Anal. Chem. 1999, 71, 3821-3827
Characterization of Minor Site Probes for Human Serum Albumin by High-Performance Affinity Chromatography Arundhati Sengupta and David S. Hage*
Chemistry Department, University of Nebraska, Lincoln, Nebraska 68588-0304
This study used high-performance affinity chromatography (HPAC) and immobilized human serum albumin (HSA) columns to examine the specificity and cross-reactivity of various compounds that have been proposed as markers for the minor binding sites of HSA. These agents included acetyldigitoxin and digitoxin as probes for the digitoxin site, phenol red as a probe for the bilirubin site, and cisor trans-clomiphene as markers for the tamoxifen site. None of these probes showed any significant binding at HSA’s indole-benzodiazepine site. However, phenol red did bind at the warfarin-azapropazone site of HSA, and cis/trans-clomiphene gave positive allosteric effects caused by the binding of warfarin to HSA. Digitoxin and acetyldigitoxin were found to bind to a common, unique region on HSA; cis- and trans-clomiphene also appeared to interact at a unique site, although trans-clomiphene displayed additional direct competition with phenol red. From these results it was possible to develop a model that described the general relationship between these binding regions on HSA. This information should be useful in future studies that employ HPAC for characterizing the binding of HSA to other drugs or clinical agents. The binding of drugs with proteins in blood is an important process in controlling the distribution, excretion, therapeutic activity, and toxicity of these compounds in the body.1 Human serum albumin (HSA) is one protein that is particularly important in the binding and transport of many low-molecular-weight compounds in blood.2 It is believed that compounds that bind with HSA interact at a series of relatively well-defined regions on this protein. The warfarin-azapropazone site (Sudlow site I) and indole-benzodiazepine site (Sudlow site II) are the two bestcharacterized of these binding regions. The presence of these two sites has been confirmed in many studies,3-7 including crystallographic work that has identified the location of these two regions as being in the IIA and IIIA subdomains of HSA, respectively.8 Although the warfarin and indole sites of HSA are believed to be responsible for most of HSA’s drug interactions,8 there is some (1) van Os, G. A. J.; Ariens, E. J.; Simonis, A. M. In Molecular Pharmacology; Ariens, E. J., Ed.; Academic Press: New York, 1964; Vol. 1, p 29. (2) Carter, D. C.; Ho, J. X. Adv. Prot. Chem. 1994, 45, 153. (3) Mu ¨ ller, W. E.; Fehske, K. J.; Schla¨fer, S. A. C. In Drug-Protein Binding; Reidenberg, M. M., Erill, S., Eds.; Praeger Publishers: New York, 1986; Chapter 2. (4) Sjo ¨holm, I. In Drug-Protein Binding; Reidenberg, M. M., Erill, S., Eds.; Praeger Publishers: New York, 1986; Chapter 4. 10.1021/ac9903499 CCC: $18.00 Published on Web 07/24/1999
© 1999 American Chemical Society
evidence that several other minor sites may also take part in the binding of certain drugs to HSA.3,4,9,10 Examples include separate sites that have been proposed for the binding of bilirubin, tamoxifen, and digitoxin, or related compounds, to HSA.2,4 Although the exact locations of these other regions have not been determined and their very existence is still the subject of some debate, there is evidence from both solution-phase experiments2,4 and recent chromatographic studies11,12 that such regions might be present on HSA. The general goal of this study is to confirm or disprove the existence of these other binding regions by using immobilized HSA columns and high-performance affinity chromatography (HPAC). Zonal elution will be performed with the HSA column by injecting a small concentration of several proposed probe compounds for HSA’s minor binding regions. As this is done, a known concentration of warfarin (which interacts at HSA’s warfarin site), L-tryptophan (which binds at HSA’s indole site), or some other competing agent will be continuously applied to the column in the mobile phase. The probe compounds to be used in this work (see representative structures in Figure 1) will include (a) digitoxin and acetyldigitoxin, which have been proposed to bind at the digitoxin site of HSA, (b) cis- and trans-clomiphene, which are thought to interact at the tamoxifen site of HSA, and (c) phenol red, which is believed to specifically displace bilirubin from the bilirubin site of HSA.2,4,11,12 These experiments should generate information on the site specificity for each of these probe compounds and the corresponding association equilibrium constants for their interactions with HSA. These data, in turn, should provide a more detailed picture of the binding regions on HSA and on the overall usefulness of these probe compounds for studying HSA-drug interactions. THEORY The theory behind the zonal elution experiments that were used in this study is described in detail in refs 12 and 13. In (5) Mu ¨ ller, W. E.; Wollert, U. Pharmacology 1979, 19, 59. (6) Sudlow, G.; Birkett, D. J.; Wade, D. N. Mol. Pharmacol. 