Selection of Ligands for Affinity Chromatography Using Quartz Crystal

Anal. Chem. 2005, 77, 4248-4256

Selection of Ligands for Affinity Chromatography Using Quartz Crystal Biosensor Yang Liu,† Xiaoling Tang,‡ Feng Liu,† and Ke′an Li*,†

The Key Laboratory of Bioorganic Chemistry and Molecular Engineering, Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China, and Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia 30322

This paper described a new strategy for rapid selecting ligands for application in affinity chromatography using a quartz crystal microbalance (QCM) biosensor. An aminoglycoside antibiotic drug, kanamycin (KM), was immobilized on the gold electrodes of the QCM sensor chip. The binding interactions of the immobilized KM with various proteins in solution were monitored as the variations of the resonant frequency of the modified sensor. Such a rapid screen analysis of interactions indicated clearly that KM-immobilized sensor showed strong specific interaction only with lysozyme (LZM). The resultant sensorgrams were rapidly analyzed by using a kinetic analysis software based on a genetic algorithm to derive both the kinetic rate constants (kass and kdiss) and equilibrium dissociation constants (KD) for LZM-KM interactions. The immobilized KM showed higher affinity to LZM with a dissociation constant on the order of 10-5 M, which is within the range of 10-4-10-8 M and suitable for an affinity ligand. Therefore, KM was demonstrated for the first time as a novel affinity ligand for purification of LZM and immobilized onto the epoxy-activated silica in the presence of a high potassium phosphate concentration. The KM immobilized affinity column has proved useful for a very convenient purification of LZM from chicken egg white. The purity of LZM obtained was higher than 90%, as determined by densitometric scanninng of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified fraction. These results confirmed that the selected KM ligand is indeed a valuable affinity ligand for purification of LZM. The new screening strategy based on a QCM biosensor is expected to be a promising way for rapid selecting specific ligands for purifying other valuable proteins. High-performance affinity chromatography (HPAC) is one of the most effective methods for the purification of biological macromolecules.1 The search for an affinity ligand specifically for a target protein is the most important step in development of * Corresponding author. Tel: 86-10-62761187. Fax: 86-10-62751708. E-mail: [email protected]. † Peking University. ‡ Emory University School of Medicine. (1) Burnouf, T.; Radosevich, M. J. Biochem. Biophys. Methods 2001, 49, 575586.

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affinity protein purification processes.2,3 Various techniques, such as chromatography,4 electrophoresis,5 NMR,6 mass spectrometry,7 ultracentrifugation,8 scintillation proximity assay,9 computer-aided molecular modeling,10 and biosensors,11 have been used for the selection of a specific ligand. Compared with other techniques, biosensors appear to be more promising for ligand selection, because they allow the real-time analysis of interaction without labeling requirements and provide expediently quantitative information on the rate and equilibrium binding constants, which is required to assess whether an affinity ligand is suitable for use in a commercial process.12 In recent years, the applications of biosensors for ligand selection are expanding rapidly.13,14 The quartz crystal microbalance (QCM) is a rather sensitive device among the biosensing methods.15-20 QCM biosensor integrated in a flow injection analysis (FIA) system has the advantage being able to work continuously and to monitor online the binding of the analyte.21-23 Some reports have proved that (2) Labrou, N. E. J. Chromatogr., B 2003, 790, 67-78. (3) Fassina, G.; Ruvo, M.; Palombo, G.; Verdoliva, A.; Marino, M. J. Biochem. Biophys. Methods 2001, 49, 481-490. (4) Evans, D. M.; Williams, K. P.; McGuinness, B.; Tarr, G.; Regnier, F.; Afeyan, N.; Jindal, S. Nat. Biotechnol. 1996, 14, 504-507. (5) Belenky, A.; Hughes, D.; Korneev, A.; Dunayevskiy, Y. J. Chromatogr., A 2004, 1053, 247-251. (6) Zartler, E. R.; Hanson, J.; Jones, B. E.; Kline, A. D.; Martin, G.; Mo, H.; Shapiro, M. J.; Wang, R.; Wu, H.; Yan, J. J. Am. Chem. Soc. 2003, 125, 10941-10946. (7) Xu, J.; Szakal, C. W.; Martin, S. E.; Peterson, B. R.; Wucher, A.; Winograd, N. J. Am. Chem. Soc. 2004, 126, 3902-3909. (8) Wieboldt, R.; Zweigenbaum, J.; Henion, J. Anal. Chem. 1997, 69, 16831691. (9) Bosworth, N.; Towers, P. Nature 1989, 341, 167-168. (10) Villanueva, J.; Fernandez-Ballester, G.; Querol, E.; Aviles, F. A.; Serrano, L. J. Mol. Biol. 2003, 330, 1039-1048. (11) Durick, K.; Negulescu, P. Biosens. Bioelectron. 2001, 16, 587-592. (12) Lahiri, J.; Isaacs, L.; Tien, J.; Whitesides, G. M. Anal. Chem. 1999, 71, 777-790. (13) Morrill, P. R.; Millington, R. B.; Lowe, C. R. J. Chromatogr., B 2003, 793, 229-251. (14) Bertucci, C.; Cimitan, S. J. Pharm. Biomed. Anal. 2003, 32, 707-714. (15) Wohltjen, H.; Dessy, R. Anal. Chem. 1979, 51, 1458-1475. (16) Theisen, L. A.; Martin, S. J.; Hillman, A. R. Anal. Chem. 2004, 76, 796804. (17) Nishino, H.; Murakawa, A.; Mori, T.; Okahata, Y. J. Am. Chem. Soc. 2004, 126, 14752-14757. (18) Nishino, H.; Nihira, T.; Mori, T.; Okahata, Y. J. Am. Chem. Soc. 2004, 126, 2264-2265. (19) Bonroy, K.; Friedt, J. M.; Frederix, F.; Laureyn, W.; Langerock, S.; Campitelli, A.; Sara, M.; Borghs, G.; Goddeeris, B.; Declerck, P. Anal. Chem. 2004, 76, 4299-4306. (20) Xie, H.; Yu, Y. H.; Xie, F.; Lao, Y. Z.; Gao, Z. Q. Anal. Chem. 2004, 76, 4023-4029. 10.1021/ac050026e CCC: $30.25

