Anal. Chem. 2006, 78, 7253-7258
L-Histidine
Imprinted Synthetic Receptor for Biochromatography Applications
Ayc¸ a Atılır O 2 zcan,† Rıdvan Say,†,‡ Adil Denizli,§ and Arzu Erso 1 z*,†
Department of Chemistry and BI˙ BAM (Plant, Drug and Scientific Researches Center), Anadolu University, Eskis¸ ehir, Turkey, and Department of Chemistry, Hacettepe University, Ankara, Turkey
We have proposed novel surface-imprinted beads for selective separation of cytochrome c (cyt c) by N-methacryloyl-(L)-histidine-copper(II) [MAH-Cu(II)] as a new metal-chelating monomer via metal coordination interactions and histidine template. We have combined molecular imprinting with the ability of histidine to chelate metal ions to create ligand exchange beads suitable for the binding of cyt c (surface histidine exposed protein). The histidine imprinted beads were produced by suspension polymerization of MAH-Cu(II)-L-histidine and ethylene glycol dimethacrylate. After polymerization, the template (L-histidine) was removed from the beads using methanolic KOH, thus getting histidine imprinted metal-chelate beads. L-Histidine imprinted metal-chelate beads can be used several times without considerable loss of cyt c adsorption capacity. The association constant (Ka) for the specific interaction between the template imprinted polymer and the template (L-histidine) itself were determined by Scatchard plots using L-histidine imprinted beads and found as 58 300 M-1. Finally, we have used these histidine imprinted beads for cyt c and ribonuclease A (surface histidine exposed proteins) and enantiometric separation of D- and L-histidine by FPLC. Molecular imprinting is a method for making selective binding sites in synthetic polymers using a molecular template. Target molecules (i.e., histidine) can be used as templates for imprinting cross-linked polymers. After the removal of template, the remaining polymer is more selective. The selectivity of the polymer depends on various factors such as the size and shape of the cavity and rebinding interactions. Covalent interactions,1-4 noncovalent interactions,5-7 electrostaticinteractions,8 andmetalioncoordination9-11 can be exploited to organize the functional monomers around the template. The bioimprinting approach has been applied to the realization of recognition and specificity to biomaterials for bioseparations, * To whom correspondence should be addressed. Tel: (90) 222 3350580/ 5821. Fax: (90) 222 3204910. e-mail:
[email protected]. † Department of Chemistry, Anadolu University. ‡ BI˙ BAM (Plant, Drug and Scientific Researches Center), Anadolu University. § Hacettepe University. (1) Wulff, G.; Grobe-Einsler, R.; Vesper, W.; Sahran, A.. Makromol. Chem. 1977, 178, 2817. (2) Shea, K. J.; Doughertly, T. K. J. Am. Chem. Soc. 1986, 108, 1091. (3) Sellergren, B. Anal. Chem. 1994, 66, 1578. (4) Whitcombe, M. J.; Rodriquez, M. E.; Villar, P.; Vulfson, E. N. J. Am.. Chem. Soc. 1995, 117, 7105. 10.1021/ac060536o CCC: $33.50 Published on Web 09/19/2006
© 2006 American Chemical Society
diagnostic assays, biosensors, and biocatalysis.12 Numerous applications in separations can be envisioned for materials that specifically bind a given protein. Template imprints of proteins as an approach toward protein recognition materials are challenging because of the fragile nature, chemical complexity, and geometric complexity of proteins.13-15 Three techniques, protein entrapment in a bulk polymer, surface imprinting on to a bead, and flat surface imprinting, can be categorized in order to make template imprinting of proteins.14 Surface imprinting beads with narrow size distribution are well suited for chromatographic separation.16 In one early attempt toward protein imprinting, the glycoprotein transferin was allowed to interact with boronate silane, followed by polymerization onto the surface of silica particles.17 Another approach was based on metal coordination interactions onto methacrylate or acrylamide derivatized silica particles.18,19 On the other hand, steric20 and thermodynamic considerations21 indicate that the use of protein templates yields less-well-defined recognition sites in molecularly imprinted polymers (MIPs). If a short peptide or amino acid residue (histidine) representing only a small exposed fragment of a protein structure is used as a template, then the resultant macroporous MIP recognizing the imprinted peptide or amino acid will also be able to recognize the protein molecule.17,22-24 In terms of strength, specificity, and directionality, the metal coordination interaction is more like a covalent interaction than (5) Arshady, R. K.; Mosbach, K. Macromol. Chem. 1981, 182, 687. (6) Ramstrom, O.; Andersson, L. I.; Mosbach, K. J. Org. Chem. 1993, 58, 7562. (7) Spivak, D. A.; Shea, K. J. Macromolecules 1998, 31, 2160. (8) Sellergren, B.; Shea, K. J. J. Chromatogr. 1993, 654, 31. (9) Kuchen, W.; Shram, J. J. Angew. Chem., Int. Ed. Engl. 1988, 27, 1695. (10) Dhal, P. K.; Arnhold, F. H. Macromolecules 1992, 25, 7051. (11) Say, R.; Birlik, E.; Erso ¨z, A.; Yılmaz, F.; Gedikbey, T.; Denizli, A. Anal. Chim. Acta 2003, 480, 251. (12) Dhal, P. K.; Kulkarni, M. G.; Mashelkar, R. A. In Molecularly imprinted polymers: Man-made mimics of antibodies and their application in analytical chemistry; Sellergren, B. Ed.; Elsevier: Amsterdam, 2001; p 271. (13) Bossi, A.; Piletsky, S.; Piletska, A. E. V.; Righetti, P. G. A.; Turner, P. F. Anal. Chem. 2001, 73, 5281. (14) Ratner, B. D.; Shi, H. Curr. Opin. Solid State Mater. Sci. 1999, 4, 395. (15) Malik, S. J.; Plunkett, S. D.; Dhal, P. K.; Johnson, R. D.; Pack, D.; Shnek, D.; Arnhold, F. H. New J. Chem. 1994, 18 (3), 299. (16) Joshi, V. P.; Karode, S. K.; Kulkarni, M. G.; Mashelkar, R. A. Chem. Eng. Sci. 1997, 53 (13), 2271. (17) Glad, M.; Norrlow, O.; Sellergren, B.; Siegbahn, N.; Mosbach, K. J. Chromatogr. 1985, 347, 11. (18) Kempe, M.; Glad, M.; Mosbach, K. J. Mol. Recogn. 1995, 8 (1/2), 35. (19) Hirayama, K.; Burow, M. Chem. Lett. 1998, 8, 731. (20) Flam, F. Science 1994, 263, 1221. (21) Nicholls, I. A. Chem. Lett. 1995, 11, 1035.
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Figure 1. Schematic representation of L-histidine template formation.
hydrogen bonding or electrostatic interactions in water.10 These features make metal coordination a promising binding for the preparation of highly specific templated polymers for the recognition of proteins, via the arrangements of metal coordinating ligands on their surface. The separation is based on the interaction of a Lewis acid (electron pair donor), i.e., a chelated metal ion, with an electron acceptor group on the surface of the protein. Proteins are assumed to interact mainly through the imidazole group of histidine and, to a lesser extent, the indoyl group of tryptophan and the thiol group of cysteine. Cooperation between neighboring amino acid side chains and local conformations plays an important role in protein binding. Aromatic amino acids and the amino terminus of the peptides also have some contributions.25 The imidazole group of N-methacryloyl-(L)-histidine (MAH) has a chelating property with transition metal ions. MAH-Cu(II) and MAH-Cu(II)-L-histidine metal chelating monomers were synthesized by our group. Then, L-histidine imprinted beads were produced by suspension polymerization of MAH-Cu(II)-histidine and ethylene glycol dimethacrylate (EDMA). Then, L-histidine imprinted beads were treated with methanolic KOH to remove L-histidine templates (Figure 1). The adsorption of horse heart cyt c (surface exposed histidine) and L-histidine adsorption on the imprinted beads from aqueous solutions containing different amounts of cyt c were performed. Finally, this study was completed with the chromatographic use of L-histidine imprinted beads (HIBS) by the fast protein liquid chromatography (FPLC) system and investigation of the histidine recogniton ability of HIBS. EXPERIMENTAL SECTION Materials. Cyt c (from horse heart, Mr 12.384, pI 10), L-histidine, and methacryloyl chloride were supplied by Sigma (St. (22) Andersson, L. I.; Mu ¨ ller, R.; Mosbach, K. Macromol, Rapid Commun. 1996, 17, 65. (23) Rachkov, A.; Minoura, N. Biochim. Biophys. Acta 2001, 1544, 255. (24) Hart, B. R.; Shea, K. J. Macromol/ 2002, 35, 6192.
