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J. Phys. Chem. B 2002, 106, 452-459
Interaction of Ochratoxin A with Human Serum Albumin. Preferential Binding of the Dianion and pH Effects Yuri V. Il’ichev,† Jennifer L. Perry,† and John D. Simon*,†,‡ Department of Chemistry, Duke UniVersity, Durham, North Carolina 27708, and Department of Biochemistry, Duke UniVersity Medical Center, Durham, North Carolina 27710 ReceiVed: June 19, 2001; In Final Form: October 15, 2001
Ochratoxin A (OTA), a fungal metabolite produced by several strains of Aspergillus and Penicillium, binds to serum albumin with high affinity only in the completely deprotonated form (dianion). The pKa of the phenolic group of OTA decreased by more than three units when it was bound to human serum albumin (HSA). Optical spectroscopy provided evidence that HSA has at least two binding sites for OTA, each being able to accommodate one dianion. These two sites were characterized by the binding constants of 5.2 × 106 and 1.0 × 105 M-1. The binding constant for the monoanion of OTA was estimated to be ∼103 M-1. Fluorescence polarization spectroscopy confirmed weak interaction of the monoanion with the protein in the F and E forms (pH < 4) and showed lower affinity of the dianion to the B form of HSA (pH > 8) compared to the N form (pH ∼ 7). Fluorescence anisotropy decay of the dianion of OTA bound to HSA (36.6 ns) was much longer than its emission lifetime (5.2 ns) and was close to reported values for the rotational tumbling time of the HSA molecule. Results of chemical denaturation with 9 M urea or 5 M guanidine hydrochloride established that high-affinity binding of OTA only occurred to the native protein. Efficient energy transfer from the single tryptophan residue of HSA (Trp214) to bound OTA was observed. Analysis of fluorescence data provided an estimate of the distance between the dianion of OTA in its highest-affinity site and the Trp214 residue, which was on the order of 16 Å. The results are discussed with respect to recent crystallographic data for HSA-ligand complexes and pH-dependent conformation of HSA.
Introduction
CHART 1: Molecular Structure of OTA
Ochratoxins are a class of naturally occurring compounds produced by several strains of Aspergillus and Penicillium fungi.1-4 These toxins are widely spread contaminants of improperly stored feed and food.2-6 Ochratoxin A (OTA in Chart 1), which is the most toxic of this class, is found to cause various adverse effects in animals and in cell cultures (for reviews, see refs 3, 4, and 7-12). In mammals, a main target of OTA toxicity is the kidney. Several experimental studies demonstrate that OTA causes porcine nephropathy.13 Its implication in human renal diseases is still not completely proven. However, occurrence of some forms of human nephropathies and tumors seems to be correlated with enhanced OTA exposure.3,4,8,11,12,14,15 In addition, OTA and other mycotoxins are suggested as major environmental factors in the occurrence of human end-stage renal disease.8b,11 Recent studies of human blood and sera from different countries demonstrate that OTA can be detected in the vast majority of human samples provided that sensitive analytical techniques with the detection limit 2, only the OTA dianion was present even though in water at this pH both HA- and A2- were present in almost equal amounts. The preferential binding of the deprotonated form of the toxin is of primary importance for characterization of the binding site, since under conditions of natural exposure OTA in the body exists almost exclusively as a complex with serum albumin. Although this change in acidity of OTA with binding has been noticed in previous studies,28,29 its relationship to the