© 2005 American Chemical Society Published on Web 05/12/2005

Scheme 1. Comparison of the New Screening Strategy and the Conventional Screening Strategya

a The drug ligand (key) or the target protein (lock) was immobilized respectively for screening corresponding target protein (lock) or drug ligand (key).

the QCM-FIA system is quite suitable for rapid affinity ranking of different receptor proteins.24,25 Such a rapid screen analysis of interactions by the QCM biosensor may also open a new way for the selection of a specific ligand of potential use in HPAC.25,26 Conventional high-throughput screening for ligand selection is known as target-based drug discovery screening; the target protein with defined binding properties is immobilized for screening a drug library. This ligand selection procedure is mainly based on the lock and key mechanism of molecular recognition. However, ligand selection by screening interactions between soluble ligands and the target protein is not representative of the complex environment created by the chemical, geometrical, and steric constraints imposed by the three-dimensional matrix.13 Furthermore, the immobilized target protein is quite sensitive to environment change and easily loses its activity, causing problems with interpretation of the screening results. Here, a new screening strategy for rapid selection of ligand for affinity chromatography using a QCM biosensor is proposed (Scheme 1). Drug ligand with defined properties is immobilized on the QCM sensor chip, and the pooled proteins are screened rapidly to find out the specific receptor, which means that a key with excellent properties in every aspect is used for the search for a corresponding lock from the pooled ones. As covalent coupling is involved for immobilization of a drug ligand, either on the sensor chip of the QCM or on the matrix for HPAC, the rapid association in the loading solution and fast dissociation in the elution solution as shown in the measurement of the QCM would be a good indicator for HPAC. This alternating screening strategy using the QCM biosensor will also make feasible the rapid and efficient generation of a specific ligand for affinity chromatography. Kanamycin (KM) is a water-soluble aminoglycoside antibiotic produced by fermentation of Streptomyces kanamyceticus.27 A number of reports have provided strong evidence that lysozyme (LZM) could interact with the antibiotic drug.28,29 However, the (21) Su, X. D.; Robelek, R.; Wu, Y. G.; Wang, G. Y.; Knoll, W. Anal. Chem. 2004, 76, 489-494. (22) Wang, H.; Zeng, H.; Liu, Z. M.; Yang, Y. H.; Deng, T.; Shen, G. L.; Yu, R. Q. Anal. Chem. 2004, 76, 2203-2209. (23) Liu, Y.; Yu, X.; Zhao, R.; Shangguan, D. H.; Bo, Z. Y.; Liu, G. Q.Biosens. Bioelectron. 2003, 18, 1419-1427. (24) Liu, Y.; Zhang, W.; Yu, X.; Zhang, H. W.; Zhao, R.; Shangguan, D. H.; Li, Y.; Shen, B. F.; Liu, G. Q. Sens. Actuators, B 2004, 99, 416-424. (25) Liu, Y.; Yu, X.; Zhao, R.; Shangguan, D. H.; Bo, Z. Y.; Liu, G. Q. Biosens. Bioelectron. 2003, 19, 9-19. (26) Liu, Y.; Zhao, R.; Shangguan, D. H.; Zhang, H. W.; Liu, G. Q. J. Chromatogr., B 2003, 792, 177-185.