1975, 11, 824. (7) Fehske, K. J.; Mu ¨ ller, W. E.; Wollert, U. Biochem. Pharmacol. 1981, 30, 687. (8) He, X. M.; Carter, D. C. Nature 1992, 358, 209. (9) Tillement, J. P.; Houin, G.; Zini, R.; Urien, S.; Albengres, E.; Barre`, J.; Lecomte, M.; D’Athis, P.; Sebille, B. Adv. Drug Res. 1984, 13, 59. (10) Mu ¨ ller, W. E.; Wollert, U. Nauyn-Schmiedeberg’s Arch. Pharmacol. 1975, 288, 17. (11) Hage, D. S.; Sengupta, A. J. Chromatogr., B 1999, 724, 91. (12) Hage, D. S.; Sengupta, A. Anal. Chem. 1998, 70, 4602. (13) Noctor, T. A. G.; Wainer, I. W.; Hage, D. S. J. Chromatogr. 1992, 577, 305.
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In the case where a solubilizing agent (S) is added to the mobile phase to bind to A and/or I, then the following modified version of eq 1 can be used to describe the elution of A in the presence of both I and S in the mobile phase.12
1/k′A ) [(VmKILCI)(1 + KASCS)]/[(KALmLtot)(1 + KISCS)] + [Vm(1 + KASCS)(1 + KISCS)]/[(KALmLtot)(1 + KISCS)] (2) In eq 2, KAS and KIS are the association constants for the binding of A and I to S, and CI is the total mobile-phase concentration of the competing agent. All other terms are the same as defined earlier. One assumption made in eq 2 is that the concentration of S is much larger than that of I, so that the term [S] will be approximately equal to the total concentration of S that was originally added to the mobile phase (CS). For the direct competition of A and I for a single type of site on L, eq 2 indicates that a plot of 1/k′A versus CI should give a linear relationship. The values for some of the individual equilibrium constants in eq 2 can be determined by preparing plots of 1/k′A versus CI in the presence of several different levels of excess solubilizing agent and measuring the intercepts and slopes in the linear region of these graphs. A plot of the intercept/slope ratios obtained from these graphs is then made versus CS according to eq 3.
intercept/slope ) 1/KIL + (KISCS)/KIL
Figure 1. Structures of several proposed probe compounds for the major and minor binding regions of HSA. Acetyldigitoxin is similar to digitoxin but has an acetyl group in the place of one of the hydroxyl groups in the carbohydrate portion of its structure.
general, this method involves continuously applying a fixed concentration of a competing agent (I) to a column that contains an immobilized ligand (L) while injections of the probe or analyte (A) are made. If I and A compete at a single type of site on L, and A binds to no other sites on the ligand or the support, then the following equation can be used to describe how the observed retention of A will change as a function of the concentration of I that is present in the mobile phase.13
1/k′A ) (VmKIL[I])/(KALmLtot) + Vm/(KALmLtot)
(1)
In this equation, [I] is the mobile-phase concentration of the competing agent, mLtot is the total moles of common binding sites in the column for I and A, Vm is the column void volume, and KAL and KIL are the association equilibrium constants for the binding of A and I to their common binding region on L. The term k′A is the capacity factor for A, as given by the relationship k′A ) (tR tM)/tM, where tR is the mean retention time for A and tM is the column void time. For the 1:1 direct competition of A with I, eq 1 predicts that a plot of 1/k′A versus [I] should yield a linear relationship with a slope of (VmKIL)/(KALmLtot) and an intercept of Vm/(KALmLtot). By calculating the ratio of the slope to the intercept for such a plot, the value of KIL (i.e., the association equilibrium constant for I at the site of competition with A) can be determined. 3822 Analytical Chemistry, Vol. 71, No. 17, September 1, 1999
(3)
This type of plot should provide a second linear relationship for a system with 1:1 binding. The intercept for this new plot is equal to 1/KIL and the slope is equal to KIS/KIL, thus providing the association equilibrium constants for the binding of I to both L and S.12 MATERIALS AND METHODS Reagents. The individual cis- and trans-clomiphene isomers were kindly supplied by the Marion Merrell Dow Research Institute (Cincinnati, OH). The acetyldigitoxin (2:1 ratio of R and β forms), digitoxin, L-tryptophan, racemic warfarin (i.e., 3-(Racetonylbenzyl)-4-hydroxycoumarin), phenol red (0.5% solution), β-cyclodextrin, and HSA (Cohn fraction V, 99% pure, fatty acid free) were obtained from Sigma (St. Louis, MO). The HSA and diol-bonded silica supports were prepared using