼ 110:0 s1 , t1=2 ¼ 0:006 s. D12 and warfarin concentrations were 2 lM. (C) Variation of k2 with phenylbutazone concentration for the displacement of warfarin from D12. All solutions were made up in 0.1 M sodium phosphate buffer (0.14 M NaCl), pH 7.4. Excitation wavelength was 330 nm and emission wavelength was selected using a 360-nm cutoff filter.
expected these domain fragments bind racemic and R and S enantiomers of warfarin (Tables 1 and 2). The binding of warfarin enantiomers to HSA has been studied using a number of methods, yielding different results. Two warfarin-binding sites have been reported by groups using circular dichroism or HSA immobilized on a Sepharose column to monitor warfarin binding [4]. Other research groups [23–26] have also utilized immobilized HSA and have shown that the S enantiomer of warfarin has marginally higher affinity for HSA than the R enantiomer [25]. This finding was in agreement with others studies using HSA in solution [21]. Some studies report no stereoselectivity in the binding of warfarin enantiomers [27–29], while others have again suggested two binding sites for warfarin, with only the secondary sites demonstrating stereose-
lectivity [30]. Discrepancies in the reported stereospecificity of albumin for warfarin may be attributed to differences in experimental conditions. The albumin molecule is highly sensitive to factors such as pH [1], temperature, fatty acid content [31], and chloride ion concentration [31,32]. HSA undergoes conformational changes between pH 7.4 and 9.0 (the N–B transition), which is known to have marked affects upon the binding characteristics of warfarin [31]. Chloride ions have been shown to displace warfarin from HSA, with variable effects on the binding of warfarin enantiomers [32]. Studies conducted at extremes of pH [24], without removal of bound fatty acids [21] and with variations in chloride ion concentration, are therefore, difficult to compare. In the studies reported here conditions have been maintained at pH 7.4, by 100 mM sodium phosphate buffer with 0.14 M NaCl, initially to reflect the ligand binding properties in vivo. We have shown R-warfarin to bind to HSA and rHA with higher affinity than Swarfarin (Table 2). Although D21 and D23 show higher affinity for binding of both enantiomers than rHA and HSA, the stereoselectivity is comparable to that of the full-length protein. Plots of kobs against ligand concentration, shown in Fig. 3, show an increase in the rate of warfarin binding until a plateau is reached, where kobs becomes independent of warfarin concentration. This is observed in the binding of warfarin to the domain fragments and to the full-length protein, indicating that warfarin binds to both the full-length protein and the domain fragments by a similar mechanism. This is consistent with the findings of Bos et al. [2] who used HSA domain fragments produced by proteolysis. The rate of dissociation of warfarin from the binding site, k2 , was estimated from the y intercept of the plots shown in Fig. 3 and confirmed by displacement studies. Phenylbutazone is known to bind within site I with high affinity (Kd ¼ 1:4 lM) [1] and is nonfluorescent. When mixed with warfarin–albumin complexes, the phenylbutazone readily displaces warfarin, and this process can be monitored by the decrease in warfarin fluorescence. The experimental values of k2 were found to be in reasonable agreement with the calculated values (Tables 1 and 2) and, in the case of rHA and D23, k2 was found to be independent of phenylbutazone concentration (Fig. 4A, inset table). The rate of displacement of warfarin from D12 was, however, found to be dependent upon the concentration of phenylbutazone. This suggests that the phenylbutazone is not simply displacing the warfarin from site I, but that a more complex process is involved. From Fig. 3 the differences in kobs in the binding of racemic warfarin to albumin and the domain fragments is apparent. The kobs for the binding of warfarin to the domain fragments is markedly greater than that seen
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with the full-length protein. The differences in kinetics are reflected in k2 , k2 , and K1 (Table 1). K1, the equilibrium constant for the first step in the binding of warfarin, is 27- and 19-fold greater for the binding of racemic warfarin to D12 and D23, respectively, in comparison to the full-length protein. This could result from changes in k1 or k1 . This first binding step is diffusion controlled and even at low ligand concentrations occurs within the mixing time of the stopped-flow apparatus, so differences in k1 and k1 cannot be determined experimentally. Wilting et al. [9], determined K1 to be 1:2 105 M1 and estimated k1 to be in the region of 3 108 M1 s1 for HSA. Factors influencing K1 include the size of the proteins, which will influence the rate of diffusion, and the charge distribution, which could result in a greater or lesser degree of electrostatic repulsion. The encounter frequency will be determined by the radius of the respective particles, the diffusion constants, and the charges on the molecules. The differences in the kinetic constants of the second step in the warfarin binding process, k2 and k2 , likely result from differences in the overall structure of the domain fragments. While the circular dichroism data have shown D12 and D23 to be predominantly a-helical, both domain fragments have structures that differ from that of the full-length protein, due to the absence of a third domain. Both D12 and D23 have notably faster k2 and k2 rates for the binding of racemic R- and S-warfarin, in comparison to those observed for binding to the full-length protein. In particular, the rate of the second step, k2 , is markedly faster for the binding of racemic, Rand S-warfarin to