Anal. Chem. 2007, 79, 9060-9065
Enhanced Protein Affinity and Selectivity of Clustered-Charge Anion-Exchange Adsorbents Joseph Y. Fu,† Sindhu Balan,‡ Ajish Potty,§ Van Nguyen,‡ and Richard C. Willson*,†,‡,§
Biomedical Engineering Program, Department of Biology and Biochemistry, and Department of Chemical Engineering, University of Houston, 4800 Calhoun, Houston, Texas 77204-4004
Ion-exchange chromatography is widely used for analytical and preparative separation of biomolecules because of the strong and easily reversible protein adsorption affinity of ion-exchange matrixes. Efforts to improve ion-exchange adsorbents have primarily focused on their mass transfer characteristics, on increasing overall ligand density through the use of tentacular adsorbents,1-4 and on immobilization of polyions such as polyethyleneimine, polysulfonic acid, and polylysine.5,6 Traditional ion-exchange adsorbents with dispersed-charge distributions display a heterogeneous landscape of adsorption sites due to variations in local charge density. Electrolytes decrease the range of electrostatic interactions (the Debye length in 100 mM of a 1:1 electrolyte such as NaCl is 0.96 nm), limiting the degree of “averaging” over local variations and heightening the
effects of surface charge heterogeneity.7 It might also be anticipated that capacity would be limited by random ligand dispersion, as those charges isolated in regions of low charge density will not be included in sites of sufficient affinity to be occupied under typical operating conditions. We speculated that an adsorbent with a nanostructured, clustered-charge distribution might exhibit higher binding affinity and capacity than one with the same density of charges presented in a dispersed fashion. Adsorbent charge dispersion may also limit the opportunity for selectivity based on clusters of concentrated protein surface charge, thus reducing resolution. Many proteins display a conserved patch or sequence of amino acids involved in proteinprotein or protein-nucleic acid recognition, and many of these recognition domains are of relatively high local charge density.8-12 Earlier work using site-directed mutagenesis has established the dominant role of such charge clusters in ion-exchange adsorption of proteins.13-16 Multivalent contact with a clustered-charge ionexchange adsorbent through these high charge density patches might improve not only capacity but also selectivity for proteins displaying charge clusters. In the present work we tested the capacity and selectivity of clustered-charge anion exchangers for the anionic proteins R-lactalbumin and cytochrome b5, each of which displays a modifiable charge cluster. Clustered- and dispersed-charge adsorbents of matched charge density were prepared using pentalysinamide/ pentaargininamide and lysinamide/argininamide. We found that clustered-charge adsorbents retain proteins with inherent charge clusters relatively better than dispersed-charge adsorbents. The clustered-charge adsorbent was also highly sensitive to small structural perturbations in proteins’ high charge-density clusters, suggesting an inherent selectivity of clustered-charge adsorbents for clustered-charge proteins.
* To whom correspondence should be addressed. Phone: 713-743-4308. Fax: 713-743-4323. E-mail:
[email protected]. † Biomedical Engineering Program. ‡ Department of Biology and Biochemistry. § Department of Chemical Engineering. (1) Mueller, E.; Harders, H. D.; Lubda, D. Merck Patent GmbH, Germany. Method for removing endotoxins, U.S. Patent 6,617,443, Sept 9, 2003. (2) Mueller, E.; Harders, H. D.; Lubda, D.; Merck Patent GmbH Polymerisable Polyamide Derivatives. U.S. Patent 6,149,994, Nov 21, 2000. (3) Mueller, W. J. Chromatogr. 1990, 510, 133-140. (4) Xie, J.; Aguilar, M.-I.; Hearn, M. T. W. J. Chromatogr., A. 1995, 711, 4352. (5) Etheve, J.; Dejardin, P.; Boissiere, M. Colloids Surf., B 2003, 28, 285-293. (6) Muranaka, K.; Trieda, T. Tosoh Corp., Japan. Anion exchanger, process for producing same, and its use. U.S. Patent 6,689,820. Feb 10. 2004.