Nucleosil Si-300 silica (7-µm particle diameter, 300-Å pore size) from MachereyNagel (Du¨ren, Germany). Other chemicals and biochemicals used were of the purest grades available. All aqueous solutions in this study were prepared using water purified by a Nanopure water system (Barnstead, Dubuque, IA). Apparatus. The chromatographic system consisted of a CM3000 solvent delivery system from LDC/Thermoseparations (Riviera Beach, FL), a Rheodyne 7010 injection valve (Cotati, CA) equipped with a pneumatic actuator from Valco (Houston, TX), a SM3100 UV-Vis variable-wavelength detector from LDC, and a WOW data acquisition interface from LDC. Chromatograms were processed by programs written in Microsoft QuickBASIC (Redmond, WA). All columns and mobile phases were maintained at 37 ( 0.2 °C using an Isotemp 9100 water bath (Fisher Scientific, Pittsburgh, PA). The columns were downward slurry-packed using an HPLC column slurry packer from Alltech (Deerfield, IL).
Methods. Diol-bonded silica was prepared as described previously.14 The diol coverage of the Nucleosil was 230 ( 3 µmol ((1 SD) per gram of silica, as determined in duplicate by an iodometric capillary electrophoresis assay.15 HSA was placed onto this support by using the Schiff base immobilization method.16,17 A small portion of the silica was washed with deionized water, dried, and assayed in triplicate for its protein content by the bicinchoninic acid (BCA) assay, using HSA as the standard and diolbonded silica as the blank;18 this gave a protein content for the support of 426 ( 2 nmol/g of silica. The HSA support and a portion of the original diol-bonded silica were downward slurry-packed at 3500 psi into two separate 3 cm × 2 mm i.d. columns. Both columns were enclosed in water jackets for temperature control. All mobile phases and packing solvents were prepared using pH 7.4, 0.067 M potassium phosphate buffer. Prior to use, the mobile phases were filtered through 0.22-µm nylon filters and degassed under vacuum for 15 min. The mobile phases for the competitive binding studies were made by adding 0-10 µM of the desired competing agent to the pH 7.4, 0.067 M phosphate buffer. In the case of experiments that used cis- or transclomiphene, acetyldigitoxin, or digitoxin, 2.5 mM β-cyclodextrin was first added to the phosphate buffer to act as a solubilizing agent. Proton NMR studies indicated that the cis- and transclomiphene solutions were stable for at least two weeks when stored in an aqueous solvent at 4-37 °C.12 Through UV-visible absorbance measurements, it was found that the acetyldigitoxin, digitoxin, and phenol red solutions were also stable for several weeks when stored in pH 7.4, 0.067 M phosphate buffer at 4-37 °C. The warfarin solutions demonstrated similar stability, but the L-tryptophan solutions had to be prepared fresh daily before use. Experiments were performed at flow rate of 0.1-0.2 mL/min, with the desired probe being applied in replicate 20-µL injections. Elution of the probe compounds was monitored at the following wavelengths: 290 nm, L-tryptophan; 310 nm, warfarin; 221 nm, digitoxin and acetyldigitoxin; 268 nm, cis- and trans-clomiphene; and 558 nm, phenol red. The retention time of each injected probe was calculated by using the first statistical moment of its corresponding peak.19 The void time of the column was determined by making similar injections of a nonretained compound (e.g., sodium nitrate). The capacity factor (k′) for the probe compound was then calculated and evaluated over a series of competing agent concentrations, as described in refs 17 and 20. The typical concentrations of the probe samples were 65 µM warfarin, 1.5 µM L-tryptophan, 1 µM phenol red, 0.5 µM digitoxin, 0.5 µM acetyldigitoxin, or 100 µM cis- or trans-clomiphene. The column back pressure throughout these experiments was less than 250 psi. There was no noticeable shift in the capacity factors for the injected probes when going to slightly higher or lower sample concentrations, thus indicating that linear elution conditions were present during these experiments, as assumed in eqs 1 and 2. There were also no significant changes in the capacity factors that were noted under the range of flow rates and column back pressures that were sampled in this study. RESULTS AND DISCUSSION Competition Studies at the Indole and Warfarin Sites of HSA. The first item addressed in this study was to determine whether the proposed probe compounds for the minor binding regions of HSA also had any interactions at the major binding
Figure 2. Typical chromatograms obtained in zonal elution experiments for the injection of cis-clomiphene into the presence of racemic warfarin in the mobile phase. The mobile-phase concentrations of warfarin (left to right) were 0, 5 × 10-7, 1 × 10-6, and 7.5 × 10-6 M.