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Louis, MO). EDMA was obtained from Fluka A.G. (Buchs, Switzerland), distilled under reduced pressure in the presence of hydroquinone inhibitor, and stored at 4 °C until use. Poly(vinyl alcohol) (MW 100.000, 98% hydrolyzed) and azobisisobutyronitrile (AIBN) were supplied from Aldrich Chemical Co. (Milwaukee, WI). All glassware were extensively washed with dilute nitric acid before use. All other chemicals were of analytical grade purity and purchased from Merck AG (Darmstadt, Germany). All water used in the experiments was purified using a Barnstead (Dubuque, IA) ROpure LP reverse osmosis unit with a high-flow cellulose acetate membrane (Barnstead D2731) followed by a Barnstead D3804 NANO pure organic/colloid removal and ion exchange packed-bed system. Buffer and sample solutions were prefiltered through a 0.2µm membrane (Sartorius, Go¨ttingen, Germany). Histidine functional monomer, MAH, and metal-chelate monomer, MAHCu(II), were synthesized according to the published procedures.11,26 Methods. Preparation of L-Histidine Imprinted Polymeric Beads. Methacryloyl-Cu(II)-L-histidine ligand exchange monomer was preorganized using MAH-Cu(II) and template, Lhistidine. MAH-Cu(II) (1.0 mmoL) and L-histidine (1.0 mmoL) were dissolved in a vial containing 3.0 mL of ethanol and stirred for 20 min. Then, HIBS were prepared by suspension polymerization: The dispersion medium was prepared by dissolving 0.2 g of poly(vinyl alcohol) in 60 mL of water. MAH-Cu(II)-L-histidine preorganized monomer complex (1.0 mmol) solution was mixed into 8.0 mL of EDMA/12.0 mL of toluene/acetonitrile (80/20) mixture, and 0.09 g of AIBN was dissolved in the monomer mixture. This solution was then transferred into the suspension medium placed in a magnetically stirred (at a constant stirring rate of 600 rpm) glass polymerization reactor (100 mL), which was in a thermostatic water bath. The reactor was flushed by bubbling nitrogen and then was sealed. The reactor temperature was kept constant at 75 °C for 6 h. The polymerization was completed at 90 °C in 3 h. After polymerization, the HIBS were separated from the polymerization medium. The residuals (e.g., unconverted monomer, initiator, and solvent) were removed by an extensive cleaning procedure. The resulting beads were treated with 60/40 (v/v) ethanol/ water containing 3 M KOH (methanolic) for 24 h to remove the template. The beads were cleaned by water/ethanol mixture and then dried in a vacuum oven at 70 °C for 48 h. In the same way, NIBS were also prepared by using MAH-Cu(II) and EDMA. Equilibrium Binding Analyses and Selectivity Studies. The capacity of the adsorbent for cyt c and L-histidine was determined in batch mode. In short, 20 mg of the adsorbent prepared as described above was equilibrated with 25 mM phosphate buffer. Then, the beads were incubated with 10 mL of cyt c or L-histidine solution for 2 h, in flasks agitated magnetically at 150 rpm. The effects of initial concentration and pH of cyt c or L-histidine on the adsorption capacity were studied. The concentration was changed between 10 and 500 µM to observe the effects of the initial concentration of cyt c on adsorption. Cyt c concentration was determined by measuring the absorbance at 410 nm, and (25) Emir, S.; Say, R.; Yavuz, H.; Denizli, A. Biotechnol. Prog. 2004, 20 (1), 223228. (26) Say, R.; Garipcan, B.; Emir, S.; Patır, S.; Denizli, A. Macromol. Mater. Eng. 2002, 287, 539.