(7) Heimenz, P. C. Principles of Colloid and Surface Chemistry, 2nd ed.; Marcel Dekker: New York, 1986. (8) Bibak, N.; Paul, R. M. J.; Freymann, D. M.; Yaseen, N. R. Anal. Biochem. 2004, 333, 57-64. (9) Brendel, V.; Karlin, S. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 5698-5702. (10) Diamond, G.; Scanlin, T. F.; Zasloff, M. A.; Bevins, C. L. J. Biol. Chem. 1991, 266, 22761-22769. (11) Hu, Y.; Chen, S.; Xu, M.; Zhang, S. Biotechnol. Appl. Biochem. 2004, 40, 89-94. (12) Schwartz, R. M.; Dayhoff, M. O. Science 1978, 199, 395-403. (13) Chicz, R. M.; Regnier, F. E. J. Chromatogr. 1988, 443, 193-203. (14) Chicz, R. M.; Regnier, F. E. Anal. Chem. 1989, 61, 2059-2066. (15) Gill, D. S.; Roush, D. J.; Willson, R. C. J. Chromatogr., A. 1994, 684, 5563. (16) Roush, D. J.; Gill, D. S.; Willson, R. C. J. Chromatogr. 1993, 653, 207-218.
In this work, we examined the possibility of improving ionexchange adsorbent performance by nanoscale structuring of ligands into clusters of fixed size rather than a random distribution of individual charges. The calcium-depleted form of the protein r-lactalbumin, which displays a cluster of acidic amino acid residues, showed enhanced adsorption affinity and capacity on clustered-charge pentalysinamide and pentaargininamide adsorbents as compared to single-charge lysinamide and argininamide adsorbents of matched total charge. Two differently charge-clustered mutants of rat microsomal cytochrome b5, E11Q and E44Q, with the same total charge also were well differentiated by clustered-charge adsorbents. Thus, an organized rather than random distribution of charges may produce adsorbents with higher capacity and selectivity, especially for biomolecules with inherent charge clustering.
9060 Analytical Chemistry, Vol. 79, No. 23, December 1, 2007
10.1021/ac070695n CCC: $37.00
© 2007 American Chemical Society Published on Web 11/01/2007
EXPERIMENTAL SECTION Materials. AminoLink Plus Immobilization matrix (45-165 µm aldehyde-agarose) and Micro BCA protein assay kits were from Pierce. Cyanogen bromide-activated Sepharose 4B (45-165 µm) was from GE Healthcare. Pentalysinamide and pentaargininamide were from the peptide synthesis service of the University of Texas Medical Branch, Galveston, TX. Recombinant rat microsomal cytochrome b5 and its E11Q and E44Q mutants conserving total surface charge were prepared as described elsewhere.16 All other reagents were from Sigma. Adsorbent Preparation. AminoLink Aldehyde Adsorbent. One column (2 mL) was washed with 5 mL of coupling buffer (0.1 M Na2PO4, 0.15 M NaCl, pH 7.2) to remove preservatives. The gel was then incubated with 0.04 mL of sodium cyanoborohydride solution (5 M NaCNBH3 in 0.01 M NaOH), immediately followed by 2 mL of 1.5 mM pentalysinamide or pentaargininamide in a 15 mL centrifuge tube on a Cole-Parmer Rototorque gyratory rotator at room temperature for at least 4 h. The peptide supernatant was then drained, the adsorbent washed with 5 mL of coupling buffer to remove unbound peptide, and the supernatant saved for uncoupled ligand determination. Residual activated groups remaining after quenching as recommended by the manufacturer (1 M Tris-HCl, 0.05% NaN3, pH 7.4) initially caused irreversible protein adsorption, which was eliminated by use of sodium borohydride. The adsorbent was washed with 2 mL of 66 mM sodium borohydride in 25% ethanol/75% PBS, followed by 30 min rotation with 2 mL of the same solution to deactivate any remaining aldehyde sites, and washed with 15 mL of 1 M NaCl and 5 mL of storage buffer (10 mM Tris-HCl, pH 8.0). The concentration of peptide ligands on the adsorbent was determined by bicinchoninic acid assay (Micro BCA Protein Assay, Pierce) of residual supernatant peptide concentrations and on the modified adsorbent itself. This procedure was used to prepare amino acid adsorbents with a final ligand density of 8.8 mM lysinamide to be compared with 1.4 mM pentalysinamide and 5.0 mM argininamide to be compared with 1.0 mM pentaargininamide to match total charge while changing distribution from random to clustered. Ligand densities of the amino acid adsorbents were determined by supernatant uncoupled ligand absorbance at 205 nm using a Nanodrop spectrophotometer. CNBr-Activated Agarose. One gram of CNBr-activated Sepharose 4B was washed with 1 mM HCl to remove preservatives. The gel, now swollen to 3.5 mL, was suspended in 8 mL of 