regions of HSA (i.e., the warfarin-azapropazone and indolebenzodiazepine sites). This was analyzed by examining the competition of each probe compound with L-tryptophan and (R/ S)-warfarin.17 L-Tryptophan was used to test binding at the indolebenzodiazepine site, since this agent is known to interact 1:1 with HSA at this region and has a well-characterized association equilibrium constant for this binding process.2,3 Similarly, both (R)- and (S)-warfarin bind in a 1:1 stoichiometry at the warfarinazapropazone region and have known association constants for these interactions.2,3,21 Experiments performed with L-tryptophan in the mobile phase did not show any significant changes in the retention times for any of the tested probe compounds. In each case, only random variations in k′A of (1-7% were seen for the injected probes in the presence of mobile phases containing 0-50 µM L-tryptophan. Also, plots of 1/k′A for the probe compounds versus [L-tryptophan] gave no significant correlation (correlation coefficient, 0.00820.2689 over seven data points) and produced slopes that overlapped with zero within a range of (1 SD of the best-fit slope. Together these results indicated that none of the proposed probe compounds had any detectable competition with L-tryptophan at the indole-benzodiazepine site of HSA. Similar competitive binding studies were performed for the warfarin-azapropazone site by injecting small amounts of each probe compound into the presence of (R/S)-warfarin in the mobile phase. Examples of some chromatograms that were obtained in these studies are shown in Figure 2. Although the R and S enantiomers of warfarin are known to have different equilibrium constants for their binding site on HSA,21 it has been found that (14) Ruhn, P. F.; Garver, S.; Hage, D. S. J. Chromatogr. 1994, 669, 9. (15) Chattopadhyay, A.; Hage, D. S. J. Chromatogr. 1997, 758, 255. (16) Larsson, P. O. Methods Enzymol. 1984, 104, 212. (17) Loun, B.; Hage, D. S. J. Chromatogr. 1992, 579, 225. (18) Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C. Anal. Biochem. 1985, 150, 76. (19) Grushka, E.; Myers, M. N.; Schettlez, P. D.; Giddings, J. C. Anal. Chem. 1969, 41, 889. (20) Yang, J.; Hage, D. S. J. Chromatogr. 1996, 725, 273. (21) Loun, B.; Hage, D. S. Anal. Chem. 1994, 66, 3814.
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Figure 3. Zonal elution plots for the injection of (a) phenol red and (b) cis-clomiphene in the presence of racemic warfarin as a competing agent.