properties of this binding interaction have not been characterized and the interactions between the antibiotic drug and other proteins still remain unclear. In this work, we described the details of a new strategy for rapid selection of a specific ligand for affinity chromatography using the QCM biosensor. A QCM-FIA system was used for rapid screen analysis of the interactions between immobilized KM drug ligand and various receptor proteins in solution. Results obtained from this screening strategy clearly demonstrated that KMimmobilized sensor showed strong specific interaction only with LZM. Rapid kinetic analysis using the kinetic analysis software based on a genetic algorithm (GA) indicated that immobilized KM showed higher affinity to LZM. Thus, KM was selected first as a novel ligand for the purification of LZM. The application of KM immobilized affinity column for separation and purification of LZM from chicken egg white (CEW) was also investigated in detail. EXPERIMENTAL SECTION Apparatus. A homemade QCM biosensor setup mainly consisted of two partssQCM system and FIA system as described previously.23 The QCM sensor chips employed were commercially available 10-MHz AT-cut quartz crystals (14-mm diameter) with gold electrodes (8-mm diameter; geometric area 50.2 mm2 × 2) on both sides purchased from Beijing 707 Factory (Beijing, China). The crystals were polished to a surface roughness of less than 5 µm, which produced a mirrorlike finish on the gold electrodes. An Agilent 1100 series liquid chromatograph system (Waldbronn, Germany) consisting of a quaternary solvent delivery system, a manual sampler, and Agilent 1100 VWD detector coupled with an analytical workstation was used. Materials. KM was the chemical control reagent supplied by the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Chicken egg white LZM (EC 3.2.1.7), chicken egg albumin (CEA), human serum albumin (HSA), bovine serum albumin (BSA), chicken IgG (C IgG), hemoglobin, transferrin, γ-globin, myoglobin, pepsin, trypsin, RNase A, and DL-dithiothreitol (DTT) were all purchased from Sigma (St. Louis, MO). 3-Glycidoxypropyltrimethoxysilane (GPS), (27) Umezawa, H.; Ueda, M.; Maeda, K.; Yagishita, K.; Kondo, S.; Okami, Y.; Utahara, R.; Sato, Y.; Nitta, K.; Takeuchi, T. J. Antibiot. Ser. A 1957, 10, 181-187. (28) Kundu, A.; Vunnum, S.; Cramer, S. M. J. Chromatogr., A 1995, 707, 5767. (29) Gulla, K. C.; Gouda1, M. D.; Thakur, M. S.; Karanth, N. G. Biosens. Bioelectron. 2004, 19, 621-625.

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1,4-Butanediol diglycidyl ether and sodium borohydride were from Aldrich (St. Louis, MO). Micrococcus lysodeikticus cells used in the spectrophotometric assay for LZM activity were supplied by Institute of Microbiology, Chinese Academy of Sciences (Beijing, China). All other chemicals were analytical reagent grade, and doubly distilled water was used for preparation of aqueous solutions. Prior to use, all solutions prepared were filtered (0.45 µm). QCM Biosensor for Affinity Ligand Selection. This section described the general procedure for ligand selection by QCM biosensor. Immobilization of KM ligand on a QCM Sensor Chip. First, the gold electrodes of the sensor chip were cleaned as described previously.23 The freshly cleaned sensor chip was immersed in a 20 mM ethanol solution of DTT. The unstirred solution was kept at room temperature in the dark for 24 h. The crystal was then washed with ethanol and doubly distilled water and sonicated for 10 min in ethanol to remove the excess of thiols. The modified crystal was immersed in the mixture solution consisting of 1 mL of ethanediol diglycigyl ether and 4 mL of 0.1 M Na2CO3 buffered solution (pH 8.5) containing 8 mg of sodium borohydride. The reactions were performed at 30 °C for 4 h and stopped by washing the surfaces with a large volume of water. The prepared crystal was immersed for 2 h in the basic solution containing 0.1 mg of KM in 5 mL of 0.1 M Na2CO3 buffered solution (pH 11.0), and the reaction temperature was kept at 40 °C. Finally, 5 mL of TrisHCl (pH 8.5, 1.0 M) was used as a blocking agent and applied to block the residual reacting sites for 30 min. QCM Biosensor for Rapid Screen Analysis of Interactions between Proteins and Immobilized KM. The fresh-coated quartz crystal was mounted in the flow-through cell and rinsed with the loading solution (50 mM potassium phosphate buffer containing 100 mM NaCl, pH 7.0) continuously until the frequency had stabilized under flow conditions (60 µL/min). By means of an injection valve, 200 µL aliquots of various protein solutions (0.5 µg/µL) were injected into the fluid system, respectively. The permanent frequency shifts versus time curves were recorded, and the binding process was monitored in real time. After one binding measurement, 600 µL of glycine hydrochloride solution (100 mM, pH 2.0) was injected into the system with the injection valve to dissociate the bound protein and free binding sites in the receptor layer for the next binding. Rapid Kinetic Data Analyses Using Kinetic Software Based GA. To obtain the kinetic constants rapidly, the kinetic analysis software based on GA was developed in our previous work.24,25 The programs involve the following procedure: creation of population, evaluation of cost, mate selection, reproduction, and mutation. Using the procedures of mate selection, mate selection, reproduction, and mutation, a new population is created and the whole circle is repeated until a proper stop criterion is fulfilled. The cost function was given by

χ2 )

1

n

∑{y - f(y ,a )} n

2

i

i

i

i)1

where the yi are the experimental data, f (yi,ai) is the function chosen as model, and the ai are the model parameters. The quality of the fitted data is evaluated by comparison between calculated 4250


and experimental curves and by the magnitude of χ2. Using this special kinetic analyses software, we could calculate the apparent binding constants for the interaction between immobilized KM and specifically bound LZM rapidly. HPAC Purification Using the Selected KM Ligand. This section described the preparation procedure of the affinity packing using selected KM ligand and its potential application for the separation and purification of LZM from complex CEW solution. Salt-Induced Immobilization of KM on the Silica Matrix of HPAC. Two grams of Uetikon macroporous silica sphere (granule diameter 5 µm, pore diameter 300 Å, specific surface area 40 m2/ g) was refluxed with 10% HCl solution for 8 h at 100 °C. A 1.5-mL aliquot of GPS was mixed with 30 mL of toluene previously dried with 5-Å molecular sieves. The toluene solution of silane was added to the washed silica, and the reaction mixture was refluxed for 8 h. After the reaction, silica was extracted with methanol using a Soxhlet apparatus.30 A 0.2-g sample of KM was dissolved in 10 mL of potassium phosphate buffer (2.5 M, pH 7.9) and the resultant mixture reacted with epoxy-activated silica at 60 °C for 48 h. Finally, 10 mL of Tris-HCl (pH 7.9, 1.0 M) was used as a blocking agent and applied to block the residual reacting sites for 3 h. Blank packing was also prepared according to the immobilization procedure except for the ligand-coupling step. The above two prepared packings were respectively packed into stainless steel columns (4.0 × 70 mm i.d.) under 16 MPa pressure. Affinity Chromatographic Conditions. All chromatographic experiments were performed at room temperature. Protein solutions were prepared in 0.5 µg/µL with the loading solution (50 mM potassium phosphate buffer containing 100 mM NaCl, pH 7.0). All separations without the special description were carried out through the column by passing the loading solution for 15 min, the elution solution (50 mM potassium phosphate buffer containing 500 mM NaCl, pH 7.0) for 15 min, and then loading solution within 15 min at a flow rate of 0.5 mL/min, after injection of the protein solution (50 µL). The chromatographic profiles were monitored at 280 nm. Separation and Characterization of LZM from CEW. CEW was separated from fresh eggs and diluted to 50% (v/v) with loading buffer. The diluted CEW was homogenized in an ice bath and centrifuged at 4 °C, at 12 000 rpm for 15 min. The supernatant fluid was filtered through a 0.45-µm filter and used as a LZM source. Then 100 µL of the treated CEW solution was injected into the affinity column. Unbound proteins were washed from the column with 50 mM potassium phosphate buffer (pH 7.0) containing 100 mM NaCl and 200 mM NaCl successively. The bound LZM was eluted out with 50 mM potassium phosphate buffer (pH 7.0) containing 1.0 M NaCl. After elution was complete, the column was washed with the loading buffer to restore it to its initial baseline for carrying out the next experiment. LZM purity characterization was carried out on a Bio-Rad electrophoresis system (Richmond, CA) with a Mini-Protein II electrophoresis cell (gel size 7 × 8 cm) and PAC 300 power. The fraction was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% gels using Coomassie Blue R-250 to stain the separated bands and protein markers from 97.4 to 14.4 kg/mol (Amresco, Solon, OH). The gels were scanned (30) Tsubokawa, N.; Kobure, A.; Maruyama, K.; Sone, Y.; Shimomura, M. Polym. J. 1990, 22, 827-833.

Chart 1. Structure of Biorecognition Layers of KM Modified Sensor

Figure 1. Interaction of KM immobilized sensor with various proteins in solution (0.5 µg/µL).

using a Shimadzu dual-wavelength flying spot scanning densitometer (Tokyo, Japan) for quantification of protein bands. LZM activity was determined by measuring the clearing of turbidity of M. lysodeikticus suspension (substrate solution) as described.31,32 The decrease in absorbance at 450 nm was monitored by a Shimadzu UV3010 spectrophotometer (Kyoto, Japan). One unit of LZM activity is equivalent to a change of 0.001 absorbance units over 1 min.33 The protein concentration in the experiment was determined using a Bio-Rad protein assay kit, and BSA was used as a protein standard.34 RESULTS AND DISCUSSION QCM Biosensor for Affinity Ligand Selection. A QCM biosensor integrated with a FIA system is a convenient and valuable tool for real-time monitoring of interactions between immobilized drug ligand and pooled proteins, providing a promising method to discover a specific ligand for purifying protein.25 On the other hand, information on kinetic constants could provide further evidence to assess whether the selected ligand is suitable for use as an affinity ligand of HPAC. Immobilization of KM Ligand on a QCM Sensor Chip. The functionalization of gold surfaces can be reached using a selfassembled monolayer of thiocompounds. Here, dithiothreiol was chosen as the functional reagent to modify the gold electrode of piezoelectric crystal. The sulfhydryl groups were easily activated by ethanediol diglycidyl ether under weak base conditions, and the crystal surfaces were modified by epoxide groups. KM was then bound directly to the epoxide groups on the modified surfaces through amino groups. The structure of biorecognition layer is depicted in Chart 1. To evaluate the progress of immobilization, the resonance frequencies of the dried crystals were determined after all reaction (31) Kikuchi, M.; Yamamoto, Y.; Taniyama, Y.; Ishimaru, K.; Yoshikawa, W.; Kaisho, Y.; Ikehara, M. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 9411-9415. (32) Masuda, T.; Ueno, Y.; Kitabatake, N. J. Agric. Food. Chem. 2001, 49, 49374941. (33) McCreath, G.; Owen, R. O.; Nash, D. C.; Chase, H. A. J. Chromatogr., A 1997, 773, 73-83. (34) Bradford, M. M. Anal. Biochem. 1976, 72, 248-254.

steps and the amount of bound material was determined according to Sauerbray equation,35 1 µg ≈ 457 Hz over the linear range of 0.01-25.0 µg. The amount of the immobilized KM was calculated, and ∼2.56 nmol of KM was immobilized on each surface of crystal (0.050 nmol of KM/mm2 gold electrode area). QCM Biosensor for Rapid Screen Analysis of Interactions between Immobilized KM and Proteins. Since the binding interactions of various proteins to immobilized KM were carried out in similar conditions (flow rate, temperature, concentration) and on the same sensor chip, their kinetic binding curves can be compared directly. The screening results are shown in Figure 1. The frequency decreased ∼95 Hz when LZM was injected into the cell. With rinsing by the buffer solution, the frequency did not return back to the initial level. This result confirmed that specific interaction between LZM and immobilized KM could occur. In addition, there was no nonspecific adsorption of other proteins to the KM modified sensor chip. As the residual epoxide groups reacting sites have been blocked and the gold surfaces were hydrophilic, the nonspecific adsorption of these hydrophobic proteins to the gold surfaces was almost impossible. