Anal. Chem. 1998, 70, 4235-4240
HSA-Solute Interactions, Enantioselectivity, and Binding Site Geometrical Characteristics Eric Peyrin, Yves Claude Guillaume,* and Christiane Guinchard
Laboratoire de Chimie Analytique, Faculte´ de Medecine et Pharmacie, Place Saint Jacques, 25030 Besanc¸ on Cedex, France
Recently, a theoretical model was proposed to study the existence of pockets of acetonitrile (ACN) called clusters in a hydroorganic mixture. The proposal used ACN as an interaction organic modifier between D,L-dansyl amino acids and their binding site in human serum albumin at site II. This solute binding is governed by primary and secondary interactions. The primary interactions are under the dependence of the solute solvation by ACN clusters and electrostatic interactions. Following this first step, the solute engages strong short-range interactions with the residues of site II. Using a biochromatographic approach, the solute binding, i.e., the solute retention, was divided into two dielectric constant (E) ranges. In the first range, E > Ec (Ec is the critical dielectric constant); the primary and secondary nonstereoselective electrostatic interactions were the major contributions to the variation in the solute binding with the ACN fraction in the mixture. In the second range, E < Ec, the solute retention variation with the ACN fraction was governed by its solvation by the ACN clusters and also by the secondary hydrophobic stereoselective interaction. The mathematical model developed provided the determination of the surface charge density of site II as well as the cluster number that solvates each solute. Human serum albumin (HSA) is the major soluble constituent of the circulatory system implicated in colloidal osmotic blood pressure and the transport of drugs and other small molecules.1 This is a globular protein (molecular mass ∼66 000 Da) consisting of a single chain of 585 amino acid residues which is formed into subdomains by paired 17 disulfide bonds. Only a few specific binding sites are present on HSA.2 The most important are sites I and II, which are also called warfarin-binding sites and benzodiazepine-binding sites.3 He and Carter4 have determined the three-dimensional structure of HSA, which shows that these two binding sites are located in hydrophobic cavities in subdomains IIA and IIIA. Many ligands, such as flurbiprofen, dansylsarcosine, and L-tryptophan, have been found to bind preferentially on the site II binding cavity.5,6 This cavity is accessed through a 8-10(1) Fehske, K. J.; Muller, W. E.; Wollert, V. Biochem. Pharmacol. 1981, 30, 687. (2) Behreus, P. Q.; Spiekermann, A. M.; Brown, J. R. Fed. Proc. 1975, 34, 591. (3) Sudlow, G.; Birkett, D. J.; Wade, D. N. Mol. Pharmacol. 1975, 11, 824. (4) He, X. M.; Carter, D. C. Nature 1992, 358, 209. (5) Muller, W. E.; Wollert, V. Res. Commun. Chem. Pathol. Pharmacol. 1975, 10, 565. S0003-2700(98)00370-9 CCC: $15.00 Published on Web 09/19/1998
© 1998 American Chemical Society
Å-diameter opening between two helicoidal structures.4,7 The distribution of hydrophobic and hydrophilic residues in the binding crevice is distinctly asymmetric. The principal nonpolar residues are sequestered into the hydrophobic cavity inside the protein core and the polar residues onto the surface.4 Many previous investigations of ligand binding to the HSA site II cavity have been reported in the literature. These studies have been based on a variety of experimental techniques including equilibrium dialysis, fluorescence circular dichroism, and biochromatography.4,8-10 Maruyama et al.11 studied the mechanistic aspects of suprofen binding to site II using dialysis and spectroscopic techniques. Thermodynamic analysis and proton relaxation rate measurements indicated that the hydrophobic side chain of suprofen was deeply inserted into the hydrophobic crevice while