racemic warfarin can still be used effectively as a probe in the initial screening of solute interactions at the warfarin-azapropazone region of HSA.11,12 Neither acetyldigitoxin nor digitoxin gave any detectable change in its capacity factors during the experiments with warfarin. For example, only a random variation in k′A of (3-4% was seen for these agents in the presence of 0-10 µM warfarin, and correlation coefficients of only 0.1865-0.1942 were obtained for these compounds over seven data points in plots of 1/k′A versus [warfarin]. The same type of independent behavior has been reported for competitive binding studies performed between L-tryptophan and warfarin on immobilized HSA columns.17 These results indicate that acetyldigitoxin and digitoxin do not have any appreciable binding to the warfarin-azapropazone site of HSA under the conditions that were used in this study.11 This agrees with previous solution-phase work, in which it was found that digitoxin was not displaced by warfarin from HSA.4 Different behavior was seen when phenol red was injected into the presence of warfarin as a competing agent. In this case, there was a noticeable decrease in the k′A value for this probe as the mobile-phase concentration of warfarin was increased. When 1/k′A for phenol red was plotted versus [warfarin] (see Figure 3a), the result was a linear relationship (correlation coefficient, 0.9989 for seven data points), indicating that phenol red and warfarin shared a single common binding site on HSA (i.e., the warfarinazapropazone region). The same type of behavior was found in the reverse experiment, in which racemic warfarin was injected into the presence of phenol red as a mobile-phase additive (see 3824 Analytical Chemistry, Vol. 71, No. 17, September 1, 1999
later section on the use of phenol red as a competing agent). On the basis of eq 1, the intercept and slope of the best-fit line in Figure 3a were used to determine the association equilibrium constant for warfarin at its site of competition with phenol red. The resulting value of 3 × 105 M-1 gave good agreement with the average association constant of (2.3-3.3) × 105 M-1 that was previously reported for the binding of racemic warfarin and (R)or (S)-warfarin to HSA,21-25 thus confirming that the site of warfarin/phenol red competition is the warfarin-azapropazone site of HSA. The capacity factors for cis- and trans-clomiphene also showed a significant change when the mobile-phase concentration of warfarin was varied. But for these probes, a plot of 1/k′A versus [warfarin] gave a relationship in which 1/k′A decreased (or k′A increased) as the warfarin concentration was raised (see Figure 3b). This type of behavior indicates that cis- and trans-clomiphene have indirect, or allosteric, competition with warfarin and that these agents do not bind directly at the warfarin-azapropazone site of HSA.12 Similar allosteric effects on HSA have been reported in solution-phase studies examining the competition between warfarin and tamoxifen, a compound that is closely related to cis/ trans-clomiphene in structure.4 Competition Studies Using cis- and trans-Clomiphene as Mobile-Phase Additives. Competitive binding studies were next performed by injecting small amounts of each probe compound into the presence of cis- or trans-clomiphene, which was employed as a probe for the proposed tamoxifen binding site of HSA.4 Clomiphene was used instead of tamoxifen for this study since it has been reported to be more specific than tamoxifen in its binding to HSA.4 Although most earlier studies that have examined the binding of clomiphene to HSA have used a mixture of the cis and trans isomers, in this work the separate isomers were used in order to provide more specific information on the protein interactions of these solutes.12 Previous zonal elution experiments on the same type of immobilized HSA column as used in this study have shown that both cis- and trans-clomiphene have 1:1 interactions at a single, common binding site on HSA. The average association constant of this site for cis-clomiphene is 7.5 ((0.2) × 106 M-1, and the average association constant for trans-clomiphene is 1.3 ((0.2) × 106 M-1.12 Also, it was shown earlier in this current report that cis- and trans-clomiphene did not have any competition with L-tryptophan and that they had only allosteric interactions with the warfarin-azapropazone site of HSA. This means that the binding regions for cis- and trans-clomiphene are separate from the major binding sites of HSA.12 The interactions of cis- and trans-clomiphene with HSA were studied further by using acetyldigitoxin and digitoxin as injected probes. No competition was seen between cis-clomiphene and either of these test solutes, with both analytes showing only small, random variations in their k′A values ((1-6%) when present in mobile phases that contained 0-10 µM cis-clomiphene. Furthermore, plots of 1/k′A versus [cis-clomiphene] yielded best-fit lines (22) Larsen, F. G.; Larsen, C. G.; Jakobsen, P.; Brodersen, R. Mol. Pharmacol. 1985, 27, 263. (23) Lagercrantz, C.; Larsson, T.; Denfors, I. Comp. Biochem. Physiol. 1981, 69C, 375. (24) Lagercrantz, C.; Larsson, T.; Karlsson, H. Anal. Biochem. 1979, 99, 352. (25) O’Reilly, R. A. J. Clin. Invest. 1969, 48, 193.