concentration was determined using HPLC.27 The amount of adsorbed cyt c or L-histidine was calculated as
L-histidine
Q ) [(Co - C)V]/m
(1)
Here, Q is the amount of cyt c or L-histidine adsorbed onto unit mass of beads (mg g-1), Co and C are the concentrations of cyt c or L-histidine in the initial solution and in the aqueous phase after treatment for certain period of time, respectively (µM), V is the volume of the aqueous phase (mL), and m is the mass of the used beads (g). Cyt c desorption experiments were performed in a buffer solution containing 0,1 M imidazole. Cyt c adsorbed beads were placed in the desorption medium and stirred for 1 h at 25 °C, at a stirring rate of 100 rpm. The final cyt c concentration within the desorption medium was determined by spectrophotometry. The desorption ratio was calculated from the amount of cyt c adsorbed on the beads and the amount of cyt c desorbed by using the following expression:
desorption ratio (%) ) (amount of cyt c desorbed)/ (amount of cyt c adsorbed on the beads) × 100 (2) Cyt c adsorption-desorption operation was done 30 times using the same HIBS beads by 0.1 M imidazole as a desorption agent to check reusability of the HIBS. The beads were washed with 50 mM NaOH solution for 30 min after each adsorptiondesorption cycle for sterilization. After this procedure, beads were washed with water for 30 min and then equilibrated with the phosphate buffer for the next adsorption-desorption cycle. The binding interaction and equlibrium information between the imprinted polymer and histidine can be obtained by Scatchard analysis. This analysis employs the equation
Q/C ) Qmax/KD - Q/KD
(3)
where Q is the amount of histidine bound to the polymer, as calculated by the HPLC variation upon addition of analyte, and C is the concentration of free histidine. Qmax represents the apparent maximum number of binding sites, and KD is the equlibriumdissociation constant of the metal-chelate polymer based on ligand exchange. Characterization. The specific surface area of unleached (before washing with KOH) and leached (after washing with KOH) imprinted beads in the dry state was determined by the multipoint Brunauer, Emmett, and Teller (BET) method with nitrogen as sorbate with experimental error (0.2 m2 g-1. The pore diameter (Å) and pore volume (cm3 g-1) were also measured. Fast Protein Liquid Chromatography. FPLC separations were performed using an Ammersham A¨ KTA-FPLC system equipped with a UV detection system. The system includes a INV 907 pump and a Frac920 fraction collector. Separations were carried out at an empty glass column that packed with MAHCu-L-histidine imprinted polymer receptors. The mobile phase (27) Wadud, S.; Or-Rashid, M. M.; Onedera, R. J. Chromatogr., B 2002, 767, 369.
Figure 2. Effect of histidine concentration on the amount of adsorbed histidine.
was 0.05 M NaH2PO4 buffer (pH 5.0). The separations were performed at room temperature with a flow rate of 0.3 mL min-1 and monitored at 254 nm. Acetone was used as the void marker. Capacity factors (k′) and separation factors (R) were calculated as k′ ) (tR- t0)/t0, R ) k′2/ k′1, where tR is the retention time of the analyte and t0 is the retention time of the void marker. The resolution (Rs) and theoretical plate numbers (N) were calculated using the following equations:
N ) 5.54(tR/w0.5 )2 Rs ) 2(tR,2 - tR,1)/(ω2 + ω1)
where w0.0.5 is the peak weight at the corresponding peak height fraction, tR,1 and tR,2 are the retention times of two adjacent peaks, and ω1 and ω2 are the widths of the two adjacent peaks at the baseline RESULTS Characterization of L-Histidine Imprinted Beads. The specific surface area of the unleached and leached beads, which are cross-linked matrixes, was found to be 72.8 and 235.2 m2 g-1, respectively. Since MAH has two coordinating sites of nitrogen atoms, it can form a ternary complex, which is coordinated histidine molecules at vacant coordination sites of Cu2+-MAH ligand exchange monomer complexes on the HIBS. The Barrett, Joyner, and Halenda (BJH) adsorption cumulative pore volumes are higher for leached HIBS (0.2543 cm3 g-1) compared to unleached HIBS (0.1132 cm3 g-1). Further, BJH and BET pore diameter is 28 Å of leached HIBS beads, indicating that imprinted beads with mesopores and macropores (>20 A°) were obtained in the present study. The pore diameter for unleached HIBS was measured as 22 Å. Adsorption Isotherm of L-Histidine to HIBS. Adsorption of L-histidine from aqueous solutions was investigated in batch experiments. As can be seen in Figure 2, the amount of adsorbed L-histidine per unit mass of HIBS increased with the initial concentration of the L-histidine. The maximum adsorption capacity was 15.38 mg g-1 HIBS. Analytical Chemistry, Vol. 78, No. 20, October 15, 2006
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Table 1. Comparison of Langmuir and Freundlich Adsorbtion Models for L-Histidine Imprinted Beads exper
Langmuir
Freundlich
impr beads
Q (mg/g)
Qm (mg/g)
b (L/mg)
HIBS-histidine HIBS-cyt c
14.93 27.06
15.31 45.05
3.349 1.21 × 10-3
R2
Kd (L/g)
n
R2
0.9457 0.987
13.361 0.976
0.0278 0.4372
0.8718 0.8145
Figure 5. Scathard plot of cyt c rebinding by the imprinted polymers.
Figure 3. Scatchard plot of histidine rebinding by the imprinted polymers.
Figure 4. Effect of initial horse heart cyt c concentration on adsorption capacity of HIBS. pH, 7.0 (phosphate buffer); time, 2 h; T, 25 °C.
When adsorption data of histidine were fitted against Langmuir and Freundlich isotherms, satisfactory fits were found with the Langmuir isotherm. The Langmuir isotherm is a valid monolayer sorption on a surface containing a finite number of binding sites. The Langmuir constant was calculated as 3.349 L m g-1 from this plot, and the R2 value of the Langmuir plot was 0.9457 (Table 1). The association constant (Ka) for the specific interaction between the template imprinted beads and the template (Lhistidine) itself was determined by Scatchard’s plots using HIBS. Ka value can be estimated as 58 300 M-1 and maximum number of ligand exchange interaction sites, Qmax, is 101.82 µmol g-1. The R2 value was calculated as 0.9883 for HIBS from this slope (Figure 3). Adsorption of Cytochrome c to L-Histidine Synthetic Receptors. Figure 4 shows the effects of initial cyt c concentration on cyt c adsorption. As presented in this figure, the amount of cyt c adsorbed per unit mass increases almost linearly at low concentrations with increasing cyt c concentration and then 7256 Analytical Chemistry, Vol. 78, No. 20, October 15, 2006
increases less rapidly and approaches saturation. The steep slope of the initial part of the adsorption isotherm represents a high affinity between cyt c and Cu2+ chelated groups. It is clear that this increase is due to specific interactions between chelated Cu2+ ions and cyt c molecules (especially imidazole side chains of histidine residue in cyt c structure). Horse heart cyt c has a single surface histidine at position 29.28 One surface histidine is reported as sufficient for the adsorption on a Cu2+ immobilized metalchelate affinity chromatography (IMAC) adsorbent and proteins varying by only one histidine can be separated.29 When adsorption data of cyt c were fitted against Langmuir and Freundlich isotherms, an approprite model was found was also the Langmuir isotherm. The Langmuir constant is 1.21 × 10-3 L mg-1 for this plot, and the R2 value for Langmuir isotherm is 0.987 (Table 1). Figure 5 shows the Scatchard plot including two different lines. This situation suggests that the HIBS have two binding sites for cyt c. The association constant (Ka) for the first binding of cyt c to HIBS is 5200 M-1 and the maximum number of ligand exchange interaction sites, Qmax, is 9.0385 µmol g-1. The association constant (Ka) for the second binding of cyt c to MIP receptor is 55 500 M-1 and the maximum number of ligand exchange interaction sites, Qmax, 2.5495 µmol g-1. The values of Ka suggest that the affinity of the binding sites is very strong. R2 values of these plots were 0.9964 and 0.9699 for the first and second binding of cyt c to MIP receptor, respectively. The pH is possibly the most critical parameter that affects the adsorption capacity. The effect of pH value on adsorption of histidine and cyt c was determined for different pH values ranging from 4.0 to 9.0 for L-histidine and 4.0-12.0 for cyt c. Results of the study show that the adsorption capacity for the imprinted and nonimprinted beads increases with increasing pH (up to pH 7.0) and then starts decreasing. The high binding affinities at pH 6.07.0 can be explained because of the deprotonation of histidine at pKa ) 6.2. (28) O′Brien, S. M.; Thomas, O. R. T.; Dunnill, P. J. Biotechnol. 1996, 50, 13. (29) Sulkowski, E. BioEssays 1989, 10, 170.
Figure 6. FPLC Separation of D-histidine and L-histidine on a column packed with MAH-Cu-L-histidine imprinted polymers. All compounds were separated in less than 7.0 min. All peaks were detected at 254 nm with 0.05 M NaH2PO4 buffer containing 0.1 M imidazole; column length, 50-mm diameter; 5-mm empty glass Tricorn column; amount of packed material, 0.700 g.
Figure 7. FPLC separation of cyt c and ribonuclease A on a column packed with MAH-Cu-L-histidine imprinted polymers. Detection was performed at 254 nm with 0,05 M NaH2PO4 buffer containing 0.1 M imidazole; column length, 50-mm diameter; 5-mm empty glass Tricorn column; amount of packed material, 0.700 g.