the carboxyl group interacted with the cationic residue at the surface of HSA. The same behavior was observed by Rahman et al.12 for the binding of caprofen to HSA. Using chromatographic techniques, Peyrin et al.13 described a binding mode of negatively charged test solutes (dansyl amino acids) on the HSA site II cavity where the compound hydrophobic groups occupied the nonpolar interior of the cavity and carboxylate and sulfonalimido groups interacted with Arg 410 and Tyr 411 residues at the cavity rim forming electrostatic and hydrogen bonds. The role of both the structural behavior of the binding cavity and the hydrophobic longrange forces on the retention mechanism of dansyl amino acids was examined using temperature studies and differential scanning calorimetry.14 In an effort to extend the investigation of the molecular aspects of solute-site II cavity binding, the influence of acetonitrile (ACN) as an organic modifier of the bulk solvent on the interaction forces controlling the solute cavity association was studied using biochromatography. The usefulness of an equation that describes the variation of the dansyl amino acid retention factor in an ACN/water mixture with an HSA column (6) Sudlow, G.; Birkett, D. J.; Wade, D. N. Mol. Pharmacol. 1976, 12, 1052. (7) Wanwinolruk, S.; Birkett, D. J.; Brooks, P. M. Mol. Pharmacol. 1983, 24, 458. (8) . Noctor, T. A.; Wainer, I.; Hage, D. S. J. Chromatogr., B 1992, 577, 305. (9) Kohita, H.; Matsuhita, Y.; Moriguchi, I. Chem. Pharm. Bull. 1994, 42, 937. (10) Otagiri, M.; Matsuda, K., Imai, T.; Imamura, Y.; Yamasaki, M. Biochem. Pharmacol, 1989, 38, 1. (11) Maruyama, T.; Kin, C. C.; Yamasaki, K.; Miyoshi, T.; Imai, T.; Yamasaki, M.; Otagiri, M. Biochem. Pharmacol. 1993, 45, 1017. (12) Rahman, M. H.; Maruyama, T.; Okada, T.; Yamasaki, K.; Otagiri T. Biochem. Pharmacol. 1993, 46, 1721. (13) Peyrin, E.; Guillaume, Y. C.; Guinchard, C. J. Chromatogr. Sci. 1998, 36, 97. (14) Peyrin, E.; Guillaume, Y. C.; Guinchard, C. Anal. Chem. 1997, 69, 4979.
Analytical Chemistry, Vol. 70, No. 20, October 15, 1998 4235
was studied. This equation is expressed in terms of fractions of free acetonitrile (not in a cluster) and water in the ACN/water mixture and the corresponding dielectric constant of the media. This new mathematical model is explicitly derived from the respective contributions of (i) the long-range (or primary) electrostatic and hydrophobic interactions and (ii) the short-range (or secondary) interactions for ligand-receptor association. This equation was tested and used to evaluate the retention and chiral recognition mechanisms of solute-HSA interaction. EXPERIMENTAL SECTION Apparatus. The HPLC system consisted of a Merck Hitachi pump L7100 (Nogent-sur-Marne, France) and an Interchim Rheodyne injection valve (model 7125, Montluc¸ on, France) fitted with a 20-µL sample loop and a Meck L 4500 diode array detector (Nogent-sur-Marne, France). An HSA protein chiral Shandon column (150 mm × 4.6 mm) was used with controlled temperature at 25 °C in an Interchim Crococil oven (TM 701, Montluc¸ on, France). After each use, the column was stored at 4 °C. Throughout the study, the column flow rate was maintained constant at 1 mL/min. Solvents and Samples. HPLC grade acetonitrile (Merck) was used without further purification. Sodium hydrogen phosphate and sodium dihydrogen phosphate were supplied by Prolabo (Paris, France). Water was obtained from an Elgastat option water purification system (Odil, Talant, France) fitted with a reverseosmosis cartridge. D,L-dansylnorvaline and D,L-dansyltryptophan were obtained from Sigma-Aldrich (Saint Quentin, France) and were made fresh daily at a concentration of 20 mg/L in acetonitrile. Sodium nitrate was used as a dead time marker (Merck). The mobile phase consisted of a 0.01 M sodium phosphate buffer at pH 6.0 with ACN fractions varying from 0.0 to 0.20. A 20-µL sample of each solute or a mixture of these was injected and the retention times were measured. THEORY The solute retention on HSA is controlled by the classical primary (i) and secondary (ii) interactions involved in the ligand receptor binding. (i) The primary or nonspecific interactions are under the dependence of the long-range forces which permit the approach of the solute at the opening of the cavity. These interactions consist principally of two oppositely charged species in a combination of hydrophobic effect and electrostatic interactions.15 The van der Waals interactions are negligible due to the short magnitude and range of their driving forces.15 Thus, the primary Gibbs free energy change of transfer of the solute from the bulk solvent to the HSA cavity could be broken down as follows:
∆G°I ) ∆G°I,H + ∆G°I,es
Primary Hydrophobic Interactions between Solute and the HSA Cavity. Generally the main parameter determining the retention in reversed-phase liquid chromatography (RPLC) is the distribution of the solute molecule in the stationary phase, whereas the interactions with the mobile phase play a minor role.16-19 In an ACN/water mixture, the solute retention is split into two main physicochemical processes, i.e., solute solvation by ACN clusters and solute transfer from a pure aqueous mobile phase to the stationary phase. The ACN organic modifier was predicted to increase the solubility of the nonpolar solute by decreasing the hydrophobic interaction due to a direct solute solvation. If y is the fraction of free ACN and Φ the fraction of water in the mixture ACN/water, it has been previously demonstrated18 that the Gibbs primary hydrophobic (i.e., solvation) isotherm was
(∆G°)I,H ) RT ∂(ln[1 + KψP(1 - y)P(1 - ψ)P])
(2)
where K is the solvation equilibrium constant of the solute per P clusters and ψ is a constant. Primary Electrostatic Interactions between Solute and the HSA Cavity. Usually, with a decrease in the dielectric constant, the electrostatic interactions between the cavity opening charged residue (Arg 410) and the ionic dansyl amino acid increase, implying an increase in the solute binding on HSA.13 The application of the Gouy-Chapman theory gave the calculation of the binding HSA cavity surface density and its dependence on the dielectric constant value. The electrostatic contribution to the primary free energy of interaction ∆G°I,es is related to the surface potential φo, where z is the charge of the solute being adsorbed and F the Faraday constant:
∆G°I,es ) zFφo
(3)
The Gouy-Chapman theory20 relates the surface potential to the surface charge density σ which has units of charge per area:
σ ) x8kT0I sinh (∆G°I,es/2RT)
(4)
This relation accounts for a mobile phase of the dielectric constant and ionic strength I (o is the permittivity of free space and kT is thermal energy). If dACN and dH2O are the molar density of ACN and water, respectively, the molar fractions of ACN xACN and water xH2O were given by the following equations:
xH2O ) dH2OΦ/(dH2OΦ + dACN(1 - Φ))
(5)
xACN ) dACN(1 - Φ)/(dH2OΦ + dACN(1 - Φ))
(6)
(1) The dielectric constant of the ACN/water mixture was given by
where ∆G°I,H corresponds to the Gibbs free energy change due to the hydrophobic effect and ∆G°I,es corresponds to the Gibbs free energy change due to the electrostatic interactions. (15) Leckband, D. E.; Israelachvili, J. N.; Schmitt, F. J.; Knoll, W. Science 1992, 255, 1419.
4236 Analytical Chemistry, Vol. 70, No. 20, October 15, 1998
(16) Guillaume, Y. C.; Guinchard, C. J. Liq. Chromatogr. 1994, 17, 2809. (17) Guillaume , Y.C.; Guinchard, C. Anal. Chem. 1996, 68, 2869. (18) Guillaume, Y. C.; Guinchard, C. Anal. Chem. 1997, 69, 183. (19) Guillaume, Y. C.; Guinchard, C. Anal.Chem. 1998, 70, 608. (20) Bard, A. J.; Faulkner, L. R. Electrochemical Methods; J. Wiley: New York, 1981.
) xH2O°H2O + xACN°ACN
(7)
where °H2O and °ACN were the dielectric constants of pure water and ACN, respectively. As sinhx = x under typical chromatographic conditions, eq 4 leads to
∆G°I,es ) (2RTσ)/(8kT oI)1/2(1/2)
(8)
As the contributions of the free energy are additives, combining eqs 2 and 8 gives
∆G°I,X ) RT
∫∂(ln[1 + Kψ (1 - y) (1 - Φ) ]) + P
P
P
(2RTσ)/[(8kTIo)1/2(1/2)] (9)
Secondary Gibbs Free Energy Change between Solute and HSA Cavity. The secondary contributions are called ξII constants. Like the primary Coulombic interactions, the secondary electrostatic contributions are proportional to 1/1/2. These secondary electrostatic forces act exclusively in increasing the second part of the hydrophobic effect on the basis of the interconnection described below (and are denoted esfH).23 The pure hydrophobic contribution noted ξII,H is considered to be dependent on the ACN concentration as the organic modifier is known to compete for the nonpolar residues that take place in the binding of solute at site II HSA.26 ξII,H was assumed to be proportional to n, n being a constant that must be calculated. The two additive effects ξII,esfH and ξII,H for, respectively, the secondary electrostatic and hydrophobic interactions implied in the intracavitary hydrophobic inclusion process can be linked to the secondary Gibbs free energy ∆G°II,H by
∆G°II,H ) RT[(ξII,esfH)/1/2 + ξII,Hn] (ii) Following this first contact step, the solute engages secondary strong specific short-range interactions with the residues of the cavity.12,21 These were represented for the dansyl amino acids binding on the site II cavity13 by (1) van der Waals interactions between the solute apolar groups and the hydrophobic residues as the consequence of the intracavitary dehydration process of ligand receptor interface22 (secondary hydrophobic interaction) and (2) hydrogen bonding between the electron donor group of solute and electron acceptor residues at the cavity rim and/or steric repulsion for the solute with large steric bulkiness (secondary nonhydrophobic interaction). The primary Coulombic interactions were also implied in the secondary specific process. It has been demonstrated that electrostatic and hydrophobic interactions were interconnected.23 When two opposite charges present on solute and cavity residues are neutralized by the ionpairing association, then the secondary hydrophobic interactions between the ligand and the receptor are enhanced by an increase in their hydrophobic character. In a previous work,13 it was demonstrated that when Coulombic interactions between a dansyl amino acid and the site II cavity decreased with increasing pH, the decrease in the solute affinity for the binding was accompanied by an enhancement of the chiral discrimination. The solute inclusion process into the cavity interior due to the secondary hydrophobic interaction decreased when the hydrophobic character of the ligand-receptor pair decreased with pH.13 Thus, the solute interacted more favorably with residues at the cavity rim through strong stereoselective H-bonding (or steric interactions) in relation to the compound. This fact would indicate that the secondary hydrophobic interaction is not the preponderant factor in the chiral recognition process and suggests that its variation is principally governed by nonhydrophobic interactions.13 Previous examples of chiral discrimination occurring through H-bonding or steric interactions between solute and chiral selectors have been reported in the literature.24,25 (21) Ross, P. D.; Subramanian, S. Biochemistry 1981, 20, 3096. (22) Absolom, D. R., Van oss C. J. Crit. Rev. Immunol. 1986, 6, 1. (23) Van Oss, C. J.; Chaudhury, M. K.; Good, R. J. Adv. Colloid Interface Sci. 1987, 28, 35. (24) Krstulovic, A. M. J. Pharm. Biomed. Anal. 1998, 6, 641. (25) O’Brien, T.; Crocker, L.; Thompson, R.; Thompson, K.; Toma, P. H.; Conlon, D. A.; Feibush, B.; Moeder, C.; Bicker G.; Grinberg, N. Anal. Chem. 1997, 69, 1999.
(10)
However, in eq 10, no allowance has been made for the stereoselective interactions (noted ξII,X, X ) D or L). It has been shown that when the hydrophobic interaction increased in the secondary process (by increasing electrostatic interactions, for example), the nonhydrophobic stereoselective contributions decreased and inversely.13 This interdependence between these two aspects of the binding process are represented by the following stereoselective constants ξII,esfH,X and ξII, H,X corresponding respectively to ξII,esfH and ξII,H. As ξII,esfH is inversely proportional to 1/2 (eq 8), then its corresponding stereoselective contribution ξII,esfH,X is expected to be proportional to 1/2 since these two interactions behave in opposite directions. In the same way, ξII,H,X was considered to be proportional to 1/n. The Gibbs free energy change of the chiral recognition process is determined by the following equation:
∆G°II,X ) RT[(ξII,esfH,X) 1/2 + (ξII,H,X)/n]
(11)
Combining eqs 10 and 11, the Gibbs free energy of the secondary process was obtained:
(∆G°II,H,X)T ) RT[(ξII,esfH)/1/2) + (ξII,esfH,X)1/2 + (ξII,H)n + (ξII,H,X)/n] (12) The total Gibbs free energy change that occurs during the HSA-solute interaction process was determined by combining eqs 9 and 12
∫
(∆G°I,II,X)T ) RT( ∂(ln[1 + KψP(1 - y)P(1 - Φ)P]) + [(ξI,es + ξII,esfH)/1/2 + (ξII,esfH,X 1/2) + (ξII,H) n + (ξII, H,X)/n] (13) where ξI,es, equal to σ/(8kT oI)1/2 represents the primary contribution of electrostatic forces. It is known that the retention factor at temperature T, for the enantiomer X noted k′X,T is related (26) Yang, J.; Hage, D. S. J. Chromatogr., A 1997, 766, 15.
Analytical Chemistry, Vol. 70, No. 20, October 15, 1998
4237
to the change in free energy ∆G°T,X incurred during the transfer between the mobile and stationary phases. This relationship was related by
ln k′X,T ) -∆G°T,X/(RT) + ln φ
(14)
where φ represents the phase ratio (volume of the stationary phase divided by the volume of the mobile phase). Substitution of eq 13 into eq 14 leads to
∫
ln k′X,T ) -[ ∂(ln[1 + KψP(1 - y)P(1 - Φ)P]) + (ξI,es + ξII,esfH)/1/2 + (ξII,esfH,X 1/2) + (ξII,H)n + (ξII, H,X)/n] + ln φ (15)
This equation links the variation of ln k′X,T with . It takes a general shape corresponding to
ln k′ X,T ) f() + η/1/2 + λ1/2 + δn + ξ/n + ln φ (16) where f() ) -ln [1 + KψP(1 - yP(1 - Φ)P] is the primary hydrophobic effect contribution (solvation process); y and Φ obviously depend on the dielectric constant of the medium; η/1/2 is the primary and secondary nonstereoselective Coulombic contribution; λ1/2 is the secondary stereoselective Coulombic contribution; δn is the secondary nonstereoselective hydrophobic contribution; and ξ/n is secondary stereoselective hydrophobic contribution. As well, for X ) D or L, the constant of equilibrium exchange process
Figure 1. Variations of the experimental logarithm of the retention factor for D-tryptophan as a function of the dielectric constant.
where ν1/2 is the secondary stereoselective Coulombic contribution and µ/n is the secondary stereoselective hydrophobic contribution.
This equation links the variation of R with . It takes a general shape corresponding to
RESULTS AND DISCUSSION To obtain the coefficients of eqs 16 and 19, the k′ and R values for the D and L enantiomers were determined for a range of water fractions 0.8-1; 11 Φ values were included in this range. For each Φ value, the corresponding value of the fraction of free ACN, y, in the ACN/water mixture had been previously determined,17,18 and the dielectric constant values were calculated using eqs 5-7. All the experiments were repeated three times. The coefficients of variation of the k′ and R values were 0.97. This good correlation between the predicted and experimental k′ and R values can be considered adequate to verify the theoretical model. All the dansyl amino acids exhibited similar variation for ln k′ with . Figure 1 represents the experimental curves obtained for the D-dansyltryptophan at T ) 25 °C. It was shown that the solute binding was maximal for an c value equal to ∼76. (a) For a high dielectric constant ( > ) range, the preponderant contribution to the variation in solute binding when decreased was attributed to the increasing primary and secondary electrostatic interactions, implying an enhancement of solute binding. (b) For a low dielectric constant range ( < ), the dominant effect of the ACN organic modifier was on the solvent properties
ln R ) µ/n + ν1/2
(27) Bevington, P. R. Data reduction and error analysis for the physical sciences; McGraw-Hill: New York, 1969.
HSAII + D T HSAII D HSAII + L T HSAII L HSAIID + L T HSAII L + D
was represented by
R ) (k′D/k′L)T
(17)
This constant reflects the enantioselectivity between D and L enantiomers, i.e., the chiral recognition properties of the site II cavity for these compounds. Combining eqs 15 and 17, gives
ln R ) (ξII,H,L - ξII,H,D)/n + (ξII,esfH,L - ξII,esfH,D)1/2 (18)
(19)
4238 Analytical Chemistry, Vol. 70, No. 20, October 15, 1998
Figure 3. Chromatograms of a racemic mixture of dansylnorvaline (see the conditions in the text)
Figure 2. Variations of the experimental logarithm of the enantioselectivity between D- and L-norvaline as a function of the dielectric constant.
of the medium.28 The ACN organic modifier was predicted to decrease the hydrophobic effect by increasing the direct solute solvation by its clusters. Thus, it can be said that the solute binding variation when decreased was governed by the decrease of the primary hydrophobic effect f() and also by the ACN competition for the apolar residues of the binding cavity. The sum of these two contributions implies a decrease in the solute inclusion when decreases. Figure 2 represents the variation of ln R with for the D- and L-norvaline enantiomers. (a) For a high dielectric constant range ( > ), the decrease in the enantioselectivity between the D and L enantiomers when increased was attributed to a reduction of the ξII,esfH,X term. This corresponded to a decrease in the stereoselective H-bonding between the electron donor group of the solute (sulfonilamido group) and the electron acceptor residue of the binding cavity represented by Tyr 411 or the steric repulsion due to the large bulkiness groups of dansyl amino acids. For a low dielectric constant range ( < ), the chiral discrimination increases due to the preponderance of the ξII,esfH,X term, which was related to the favorable stereoselective interactions following the ACN competition for apolar residues. An example illustrating this type of enantioselectivity behavior is shown in Figure 3. In the chromatographic system used for ) c ) 76, chiral discrimination for dansylnorvaline provided a minimum R value equal to 1.01 (Figure 3A). Based on the trends noted in Figure 2, it was possible to adjust the dielectric constant of the medium to adjust the conditions in order to obtain a greater (28) Morrisson T. J. Chem. Soc. 1952, 3, 3814. (29) Kabat E. A. Structural concepts in immunology and immunochemistry, 2nd ed.; Holt, Rinehart and Winston: New York, 1976.
Figure 4. Schematic representation of the theoretical binding cavity and its accessible surface area s with a depth of d ) ∼16 Å, a width of w ) 8 Å, and a curvature radius of r ) 8.5 Å.
enantiomeric separation on the HSA stationary phase without increasing the retention time. This was done by decreasing the dielectric constant ) 72 < c. This provided an enantioselectivity equal to 1.25 with a retention time equal to 11 min and then a good resolution (Figure 3B). The surface charge density σ and n values were determined using the model equations. They were found to be independent of the solute. The n value was equal to 1.12 and the σ/F value to 9.2 × 10-7 mol/m2. The maximum variation obtained was