binding properties of different forms of OTA has not been recognized. Such preferential binding of the anionic form of a ligand has also been observed previously in two different systems: Porter44a found a similar apparent pKa shift in the interaction of 2-acetylpyridine-5-[(2-chloroanilino)thiocarbonyl] thiocarbonohydrazone with HSA, and Burke and Mi44b observed a much higher affinity of HSA to the negatively charged carboxylate form of camptothecin in comparison to its neutral lactone form. Compared to the unbound molecule, the bound dianion showed a 14 nm red shift in absorption and a slight broadening with a ∼3 nm red shift in fluorescence. Fluorescence quantum yield (φ) measurements revealed a slight difference between A2- in water (φ ) 0.39) and bound to HSA (φ ) 0.42 for [HSA]/[OTA] ) 3). Consistent with the change in φ, the fluorescence lifetime (τ) was slightly longer for A2- in the presence of HSA (τ ) 5.22 ns) than in water (τ ) 5.07 ns). We used the fluorescence anisotropy decay to calculate a rotational correlation time for OTA bound to HSA. At 24 °C, this decay was described by a single-exponential function with a lifetime of 36.6 ns. Using an average anisotropy value from steady-state experiments (0.34, corresponding to P ) 0.43), the fluorescence lifetime, and the measured limiting anisotropy value at time zero (0.39, corresponding to P ) 0.49), we calculated a rotational correlation time for OTA complexed with HSA of 33.6 ns. This value is consistent with the time-resolved data and close to the rotational correlation time of the HSA molecule as a whole. Thus our results indicate that the bound dianion is essentially immobile within the protein matrix and suggest that strong localized interactions are responsible for its high affinity to HSA. Previously published data for the rotational tumbling time of HSA vary over a surprisingly broad range. For example, at 25 °C and pH ∼ 7 two values of 22 ns45 and 34 ns46 were reported. More data can be found for temperatures close to 20 °C, where the reported values practically cover the whole range between 22 and 45 ns.47,48 It remains unclear what factors are responsible for these discrepancies, but the short decay times of intrinsic fluorescence of HSA, which was mainly used in these studies, make accurate determination rather difficult. Therefore, the rotational correlation time of 41 ns48 determined
Interaction of OTA with HSA
J. Phys. Chem. B, Vol. 106, No. 2, 2002 455
Figure 3. (A) OTA Fluorescence polarization (P) plotted against the HSA-to-OTA concentration ratio. The excitation wavelengths were 375 nm (solid circles) and 410 nm (open circles). The different polarizations observed for the two wavelengths studies results from the different absorption cross sections of free and bound A2- at the two wavelengths examined. (B) Number of bound OTA dianion per HSA molecule as a function of free OTA concentration. The excitation wavelengths were 375 nm (solid circles) and 410 nm (open circles). The measurements were performed in 50 mM phosphate buffer at pH 7.4.
at 20 °C by using long-lived luminescence of transition metal complexes should be considered as the most accurate one. We quantified the binding of OTA to HSA using fluorescence polarization spectroscopy. Figure 2B shows polarization spectra for OTA in water and for OTA bound to HSA. Values of P as a function of HSA-to-OTA concentration ratio are plotted in Figure 3A. When [HSA]/[OTA] > 3 the polarization P was ≈ 0.43, independent of HSA concentration; this value reflects complete binding. Assuming a simple two-state model (e.g., two distinct P values for OTA in water and bound to the protein), the molar fraction of bound A2-, R, can be determined using eq 2.
R ) (P - Pf)/[θ(Pb - P ) + P - Pf]
(2)
In the above equation, Pf and Pb are fluorescence polarization values for free and bound toxin, respectively, and θ is approximated by the ratio of the products of molar absorptivities () and fluorescence quantum yields (φ) of the bound (b) and free (f) fluorophore, bφb/fφf. Good agreement was observed between the results obtained from experimental P values at two different excitation wavelengths. The binding constant(s) can be obtained by fitting the number of bound ligands per protein molecule to a general equation for multiple-site binding:
[Lb]/[E] )
∑niKi[Lf]/(1 + Ki[Lf])
(3)
where ni and Ki are the occupancy number and the binding constant for ith site, respectively, and [E], [Lb], and [Lf] are the total protein, bound, and free ligand concentrations, respectively. We were able to fit the data in Figure 3B to eq 3 by using two binding sites for A2-. The best fit yielded binding constants that differed by more than an order of magnitude: K1 ) 5.2 × 106 M-1 and K2 ) 1.0 × 105 M-1, and corresponding occupancy numbers close to unity: n1 ) 0.87 and n2 ) 1.18. If the values of ni were fixed to 1.0 for both sites (dashed line) the binding constants did not change substantially: K1 ) 3.7 × 106 M-1 and K2 ) 9.0 × 104 M-1. Although binding of OTA to serum albumins from several species has been studied,26,28,29 a detailed picture of the toxinprotein interaction has not yet emerged. Our optical spectroscopy results now demonstrate that HSA has two distinct binding sites for OTA with substantially different affinities. This result is in
clear contrast to previous findings suggesting the presence of one site able to accommodate two molecules of OTA.26,28,29 Simple comparison of the molecular dimensions of OTA with cavity sizes for Sudlow site I and site II on HSA49 suggests that each site may contain only one OTA. Our two quite different binding constants are consistent with the significant differences in the structures (amino acids involved in principle contacts) of these two sites. Quenching of Trp214 Emission by OTA. HSA contains a single tryptophan residue, Trp214.30 In the excitation interval where tryptophan is practically the only fluorescent amino acid to absorb43 (λex ) 295-305 nm) fluorescence spectra of OTAHSA mixtures showed emission from both OTA and Trp214. Compared to 10 µM HSA or 10 µM OTA alone, a solution containing both components (each 10 µM) clearly show quenching of the Trp fluorescence and an increase in the OTA emission (Figure 4A) due to energy transfer from electronically excited Trp214 to A2- located within the protein matrix. Figure 4B illustrates normalized integrated intensities of Trp214 and OTA fluorescence as a function of the ratio between HSA and OTA concentrations ([OTA] ) 10 µM). In the absence of the toxin, the intensity of HSA emission showed approximately linear dependence on protein concentration up to 30 µM, while at higher concentrations efficient self-quenching was observed. In the presence of OTA, tryptophan emission was strongly quenched, with the quenching efficiency decreasing as [HSA]/[OTA] increased. For an OTA-HSA mixture with 1:1 molar ratio the quantum yield of Trp fluorescence relative to that in the absence of OTA was 0.21. Conversely, the OTA fluorescence intensity increased with increasing protein concentration and the relative quantum yield reached a maximum value of 4.1 at [HSA]/[OTA] ) 2. Absorbance of HSA and OTA-HSA solutions at 295 nm is also depicted in Figure 4B. The HSA absorbance is a linear function of the protein concentration up to 100 µM. At 280 nm, we obtained a molar absorptivity of 34600 M-1 cm-1 for HSA in good agreement with a calculated value of 34500 M-1 cm-1 and reported values varying in the range of 32600-39800 M-1 cm-1.50 It is important to note that OTA absorbance at 295 nm does not change substantially with HSA concentration. Tryptophan fluorescence is known to be sensitive to environment.43 We found that HSA concentration did not affect either the shape of the Trp214 emission band or the position of its
456 J. Phys. Chem. B, Vol. 106, No. 2, 2002
Il’ichev et al. respectively. In the second equality, R is the distance between Trp214 and OTA. Ro is a characteristic distance related to the properties of donor and acceptor, and can be calculated using51-53
∫
∫
Ro(Å)6 ) 8.79 × 10-5n-4κ2φo (λ)f (λ)λ4dλ/ f (λ) dλ (5)
Figure 4. (A) Fluorescence spectra of aqueous solutions (pH 7.3) containing 10 µM OTA and 10 µM HSA (solid), 10 µM HSA (dashed), and 10 µM OTA (dashed-dot) for the excitation at 295 nm. (B) Integrated fluorescence intensities (open symbols) determined from the Trp214 (squares, HSA; down triangles, HSA-OTA mixtures) and OTA (diamonds) emission bands plotted against HSA-to-OTA concentration ratio. Fluorescence was excited at 295 nm and intensities were normalized to the maximum of OTA emission Absorbance (solid circles, HSA; solid up-triangles, HSA-OTA mixtures) at 295 nm measured in the same solutions is also presented. Solid line represents a linear fit to HSA absorbance.
maximum (341 nm). However, in the presence of OTA the tryptophan fluorescence exhibited a distinct shift to the blue, with λmax ) 327 nm when [OTA]/[HSA] was 10:1. Conversely, the polarization of Trp fluorescence was not affected by the toxin bound to the protein (P ) 0.196 for excitation at 295 nm). When we monitored scattered light at the excitation wavelength, the intensity showed the same linear dependence on HSA concentration (0-50 µM) in the presence of 10 µM OTA as in the absence of the toxin. Thus the addition of OTA caused no significant changes in the overall 3D structure of HSA. We used the binding constants determined in the previous section to calculate the distribution of OTA in HSA solution. For a pH 7.4 solution containing 10 µM HSA and 10 µM OTA, ca. 95% of the toxin was bound to the protein and only ca. 5% of bound OTA was located in the site with the smaller binding constant. Therefore, under these conditions the observed tryptophan fluorescence quenching was mainly determined by the interaction of Trp214 with the single OTA dianion located in the highest affinity site. The distance between Trp214 and OTA can be estimated from the energy transfer efficiency E:51-53
E ) 1 - φ/φo ) (1 + R6/Ro6)-1
(4)
where φo and φ are the quantum yields of Trp emission measured for the protein alone and for an OTA-HSA complex,
where n is the refractive index of the medium, κ 2 is a geometric factor related the relative orientation of the transition dipole moments on the donor and acceptor, (λ) is the molar absorptivity of OTA, and f (λ) is the normalized fluorescence intensity of Trp214. The Trp emission data for a 1:1 OTA-HSA mixture gave φo/φ ) 4.8. To calculate Ro from eq 5, we used an index of refraction n of 1.4, a measured quantum yield φo of 0.05, and a geometrical parameter κ2 of 2/3. Although a geometrical factor of 2/3 corresponds to free rotation of energy donor and acceptor, this value provides a reasonably good estimate for the system because the tryptophan chromophore rotates very rapidly in the protein, as indicated by ps and presumably subps components in its emission anisotropy decay.45-47,54 These parameters yielded a value for Ro of 20 Å, leading to an estimate for R, the apparent distance between Trp214 and A2-, of 16 Å. In view of its rapid rotation, it is reasonable to treat the Trp214 moiety as freely rotating within the volume of a cone. The ratio between the actual distance and R, the apparent distance, can then be estimated from the cone semiangle.52 For a semiangle of 30°, estimated from the reported anisotropy data45-47 for Trp residues in serum albumins, this ratio will be between 0.76 and 1.25, giving a donor-acceptor distance within the range of 12-20 Å. We used the HSA crystal structure (PDB ID 1AO6) to estimate distances from Trp214 to amino acids involved in ligand binding. For the amino acids of site I located in the same subdomain IIA as Trp the distance range (3-16 Å) is much smaller than that for those of site II (9-24 Å). On the bases of only our data for the distance between Trp214 and OTA, we cannot assign the highest-affinity site of OTA to one of these two pockets. However, the significantly larger number of basic and positively charged residues present in site I as compared to site II suggests stronger interaction and therefore a higher binding constant for the site in subdomain IIA. As described in detail in the following paper, further evidence for this assignment was obtained from competitive binding of the toxin and warfarin, a widely used drug with high affinity to serum albumin. It is well documented that the main binding site of warfarin is located in subdomain IIA,32,34b and we showed that OTA was able to displace this drug from that site.55 The recent computational study37 of the binding of OTA to HSA assumed that the toxin binds as a monoanion. According to these calculations the carboxylic group of OTA interacts strongly with Lys199, and the carbonyl group of Arg222 is hydrogen bonded to the protonated phenol group of OTA. However, our work establishes that the phenol group is deprotonated within the protein, and therefore it is likely that the calculated geometry is not correct. Examination of the residues forming site I suggests that the phenoxide ion could be stabilized by electrostatic interactions with Arg257, His242, Arg222, Arg218, Lys199, or Lys195. Crystal structures of HSA complexes with TIB32,33a show that His242 and Lys199 are principal residues involved in stabilization of anionic groups in site I. The role of His242 is confirmed by the crystal structure of warfarin bound to the HSA-myristate complex; His242 is in close contact with the O- group of this drug, while Arg222 interacts with two carbonyl groups.34b
Interaction of OTA with HSA
Figure 5. Intensity of fluorescence excitation spectrum of a 2:1 OTAHSA mixture at 334 nm (open circles) and 390 nm (solid circles) plotted against pH. Emission was collected at 530 nm. Insert shows fluorescence excitation spectra at pH 7.4 (solid), 4.2 (dashed), and 3.0 (dashed-dot).
Assuming that interactions of the carboxyl group of OTA mimic those of TIB one could expect deeper penetration of OTA into the binding cavity compared to warfarin. The crystal structure of TIB bound in site I also shows close proximity of one of the iodine atoms to the positively charged group of Arg257. It is feasible to assume that this residue is responsible for the deprotonation of OTA in the protein environment. Chemical Denaturation and pH Effects on OTA Binding to HSA. Fluorescence polarization spectroscopy was used to compare the binding of OTA to the native and denatured protein. When HSA in the presence of a 2-fold excess of OTA was denatured by 5 M guanidine hydrochloride or by 9 M urea, polarization of OTA emission excited at 375 nm decreased from 0.331 to values close to zero (0.03 and 0.05 for guanidine and urea, respectively). Recall that emission of OTA is completely depolarized in water. These data indicate that chemical denaturation of HSA destroys the high-affinity site for OTA binding. Under the same conditions polarization of Trp214 emission excited at 295 nm decreased only from 0.198 to 0.090. As mentioned above, the binding of OTA to HSA causes a substantial shift of OTA protolytic equilibrium toward the deprotonated form. To determine an apparent pKa value of OTA within the protein environment we measured absorption and fluorescence spectra for a 2:1 OTA-HSA mixture as a function of pH. Under these conditions OTA is not completely bound even at pH ∼ 7. Figure 5 presents intensities of fluorescence excitation spectra at the absorption maxima of HA- (334 nm) and bound A2- (390 nm) determined by collecting fluorescence at 530 nm from solutions with different pH values. Although rather complex pH profiles were obtained, the data clearly show two main inflection points around pH 7 and 4 corresponding to the protonation of free and bound A2-. Similar behavior was observed for the absorption spectra. Apparent pKa values of 3.6 and 4.2 were obtained from fluorescence excitation intensities at 334 and 390 nm, respectively. Combining the pKa values for free and bound OTA and the measured binding constant for the highest affinity site (K1 ∼ 5
J. Phys. Chem. B, Vol. 106, No. 2, 2002 457
Figure 6. Relative quantum yield (A), peak position (B), and full spectral width (C) for OTA (triangles) and Trp214 emission (circles) as a function of solution pH. Spectral parameters were determined from fluorescence spectra of an OTA-HSA mixture (2:1 m/m, solid symbols) and HSA (half-filled circles). Open symbols correspond to the parameters of OTA and L-tryptophan in aqueous solution.