D23. Absence of a domain may reduce the steric hindrance encountered by warfarin when entering the binding site. Interdomain interactions have long been known to play a role in ligand binding to HSA [1]. Indeed, the albumin molecule appears conformationally labile, with structural changes resulting from alterations in pH, fatty acid binding, and salt concentration. It would not seem surprising that removal of a domain would markedly affect the conformation and flexibility of the molecule. More interestingly, the removal of domain 1 in D23 appears to have a more dramatic impact upon k2 than does the removal of domain 3 in D12. These differences are not reflected in k2 , which show little difference between D12 and D23 in the case of racemic warfarin. With respect to the binding of the warfarin enantiomers to rHA, the R enantiomer is bound with a 2-fold higher affinity than the S enantiomer. Kinetically, the differences in binding affinity lay primarily in the second binding step. The values of K1 for the binding of R- and S-warfarin to rHA are similar. The differences in k2 , however, are more marked. The binding of the R enantiomer occurs with a k2 that is 1.3-fold faster than that of the S enantiomer. The k2 value, which is 1.5-fold slower in the case of R-warfarin, suggests that a tighter
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final ligand protein complex is formed. Based on these data, it can be concluded that it is the second step in the binding of warfarin to rHA in which the chiral discrimination occurs. It has been postulated by other authors that the second step in warfarin binding involves a conformational change in the protein as it adapts to accommodate the ligand [10]. The nature of this conformational change may differ depending upon the enantiomer occupying the binding site, leading to the difference in k2 values. As shown in Table 2, D12 displays a greater degree of stereoselectivity toward the warfarin enantiomers than does the full-length protein. The differences in the binding kinetics of R- and S-warfarin to D12 are similar to those observed with the binding of warfarin enantiomers to rHA. The K1 values differ slightly, but the most distinct differences are observed in the k2 values. The k2 for R-warfarin is 2.5 times more rapid than the binding of the S enantiomer. The k2 value, however, is 2.6-fold slower in the case of the R enantiomer. In fact, the S enantiomer dissociates at almost the same rate at which it associates. These data suggest that the R enantiomer binds more favorably than does the S enantiomer, forming a more stable ligand–protein complex, which dissociates less readily. The degree of stereoselectivity of D23 toward the enantiomers of warfarin is lower than that exhibited by D12 or rHA. As observed with the binding of racemic warfarin to D23, the overall binding process for both warfarin enantiomers is more rapid in comparison to the kinetic values seen for enantiomer binding to rHA. Little difference is observed in k2 for R- and S-warfarin binding to D23. However, small differences lie in the k2 and K1 values for the binding of R- and S-warfarin. These data suggest that domain 1 is of key importance in determining the stereoselectivity of binding site I toward the enantiomers of warfarin. The structure of albumin with the enantiomers of warfarin bound has been resolved [5]. From this it can be seen that the warfarin-binding pocket is formed by all six helices of subdomain IIA. The R-(+) and S-()) enantiomers were observed to bind in the pocket in a similar orientation. The main difference noted in the conformation of bound enantiomers was in the acetonyl group, which branches from the chiral carbon. This is located near the mouth of the pocket. In this position the chiral groups within the albumin are not so constrained by the binding pocket, which would explain why the differences in the binding of the stereoisomers of warfarin are relatively small. The structure of rHA containing bound R- and Swarfarin was determined in the presence of myristate [33]. Fatty acids are known to enhance the affinity of HSA for warfarin and a number of other site I ligands [31,34] and could potentially influence the binding of the individual enantiomers. Fatty acid binding causes
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a substantial change in protein conformation involving rotations of domains I and III relative to domain II [33]. The principle driving force behind this conformational change is the binding of a fatty acid to FA2 (the second fatty acid binding site) that spans the interface between subdomains IA and IIA. This interface lies close to the warfarin-binding site. Overall, the binding of fatty acid results in the removal of Tyr 150 from the warfarinbinding pocket and rotation of the aliphatic portion of Arg 257, which are thought to render the warfarinbinding pocket more hydrophobic. It has been proposed that this results in the increased binding affinity of warfarin observed in the presence of fatty acids, but the presence of myristic acid bound to the rHA may subtly alter the binding of the warfarin enantiomers.
Conclusion We have produced domain fragments of recombinant human albumin that possess a functional warfarinbinding site that retains the ability to discriminate between pairs of warfarin enantiomers. We have also demonstrated that it is the second step in the postulated warfarin-binding mechanism that is responsible for the chiral discrimination. These studies complement other binding and structural studies in unraveling the complex ligand binding behaviour of human serum albumin.
Acknowledgments S.M.T. was funded by a CASE studentship from the BBSRC. The authors thank Andrew Turner for his technical support.
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