0.5 M NaHCO3, pH 7.0 containing 0.5 M NaCl in a 15 mL Falcon tube. Lysinamide or pentalysinamide dissolved in the same buffer was added to the gel suspension and the reaction tube rotated at 4 °C overnight. The suspension was then filtered and the supernatant analyzed for amino acid content by the ninhydrin assay or peptide content using the bicinchoninic acid assay. The gel was washed with 3 volumes of 10 mM Tris-HCl buffer, pH 8.0, rotated for 2 h in 6 volumes of 0.1 M Tris-HCl, pH 8 with 0.5 M of NaCl to quench any remaining active sites, then filtered and washed with 5 volumes each of 0.1 M sodium acetate, pH 5.0 containing 0.5 M NaCl, and 0.1 M Tris-HCl buffer, pH 8.0 with 0.5 M NaCl, and suspended in 10 mM Tris-HCl, pH 8.0, the buffer for adsorption isotherm measurements. Adsorption Isotherms. Varying amounts (10-25 µL) of adsorbent were aliquoted into a set of 1.5 mL Eppendorf tubes so as to match the total charge between the clustered- and the dispersed-charge adsorbent (e.g., 23.1 µL of 8.8 mM lysinamide
adsorbent vs 25 µL of 1.4 mM pentalysinamide adsorbent). To these tubes varying amounts of 10 mM Tris-HCl, pH 8.0, plus 40 mM NaCl and protein solution (3.52-113 µM of R-lactalbumin or 3.13-100 µM cytochrome b5) were added to achieve a final volume of 1 mL in each of the tubes. The tubes were then placed on a gyratory rotator at 25 °C for 1 h, a time found in control experiments to be sufficient for equilibration. After 10 min centrifugation at 16000g in an Eppendorf microcentrifuge, R-lactalbumin or cytochrome b5 in the supernatant was quantified at 280 or 412 nm, respectively, using a Beckman-Coulter DU 530 spectrophotometer. An amount of 1 mL of 10 mM Tris-HCl, pH 8.0 was added to each of the pellets which were then vortexed and centrifuged to wash unbound protein from the interstitial space. The supernatant was discarded, and the protein was eluted with 1 mL of 10 mM Tris-HCl, pH 8.0 containing 1 M NaCl and analyzed spectrophotometrically for determination of bound protein content and calculation of mass recovery. Data Analysis. The mass balances (protein remaining in supernatant + protein eluted, divided by protein originally added) for all adsorption data closed in the range of 85-96%. Protein adsorption isotherms were fit to both Langmuir and LangmuirFreundlich (LF) isotherms using Igor Pro (WaveMetrics, Lake Oswego, OR) version 4.04, which uses the Levenburg-Marquardt algorithm for parameter estimation by minimizing the sum of the squared differences between the measured and fitted values. The initial guess vectors for the parameters were varied manually to ensure that a true global minimum was found. The two-parameter Langmuir model fit the data with similar statistical significance and generally lower standard deviations than the three-parameter LF model and was employed to obtain adsorption parameters to compare the binding affinity and capacity of the adsorbents. However, Langmuir fits of the adsorption of calcium-saturated lactalbumin on lysinamide-agarose, and of E44Q mutant cytochrome b5 on lysinamide-agarose resulted in parameters with high statistical errors (SD g 30% of the mean). To overcome this limitation, the statistically more applicable Qmax value obtained from the LF fit was used to predict Kd by reducing the twoparameter Langmuir model to a single-parameter model. The relatively small potential error in the estimation of Qmax from the LF fits was not taken into account in predicting Kd from the Langmuir equation. A plot of the logarithm of the initial slope of the isotherm vs logarithm of salt concentration is called in the chromatographic literature a z-plot.17-19 The negative of the slope obtained from z-plots gives the apparent number of interactions, z, between the protein and the adsorbent. Safety Consideration. Hazardous Materials. Sodium borohydride and sodium cyanoborohydride are toxic. Their solutions should be prepared and aliquoted in a fume hood and disposed of according to the regulations of each institution. RESULTS AND DISCUSSION Adsorbent Preparation. The amide forms of the ligands were used to avoid creating an -amino/carboxyl dipolar adsorbent. (17) Kopaciewicz, W.; Rounds, M. A.; Regnier, F. E. J. Chromatogr. 1983, 266, 3-21. (18) Boardman, N. K.; Partridge, S. M. Biochem. J. 1955, 59, 543-552. (19) Record, M. T.; Lohman, T. M.; De Haseth, P. J. Mol. Biol. 1976, 107, 14558.