× 106 M-1) into a thermodynamic cycle enables us to estimate the binding constant for the monoanion to be on the order of 103 M-1. Weak binding of the monoanion was confirmed by emission polarization. Fluorescence polarization spectra of 1:1 HSA-OTA mixture were measured at three different pH values with the excitation wavelength set at the absorption maximum of the predominant form at each pH. The P value at the maximum of OTA emission dropped from 0.351 at pH 9.0 to 0.034 at pH 2.0, with P ) 0.407 at pH 7.4. The tryptophan emission polarization was only slightly affected by pH variation. These data directly show that protonated OTA has a very low affinity to HSA. Previous studies30,31,56-58 showed that HSA undergoes several reversible conformational changes with changes in solution pH: from the extended form (E) to so-called fast (F) at pH > 3, to normal (N) at pH ∼ 4, to basic (B) at pH > 8, and to aged (A) with time if pH > 8. Ligand binding is known to depend on the conformational state of serum albumin.30,31,59-63 It is interesting to compare our data for OTA with those for warfarin which shows an approximately 3-fold increase in the affinity for the B form in comparison to the normal conformation.59,61 Higher affinity seems to be correlated with more spatially confined binding site at basic pH.61 Our polarization data demonstrated that the binding constant for the dianion decreases substantially with pH in the range corresponding to the N f B conformational transition of HSA. Considering the larger size of OTA as compared to warfarin this confinement for the B form may lead to an opposite effect of basic pH on the OTA affinity if these two ligands share the same binding site. Parameters of fluorescence spectra recorded for HSA and OTA-HSA solutions with different pH values are presented in Figure 6. In acidic solutions (pH < 3.8) Trp emission intensity was not affected by OTA, but it dropped to a ca. 3-times smaller value when the pH increased from 3 to 5. In the same pH range the OTA emission intensity showed a substantial increase. HSA
458 J. Phys. Chem. B, Vol. 106, No. 2, 2002 fluorescence shifted to the blue and narrowed upon acidification (λmax ) 338 and 329 nm, fwhm ) 58 and 55 nm at pH 6.0 and 3.0, respectively). The transition midpoint was found to be at approximately pH 4.2 for both parameters. In the presence of OTA qualitatively similar behavior of the spectral parameters of HSA emission was observed in this pH range. OTA emission showed a significant red shift and broadening around pH 4 resulting from the protonation of A2-. It is noteworthy that spectral parameters observed for the protonated form in the presence of HSA were practically the same as those for OTA in water at pH 3.0 (see Figure 6). In contrast peak position and spectral width for the deprotonated form of OTA bound to HSA were different from those measured for A2- in water. The transition midpoints calculated from OTA spectral maximum and width were at pH 3.6 and 3.8. Another transition around pH 8-9 could be detected from spectral parameters presented in Figure 6. The pH-dependent changes in spectral parameters of Trp214 emission observed in this study are in good agreement with previously reported results.30,31 A blue shift of the emission maximum suggests a less polar (or more confined) environment of Trp214 residue in F and B form than in the N form. The slight variation in the intensity of tryptophan fluorescence in the N f F transition range observed in this study agrees with the results of Dockal et al.58 For OTA-HSA mixtures the major factor determining the intensity and spectral shape of Trp214 emission is energy transfer to the bound dianion of OTA. The apparent pKa derived from the emission intensity was pKa ∼ 4.2, which is very close to the midpoint corresponding to the N f F conformational transition of HSA. The intensity decrease for OTA emission around pH 4 suggests a larger distance between OTA and Trp214 in the F form than in the N form. The apparent pKa values (3.6-3.8) derived above from excitation intensity at 334 nm and OTA emission parameters mainly reflect its protonation, which occurs at a lower pH than the N f F transition. Conclusions Our studies support the following major conclusions. First, OTA binds to HSA as a dianion; the apparent pKa of the phenolic group of OTA decreases by more than three units when bound to the protein. Second, there are two unique binding sites for OTA and each site can accommodate one dianion. The binding constants for these two sites differ by a factor of ∼50. Third, the binding of OTA is affected by pH-induced structural changes to HSA. No binding is observed between OTA and the denatured protein. Finally, bound OTA strongly quenches the fluorescence of Trp214. These data suggest that the highest affinity binding site is in subdomain IIA of the protein. Acknowledgment. Duke University and a Faculty Recruitment Grant from the North Carolina Biotechnology Center supported this work. References and Notes (1) Van Der Merwe, K. J.; Steyn, P. S.; Fourie, L. J. Chem. Soc. 1965, 7083-7088. (2) Pohland, A. E.; Nesheim, S.; Friedman, L. Pure Appl. Chem. 1992, 64, 1029-1046. (3) (a) Kuiper-Goodman, T.; Scott, P. M. Biomed. EnViron. Sci. 1989, 2, 179-248. (b) Kuiper-Goodman, T. Food Addit. Contam. 1996, 13, 5357. (4) Marquardt, R. R.; Frohlich, A. A. J. Anim. Sci. 1992, 70, 39683988. (5) Chu, F. S. Mutat. Res. 1991, 259, 291-306. (6) Scudamore, K. A. Food Addit. Contam. 1996, 13, 39-42.
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