Figure 5. Plot of the intercept/slope ratio as a function of [β-cyclodextrin] for data obtained from 1/k′A plots during the injection of phenol red in the presence of trans-clomiphene as a mobile-phase additive.
Figure 4. Zonal elution plots for the injection of phenol red in the presence of (a) cis-clomiphene and (b) trans-clomiphene as mobilephase additives.
that gave no significant correlation (correlation coefficient, 0.27700.6676 over seven data points) and that produced slopes which overlapped with zero within a range of (2 SD. A similar lack of correlation was noted when examining the change in k′A for acetyldigitoxin and digitoxin in the presence of mobile phases that contained various concentrations of trans-clomiphene. In this latter set of studies, only random variations of (3-5% were noted in k′A, and correlation coefficients of 0.7272-0.7403 were obtained over seven data points for plots of 1/k′A versus [trans-clomiphene]. Altogether, these data indicate that acetyldigitoxin and digitoxin did not have any significant competition with either cis- or transclomiphene for HSA binding regions. However, a difference in the behavior of cis- and transclomiphene was seen when phenol red was used as the injected solute (see Figure 4). For example, phenol red injected into mobile phases that contained 0-10 µM cis-clomiphene did not produce any noticeable change in the retention of phenol red ((3% variation in k′A; correlation coefficient, 0.2770 over seven data points for a plot of 1/k′A versus [cis-clomiphene]). But phenol red did show a significant change in retention when trans-clomiphene was used as a mobile-phase additive. The resulting plot of 1/k′A versus [trans-clomiphene] (see Figure 4b) was similar in appearance to that observed previously for the competition of cis/cisand trans/trans-clomiphene.12 When the competition between phenol red and trans-clomiphene was examined, plots of 1/k′A versus [trans-clomiphene] were found to be linear when using 0-1.0 µM clomiphene and
1.0-2.6 mM β-cyclodextrin as a solubilizing agent (e.g., see Figure 4b). The correlation coefficients of these plots ranged from 0.9997 to 0.9999 over four to five data points. According to eq 2, this suggested that phenol red and trans-clomiphene had direct 1:1 competition for binding sites on HSA. The ratios of the intercepts and slopes from these graphs were next plotted versus the concentration of β-cyclodextrin that was used in the mobile phase. This gave a second linear relationship, as predicted by eq 3, that had a correlation coefficient of 0.9997 over four data points (see Figure 5). By taking the reciprocal of the intercept in Figure 5, it was possible to determine the association equilibrium constant for trans-clomiphene at its site of competition with phenol red. The resulting value of 1.3 ((0.2) × 106 M-1 was statistically identical to that found earlier for the binding of trans-clomiphene at its site on HSA.12 The fact that phenol red showed competition with trans- but not cis-clomiphene suggests that all three agents are binding to the same general vicinity of HSA, but with cis-clomiphene and phenol red having no overlap in the position of their individual binding sites. On the other hand, trans-clomiphene, which has direct competition with both cis-clomiphene and phenol red, must occupy some intermediate binding region that does have overlap with the binding sites for each of these other two compounds. Competition Studies Using Acetyldigitoxin or Digitoxin as Mobile-Phase Additives. It has been proposed in the literature that digitoxin and acetyldigitoxin have a distinct site on HSA that is separate from the indole-benzodiazepine binding region4 and separate from but near to the warfarin-azapropazone site.4,26,27 This made both of these compounds appealing for use as potential probes in examining the interactions of other compounds at HSA’s digitoxin site. Previous zonal elution studies on HSA columns have confirmed that digitoxin and acetyldigitoxin share a single common binding region on HSA. The average association equilibrium constant for digitoxin at this binding site is 5.3 ((0.2) × 104 M-1 and the average association constant for acetyldigitoxin is 4.8 ((0.2) × 104 M-1.11 These numbers show good agreement with (26) Kragh-Hansen, U. Biochem. J. 1985, 225, 629. (27) Peters, T., Jr. All About Albumin; Academic Press: New York, 1996; Chapter 3.