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Table 1. Values of Langmuir Isotherm Parameters Qmax and Kd and Langmuir Freundlich Parameter nH for Adsorption of r-Lactalbumin, Wild Type Cytochrome b5, and E11Q and E44Q Mutants of Cytochrome b5 on Lysinamide-CNBr Agarose and Pentalysinamide-CNBr Agarosea ] lysinamide-CNBr agarose
protein R-lactalbumin Ca2+saturateda R-lactalbumin Ca2+ depleted cytochrome b5 wild type cytochrome b5 E11Q cytochrome b5 E44Qa a
pentalysinamide-CNBr agarose
nH
Qmax (mM)
Kd (µM)
Qmax/Kd
nH
ratio (Lys5Qmax/Kd)/ (Lys1Qmax/Kd)
2.70
1.50 ( 0.32
0.22 ( 0.02
81.3 ( 13.0
2.70
0.80 ( 0.20
1.00
58.2 ( 12.0
4.00
1.31 ( 0.50
0.48 ( 0.03
61.4 ( 7.60
7.70
1.56 ( 0.20
1.92
0.19 ( 0.03
48.0 ( 13.0
3.90
0.90 ( 0.32
0.18 ( 0.01
20.9 ( 4.20
8.80
0.57 ( 0.17
2.25
0.20 ( 0.02
51.9 ( 10.0
4.00
1.05 ( 0.21
0.16 ( 0.01
17.8 ( 3.80
8.80
0.48 ( 0.00
2.20
0.18 ( 0.06
47.9 ( 3.20
3.70
1.26 ( 0.30
0.15 ( 0.01
30.9 ( 5.80
4.90
0.85 ( 0.22
1.32
Qmax (mM)
Kd (µM)
Qmax/Kd
0.18 ( 0.04
65.4 ( 6.40
0.24 ( 0.02
Qmax and SD obtained from Langmuir-Freundlich fit.
Such adsorbents have been used in RNA separation and are commercially available,20 but in this work, it was important that the adsorbents be purely cationic and not dipolar. The low coupling pH (7.0) was chosen based on SPARC electrostatics calculations [http://ibmlc2.chem.uga.edu/sparc/], which indicated a lysinamide R-amino pKa of 6.86 as compared to 8.90 for the R-amino group of lysine. The amine densities of lysinamideCNBr agarose and pentalysinamide-CNBr agarose were matched at 5 µmol/mL gel, taking advantage of the nearly quantitative coupling to CNBr. The amine density of derivitized aldehydeagarose was more difficult to control, as coupling efficiency was typically 60-85%. The clustered-charge adsorbent was prepared first, and the dispersed-charge form was then made with an effort to match amine densities, which was achieved within 25%. For purpose of comparison it is noteworthy that the total charge on each clustered adsorbent was lower than that on the corresponding dispersed adsorbent. With the use of SPARC, the expected charges on both the pentalysinamide and pentaargininamide are +5; all five side chain amine groups are protonated at pH 8.0. Because the side chain pKas of pentaargininamide are higher (∼12) than those of pentalysinamide (∼9), pentaargininamide is expected to show higher anion affinity. The coupling of an amino group to the aldehyde results in a neutral linkage, but the coupling of an amine group to a CNBr activated matrix results in an isourea linkage which depending on conditions can be positively charged, adding to the cationic character of each anion-exchange ligand and proportionally more so to lysinamide. The enhanced adsorbent performance of the clustered-charge CNBr adsorbent thus represents a lower bound on the degree of enhancement potentially achievable by clustering of ligands. The observations in this work are further supported by our preliminary work with cationexchange adsorbents, in which clustering also enhances the performance of anionic ligands. The clustered-charge adsorbents of the present work differ from the commercially available tentacular ion exchange adsorbents prepared by grafting functionally substituted acrylamides to an adsorbent matrix.2,3 The amount of the monomer present in the polymerization reaction governs the average length of the polymer chains,3 which is 15-20 monomer units. This results in enhanced protein capacity by increasing the accessibility of the 9062