The amount of cyt c adsorbed onto the HIBS and NIBS as a function of pH exhibits two adsorption domains: (i) The amount of cyt c adsorbed onto the NIBS beads shows a maximum at pH 10, with a significant decrease at lower and higher pH values (isoelectric point of cyt c, 10.6). Specific interactions (electrostatic and coordination) between cyt c and chelated Cu2+ ions at pH 10 may have resulted both from the ionization states of several groups on amino acid side chains in cyt c and from the conformational state of cyt c molecules (more folded structure) at this pH. (ii) At
pH values lower and higher than 10, the adsorbed amount of cyt c drastically decreases. This could be attributed to the ionization state of cyt c and be caused by repulsive electrostatic forces between cyt c and the chelated Cu2+ ions. Regeneration of HIBS. Regeneration is a crucial step in all affinity chromatography techniques. It was thus necessary to evaluate the regeneration efficiency of the affinity adsorbents after each cycle. In this study, more than 90% of the adsorbed cyt c molecules was removed easily from the histidine imprinted beads Analytical Chemistry, Vol. 78, No. 20, October 15, 2006
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Table 2. Chromatographic Separation Data
D-histidine L-histidine
ribonuclease A cyt c
tR
N
k′
1.76 5.43 1.85 4.11
18.0 163.0 53.0 81.0
0.47 3.53 0.54 2.43
a
Rs
7.56
1.43
4.47
1.39
in all cases when imidazole was used as a desorption agent. Note that there was no Cu2+ release in this case, which shows that the novel metal-chelate monomer MAH-Cu(II) is polymerized on the bead’s surface by strong chelate formation. With the desorption data given above, we concluded that imidazole is a suitable desorption agent for the HIBS. To show the reusability of the HIBS, the adsorption-desorption cycle was repeated 30 times using the same imprinted beads from aqueous cyt c solution and there was no significant loss in the adsorption capacity of the beads. After 30 cycles, the capacity had decreased by ∼8%. These results demonstrated the stability of the present solid support as imprinted affinity beads. Chromatographic Usage of L-Histidine Imprinted Receptor. The glass column packed with MAH-Cu-L-histidine imprinted polymers was connected to the FPLC and then a 50 ppm D,L-histidine mixture at pH 5.0 was injected into the system. As shown in Figure 6, D-histidine and L-histidine separation was observed at 1.76 and 5.43 min, respectively. For the determination of cyt c and separation of ribonuclease A and cyt c, a MAH-Cu-L-histidine packed coloumn was used and the NaH2PO4 buffer was prepared with 0.1 M imidazole. As seen in Figure 7, cyt c and ribonuclease A separation was also observed at 1.85 and 4.11 min, respectively. The tR, R, N, k′, and R values are given in Table 2. Rs values were calculated as 1.43 and 1.39 for D-L-histidine and cyt c-ribonuclease A, respectively. Because the Rs value should be higher
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than 1.0 for a good resolution of two peaks in such a chromatography system, these results for the resolution of D-L-histidine and cyt c-ribonuclease A can be accepted as good resolution values. CONCLUSIONS MIPs based on metal coordination are better than polymer based on IMAC for histidine determination, because no metal leakage and the histidine cavities on the microbeads’ surface are good for proteins carrying histidine on the surface having suitable geometrical and selective binding. The adsorption capacity for the recognition of horse heart cyt c could be improved presumably through enhanced interaction based on metal-chelate between the functionalities of the MAH-Cu(II) monomer and L-histidine template. These features make metal coordination a promising binding mode for preparing highly specific templated polymers for recognition of horse heart cyt c via the arrangement of a metalcoordinating ligand (i.e., L-histidine) on their surfaces. This technique can be used for the separation of D-L enantiomers of histidine and also separation of ribonuclease A and cyt c (surface histidine exposed protein).We think that these tailor-made MIPs based on metal-chelate interaction will be an attractive alternative to the metal-chelate affinity that has some problems such as selectivity and adsorption restrictions and metal leakage, and this approach is the first study using FPLC for the preparative applications of the above proteins and enantiomers. ACKNOWLEDGMENT The authors acknowledge Anadolu University, Commission of Scientific Research Projects (042838), for financial support to carry out this research work.
Received for review March 24, 2006. Accepted July 13, 2006. AC060536O