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Table 1. Competition between Proposed Probe Compounds with Agents Known To Bind at HSA’s Warfarin-Azapropazone Site ((R/S)-Warfarin) or Indole-Benzodiazepine Site (L-Tryptophan) competing agent and type of competitiona probe compound
L-tryptophan
(R/S)-warfarin
cis-clomiphene trans-clomiphene digitoxin acetyldigitoxin phenol red
no competition no competition no competition no competition no competition
allosteric allosteric no competition no competition direct-1 site Ka for phenol red, 7.8 ((1.3) × 105 M-1; Ka for warfarin, 3.0 ((0.2) × 105 M-1
a These data were obtained at 37 °C in pH 7.4, 0.067 M phosphate buffer. The numbers in parentheses represent a range of (1 SD.
Figure 6. Zonal elution plots for the injection of (a) racemic warfarin and (b) phenol red into the presence of phenol red as a mobile-phase additive.
a previous value of 4 × 104 M-1 that was reported for the interaction of digitoxin with HSA in solution.28 In earlier sections of this current report, it was found that acetyldigitoxin and digitoxin did not have any observable competition with warfarin, L-tryptophan, cis-clomiphene, or trans-clomiphene. Additional zonal elution studies were next carried out to examine the competition of acetyldigitoxin and digitoxin with phenol red. Increasing the amount of acetyldigitoxin in the mobile phase did not show any significant change in the retention times for phenol red on the HSA column (random variations in k′A of (2% and a correlation coefficient of 0.4412 over seven data points for plots of 1/k′A versus [acetyldigitoxin]). Increasing the amount of phenol red in the mobile phase also did not create any significant changes in the retention times for digitoxin on the HSA column (random variation of (1.9% and a correlation coefficient of 0.0036 for seven data points for plots of 1/k′A versus [phenol red]. Thus, the binding of acetyldigitoxin and digitoxin was independent of the interactions between phenol red and HSA. Competition Studies Using Phenol Red as a Mobile-Phase Additive. Phenol red was previously suggested as a probe for the bilirubin site of HSA4 since it is known that bilirubin competitively displaces this agent from at least some of bilirubin’s binding regions.29 It was shown earlier that phenol red does not compete with L-tryptophan, cis-clomiphene, acetyldigitoxin, or digitoxin. However, it was also seen that some interactions do occur between phenol red and warfarin or trans-clomiphene. These later interactions were now examined in more detail by studying 3826 Analytical Chemistry, Vol. 71, No. 17, September 1, 1999
the change in retention for warfarin and trans-clomiphene as the mobile-phase concentration of phenol red was varied. Competitive binding studies using injected warfarin and phenol red in the mobile phase produced behavior that was consistent with a direct competition between these two agents for a single, common binding site. This is demonstrated in Figure 6a, where a linear relationship was noted for a plot of 1/k′A for warfarin versus [phenol red] (correlation coefficient, 0.9987 over seven data points). From the earlier studies using warfarin as a competing agent, it was known that this competition was occurring at the warfarin-azapropazone site of HSA. By using eq 1 along with the slope and intercept of Figure 6a, it was possible to determine the association equilibrium constant for phenol red at this site. The result was an association constant of 7.8 ((1.3) × 105 M-1 for the interactions of phenol red at the warfarin-azapropazone site. Finally, competitive binding studies were performed using phenol red as both the injected solute and mobile-phase additive. The resulting plot of 1/k′A versus [phenol red] (see Figure 6b) gave a nonlinear relationship even though no solubilizing agent was used in this experiment. This same general behavior has been observed for the self-competition of (S)-ibuprofen30 and indicates that phenol red has more than one binding region on HSA. It was shown earlier in this current study that one of the sites for phenol red on HSA is the warfarin-azapropazone region. But it was also shown previously that phenol red and trans-clomiphene have direct competition on HSA when trans-clomiphene is used as a mobile-phase additive and only a trace amount of phenol red is present in the system. To examine this situation further, another set of studies was performed using phenol red in the mobile phase and trans-clomiphene as the injected probe. Plots generated for 1/kA′ versus [phenol red] now gave a curved line with a decreasing slope similar to that seen for cis- and trans-clomiphene injected into the presence of warfarin as a competing agent (see Figure 3b). This behavior indicated that at least one of the other binding regions for phenol red has allosteric interactions with transclomiphene, in agreement with the fact that phenol red is known to bind at the warfarin-azapropazone site. Although this allosteric (28) Kragh-Hansen, U. Pharmacol. Rev. 1981, 33, 17. (29) Fleck, C.; Kunze, C.; Braunlich, H. Exp. Pathol. 1987, 32, 99. (30) Hage, D. S.; Noctor, T. A. G.; Wainer, I. W. J. Chromatogr., B 1995, 693, 23.
Table 2. Number of Common Binding Sites for the Selected Probe Compounds type of competition and association equilibrium constants for competing agent (M-1) added to mobile phase probe compound
cis-clomiphene
trans-clomiphene
phenol red
no competition
acetyldigitoxin
no competition
direct-1 site, 1.3 ((0.2) × 106 no competition
digitoxin
no competition
no competition
trans-clomiphene
direct-1 site, 7.6 ((0.2) × 106 direct-1 site, 7.4 ((0.2) × 106
direct-1 site, 1.3 ((0.2) × 106 direct-1 site, 1.2 ((0.2) × 106
cis-clomiphene
Figure 7. Proposed relative arrangement of the major and minor binding regions on HSA. The arrow indicates the presence of allosteric effects between the warfarin and tamoxifen sites.
behavior made it impractical to determine the association constants for phenol red at all of its sites on HSA, this did give further support to a model in which HSA has more than one binding region for phenol red. Similar behavior has been seen for the interactions of bilirubin with HSA, which involves at least two binding regions that appear to be linked through allosteric effects. These allosteric interactions between the bilirubin and warfarin sites are believed to be due to the relatively close location of these regions to each other in the IIA subdomain of HSA.27 SUMMARY This report used HPAC and immobilized HSA columns to examine the specificity of various compounds that have been proposed for use as probes or markers for the minor binding sites of HSA. The agents that were studied included acetyldigitoxin and digitoxin (as probes for the digitoxin site of HSA), phenol red (as
acetyldigitoxin
digitoxin
no competition
no competition
direct-1 site, 4.8 ((0.2) × 104 direct-1 site, 4.9 ((0.2) × 104 no competition
direct-1 site, 5.4 ((0.2) × 104 direct-1 site, 5.2 ((0.2) × 104 no competition
no competition
no competition
phenol red multisite; 7.8 ((1.3) × 105 at warfarin site no competition no competition allosteric no competition
a probe for the bilirubin site), and cis- or trans-clomiphene (as markers for the tamoxifen site). In studies examining the binding of these compounds to the major sites of HSA (see Table 1), none of these probes gave any significant binding at HSA’s indolebenzodiazepine site. However, phenol red was found to have direct competition with warfarin at the warfarin-azapropazone site. Also, the interactions of cis- and trans-clomiphene were enhanced through positive allosteric effects brought about by the binding of warfarin to its site on HSA. In comparing the selectivity of these marker compounds at the minor sites of HSA (Table 2), digitoxin and acetyldigitoxin were found to each bind to a unique region on this protein. Similarly, cis-clomiphene and trans-clomiphene appeared to interact at a unique site on HSA, although trans-clomiphene displayed additional direct competition with phenol red. The observation that cis- and trans-clomiphene compete with each other but that only the trans form competes with phenol red is consistent with a previously proposed model in which the binding regions of HSA are viewed as being general locations on this protein as opposed to locations confined to a few amino acid residues.3,13 Through the results obtained in this work, it was possible to develop a model that described the general relationship between the various binding regions on HSA (see Figure 7). It was clear from these studies that using digitoxin/acetyldigitoxin and cis/ trans-clomiphene as marker compounds did allow the selective sampling of binding regions on HSA that are separate from the major binding sites for this protein. The results of this study also suggest that the bilirubin site might overlap with the tamoxifen binding region; however, further studies are still needed to confirm or disprove this hypothesis. What does result from this work is a more complete picture of the specificities and binding properties for these marker compounds in their interactions with HSA. This information should be useful in future studies employing HPAC for the characterization of HSA binding to other pharmaceutical compounds or endogenous agents. Furthermore, the methods described within this report could be employed with other potential markers or proteins for examining additional soluteprotein interactions of pharmaceutical or clinical interest. ACKNOWLEDGMENT This work was supported under Grant RO1 GM44931 from the National Institutes of Health. Received for review April 6, 1999. Accepted June 16, 1999. AC9903499 Analytical Chemistry, Vol. 71, No. 17, September 1, 1999
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