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ligands. In the present work, the ligand itself is polymerized to form small clusters of charges; the premise is to immobilize small, preformed clusters of functional groups such that a complementary small “cluster” or complementary patch on a biomolecule can be selectively recognized by the adsorbent. This work is perhaps conceptually most closely related to the work of Jennissen et al. on “critical hydrophobicity” in HIC adsorption of proteins.21,22 Adsorption Isotherms. The effect of structured versus random charge distribution was first tested using the Ca2+saturated and Ca2+-depleted forms of the anionic protein bovine R-lactalbumin. This protein has a high affinity for divalent calcium ion (dissociation constant 10-7M),23 mediated by conserved Ca2+binding aspartate residues located at the interface between the two subdomains of the protein.24 In the Ca2+-depleted R-lactalbumin there is increased local mobility and slightly higher solvent accessibility at the Ca2+-binding site, largely due to the charge repulsion among the five aspartate residues in this region.25 In the presence of Ca2+, these residues are both less accessible and also partially neutralized by the cation, reducing the anionic character of this cluster. Ca2+-saturated and Ca2+-depleted (with clustered protein surface charges) R-lactalbumin were adsorbed on lysinamide and pentalysinamide-CNBr agarose of matching amine group densities. As shown in Table 1 and Figure 1, clustering of adsorbent charge slightly enhances adsorption of the protein without charge clusters but radically increases both initial affinity and capacity of adsorption of the clustered-charge protein; the clustered-charge adsorbent has greater selectivity (Figure 1, upper curve). This selectivity implies that the enhanced binding of protein to the clusteredcharge adsorbent is not merely an effect of enhanced steric accessibility but also depends on the surface charge distribution of the protein. As shown in Table 1, binding capacity more than doubles from 0.24 to 0.48 µmol/mL for the Ca2+-depleted (20) Jones, D. S.; Lundgren, H. K.; Jay, F. T. Nucleic Acids Res. 1976, 3, 15691576. (21) Jennissen, H. P.; Heilmeyer, L. M. G., Jr. Biochemistry 1975, 14, 754-760. (22) Jennissen, H. P. Biochemistry 1976, 15, 5683-5692. (23) Stuart, D. I.; Acharya, K. R.; Walker, N. P. C.; Smith, S. G.; Lewis, M.; Phillips, D. C. Nature 1986, 324, 84-87. (24) Chrysina, E. D.; Brew, K.; Acharya, K. R. J. Biol. Chem. 2000, 275, 3702137029. (25) Kronman, M. J. Crit. Rev. Biochem. Mol. Biol. 1989, 24, 565-667.
Figure 1. Adsorption isotherms of R-lactalbumin (Ca2+ saturated, “unclustered”-charge protein) on lysinamide (“Lys1”; 2) and pentalysinamide (“Lys5”; 9) CNBr agarose and R-lactalbumin (Ca2+ depleted, “clustered”-charge protein) on lysinamide (() and pentalysinamide (b) CNBr agarose at 25 °C in 10 mM Tris, pH 8 at 26 mM NaCl, where the amino group density is 5.0 µmol/mL of each gel. Error bars correspond to mean ( 1 SD.
Figure 3. Adsorption isotherms of R-lactalbumin (Ca2+ depleted) on lysinamide (Lys1; 0,∆,O) and pentalysinamide (Lys5; 9,2,b) aldehyde agarose at 25 °C in 10 mM Tris, pH 8 at 10 (0,9), 26 (∆,2), and 40 (O,b) mM NaCl; R-lactalbumin (Ca2+ saturated) on lysinamide (Lys1; )) aldehyde agarose at 25 °C in 10 mM Tris, pH 8 at 10 ()) mM NaCl; the amino group density is 7.9 ( 0.9 µmol/mL of the gel. Error bars correspond to mean ( 1 SD.
Figure 4. Adsorption isotherms of R-lactalbumin (Ca2+ depleted) on argininamide (Arg1; 0, ∆, O) and pentaargininamide (Arg5; 9, 2, b) aldehyde agarose at 25 °C in 10 mM Tris, pH 8 at 10 (0, 9), 26 (∆, 2), and 40 (O, b) mM NaCl where the amino group density is 5.0 µmol/mL of the gel. Error bars correspond to mean ( 1 SD. Figure 2. Adsorption isotherm of R-lactalbumin (Ca2+ depleted) on lysinamide (∆,2) and pentalysinamide (0,9) aldehyde (∆,0) and CNBr (2,9) agarose at 25 °C in 10 mM Tris, 26 mM NaCl, pH 8 where the amine group density is 5.0 µmol/mL of CNBr gels, 8.9 ( 0.1 µmol/ mL of aldehyde adsorbents. Error bars correspond to mean ( 1 SD.
(clustered-charge) protein in going from dispersed to the nanostructured clustered-charge adsorbent. Also, the initial affinity of binding (Qmax/Kd) for the Ca2+-depleted (clustered-charge) protein is approximately 2-fold higher with pentalysinamide as compared to lysinamide, whereas the initial binding affinity of the Ca2+saturated (unclustered) form is similar for both adsorbents. While this enhanced selectivity is evident in batch experiments with one theoretical plate of resolution, its effects would be greatly magnified in any higher-resolution column chromatography. The potentially positive isourea linkage of an amino group to activated CNBr may reduce the benefits of clustering ligands. The same ligands also were tested immobilized on aldehyde agarose in order to address this issue and to control for any effect of coupling linkage. As shown in Figure 2, lysinamide/pentalysinamide-aldehyde agarose adsorption isotherms of charge-clusterdisplaying Ca2+-depleted R-lactalbumin exhibit trends similar to CNBr agarose in 10 mM Tris, pH 8.0 with 26 mM NaCl. The enhancement effect of ligand clustering is seen with both aldehyde and CNBr matrixes suggesting that any contribution of linkage chemistry is not dominant (Figure 2).
Figures 3 and 4 show adsorption isotherms for Ca2+-depleted (clustered-charge) R-lactalbumin on aldehyde-immobilized pentalysinamide and lysinamide of matched amine group concentration (7.9 ( 0.9 mM) and argininamide and pentaargininamide (amine concentration 5.0 mM). In each case, the clustered-charge adsorbent shows higher affinity and capacity of binding at all salt concentrations tested. At the bottom of Figure 3, the adsorption isotherm for Ca2+-saturated (unclustered) R-lactalbumin on aldehyde-immobilized lysinamide shows low binding even at only at 10 mM NaCl concentration (Figure 3); adsorption of the unclustered protein was negligible at higher salt concentration. As shown in Table 2, the initial binding affinity (Qmax/Kd) of the Ca2+depleted (clustered-charge) protein increased 5-16-fold in transitioning from dispersed-charge argininamide adsorbent to the clustered-charge pentaargininamide adsorbent; for the transition of dispersed-charge lysinamide to clustered-charge pentalysinamide adsorbent, the enhancement is more modest at 1.3-2.2-fold (Table 2). The Langmuir isotherm model (eq a) in some cases predicts a higher Qmax for the dispersed-charge adsorbent compared to the clustered-charge adsorbent (Tables 1 and 2). This could be due to lack of sufficient data points in the saturation region of the isotherm for these cases. On the other hand, the Analytical Chemistry, Vol. 79, No. 23, December 1, 2007
9063
Table 2. Values of Langmuir Isotherm Parameters Qmax and Kd and Langmuir Freundlich Heterogeneity Parameter nH for Adsorption of r-Lactalbumin (Ca Depleted) on Lysinamide/Pentalysinamide-Aldehyde Agarose and Argininamide/Pentaargininamide Agarose argininamide-aldehyde agarose concn NaCl (mM)
Qmax (mM)
Kd (µM)
Qmax/Kd
10 26 40
0.85 ( 0.04 0.64 ( 0.02 0.46 ( 0.07
174 ( 12.0 160 ( 7.20 163 ( 34.0
4.00 4.00 2.80
pentaargininamide-aldehyde agarose
nH
Qmax (mM)
Kd (µM)
Qmax/Kd
nH
ratio (Arg5Qmax/Kd)/ (Arg1Qmax/Kd)
1.00 ( 0.05 0.96 ( 0.02 1.20 ( 0.12
0.47 ( 0.04 0.55 ( 0.07 0.53 ( 0.06
8.40 ( 2.70 28.0 ( 8.20 39.1 ( 8.60
56.0 20.0 14.0
0.36 ( 0.05 0.50 ( 0.08 0.65 ( 0.08
14 5.0 5.0
lysinamide-aldehyde agarose concn NaCl (mM)
Qmax (mM)
Kd (µM)
Qmax/Kd
10 26 40
0.94 ( 0.05 1.30 ( 0.13 0.95 ( 0.14
52.7 ( 5.10 178 ( 26.0 222 ( 44.0
18.0 7.30 4.30
pentalysinamide-aldehyde agarose
nH
Qmax (mM)
Kd (µM)
Qmax/Kd
nH
ratio (Lys5Qmax/Kd)/ (Lys1Qmax/Kd)
0.88 ( 0.07 1.10 ( 0.05 1.10 ( 0.11
0.83 ( 0.06 1.00 ( 0.05 1.10 ( 0.15
20.7 ( 4.00 79.7 ( 6.30 195 ( 33.0
40.0 13.0 5.60
0.66 ( 0.07 0.90 ( 0.04 0.98 ( 0.12
2.2 1.8 1.3
Figure 5. Z plot for adsorption isotherm of R-lactalbumin (Ca2+ depleted) on argininamide (0)/pentaargininamide (9) and lysinamide (O)/pentalysinamide (b) aldehyde agarose at 25 °C in 10 mM Tris, pH 8 at 10, 26, and 40 mM NaCl.
Qmax predicted from the Langmuir-Freundlich isotherm (eq b) fits in all cases is higher for the clustered-charge adsorbent, and the experimental data always show superior performance of clustered adsorbents.
Langmuir equation y )
Langmuir-Freundlich equation
QmaxX Kd + X y)
QmaxXnH KndH + XnH
(a)
(b)
As shown in Figure 5, the z-plots show a linear dependence of the logarithm of initial slope, Qmax/Kd, on log of salt concentration as predicted by the stoichiometric displacement model.17,18,26,27 For a 1:1 electrolyte such as NaCl, the slope of this plot gives the apparent number of interaction sites, z, between the protein and the adsorbent. As protein molecules can adsorb in different orientations and each orientation can have a different number of (fractional, double layer-mediated) interactions with the adsorbent (26) Gill, D. S.; Roush, D. J.; Willson, R. C. J. Colloid Interface Sci. 1994, 167, 1-7. (27) Velayudhan, A.; Horvath, C. S. J. Chromatogr. 1986, 367, 160-162.
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surface, z can have fractional values.28 For aldehyde adsorbents, clustering of lysinamide to pentalysinamide may slightly increase the z value from (1.2 ( 0.2) to (1.4 ( 0.2). Clustering of argininamide to pentaargininamide increases z from (0.3 ( 0.1) to (1.0 ( 0.1). The differences between lysine and arginine may potentially arise from a combination of the greater ability of the guanidine cation to form ideal-geometry hydrogen bonds, its larger size, and its soft-acid character,29 though a dominant role of hydrogen bonding is ruled out by the salt-reversibility of the interaction. Each of our observations, however, is consistent with the hypothesis that clustered-charge adsorbents form a larger number of intermolecular interactions with the average adsorbate. A stronger dependence of adsorption affinity on salt concentration would also imply higher resolution in elution chromatography. In a second example, recombinant rat microsomal cytochrome b5 and its matched-charge E11Q and E44Q mutant forms were adsorbed on lysinamide-CNBr agarose and pentalysinamide-CNBr agarose in separate batch experiments (Figure 6). Our work involving conservative single point mutations demonstrated that cytochrome b5 has a preferred ion-exchange active cluster of glutamic and aspartic acid residues; the mutation of Glu44 to Gln disrupts this cluster.15,30 The resultant mutant protein has a lower binding affinity for the anion-exchange adsorbent Mono Q as compared to the wild type cytochrome b5. The mutant protein E11Q, which has the same net charge, did not exhibit a significant reduction in binding affinity, indicating that Glu11 does not belong to any ion-exchange active patch in the protein. These results are consistent with our previous structural and computational electrostatics studies.30-32 As one Glu residue has been neutralized in each protein, they are equivalent in terms of net charge at pH 8.0. Clustering the charges on the adsorbent increases the capacity for the mutant protein E11Q and the wild type. (28) Whitley, R. D.; Wachter, R.; Liu, F.; Wang, N. H. L. J. Chromatogr. 1989, 465, 137-56. (29) Fromm, J. R.; Hileman, R. E.; Caldwell, E. E. O.; Weiler, J. M.; Linhardt, R. J. Arch. Biochem. Biophys. 1995, 323, 279-287. (30) Roush, D. J.; Gill, D. S.; Willson, R. C. J. Chromatogr., A. 1995, 704, 339349. (31) Roush, D. J.; Gill, D. S.; Willson, R. C. Biophys. J. 1994, 66, 1290-1300. (32) Wu, Y.; Wang, Y.; Qian, C.; Lu, J.; Li, E.; Wang, W.; Lu, J.; Xie, Y.; Wang, J.; Zhu, D.; Huang, Z.; Tang, W. Eur. J. Biochem. 2001, 268, 1620-1630.
Figure 6. Adsorption isotherm of wildtype cytochrome b5 on lysinamide ()) and pentalysinamide (() CNBr agarose, cytochrome b5 (44Q) on lysinamide (∆) and pentalysinamide (2) CNBr agarose, and cytochrome b5 (11Q) on lysinamide ([squlo]) and pentalysinamide (9) CNBr agarose at 25 °C in 10 mM Tris, pH 8 at 26 mM NaCl where the amine group density of each adsorbent is 5.0 µmol/mL. Error bars correspond to mean ( 1 SD.
The adsorption isotherms for wild type cytochrome b5 (Figure 6 and Table 1) show a higher binding affinity for clustered-charge as opposed to the dispersed-charge adsorbent, probably mediated by the cluster of anionic residues which includes Glu44. While there is a slight decrease in binding of the less-anionic mutant E11Q compared to the wild type, the anionic cluster is intact and the protein shows enhanced binding on the clustered-charge adsorbent. For the cluster-disrupted mutant protein E44Q (Figure 6), the effect of loss of the preferred ion-exchange patch is evident, as there is no substantial improvement in the binding capacity or affinity for the clustered-charge adsorbent as compared to the dispersed-charge adsorbent. The dissociation constant (Table 1) for the wild type protein and E11Q mutant for the clustered-charge adsorbent is lower than that for E44Q, which may be attributed to the disruption of the favored ion-exchange patch in E44Q. Similar enhancements were obtained for the adsorption of the matched-charge cytochrome b5 mutants on aldehyde-based clustered-charge adsorbents.
An interesting trend in the heterogeneity index (nH) (calculated from the Langmuir-Freundlich (LF) fits) was observed for dispersed- and clustered-charge adsorbents. Adsorption on dispersed-charge adsorbents generally shows either positive or neutral cooperativity (as characterized by nH g 1). On the other hand, clustered-charge adsorbents generally exhibit negative cooperativity (nH < 1) i.e., binding of the first protein reduces affinity for binding of additional protein. However, the overall avidity of clustered-charge adsorbents is evidently higher compared to dispersed-charge adsorbents. This suggests that the superior performance of clustered-charge adsorbents is derived from interactions with multiple protein-sites (also supported by higher z values). Moreover, on increasing the salt concentration, the value of nH shifts closer to unity. This decrease in binding heterogeneity may be a result of salt-mediated shielding of low affinity interactions.25 CONCLUSIONS In this work, clustered-charge anion exchangers were shown to give higher affinity, capacity, and selectivity of protein adsorption, especially for proteins displaying clusters of charged residues on their surfaces. Clustered-charge adsorbents were especially effective in recognizing minor structural perturbations that affected charge clusters in protein adsorbates. These results show that nanoscale structuring of adsorbent charges can improve the affinity and selectivity of ion-exchange adsorbents. These improvements may be especially large with multivalent adsorbates such as nucleic acids. ACKNOWLEDGMENT This research was funded in part by grants from NSF under Grant CTS-0004544 and the Robert A. Welch Foundation under Grant E-1264.
Received for review April 10, 2007. Accepted August 13, 2007. AC070695N
Analytical Chemistry, Vol. 79, No. 23, December 1, 2007
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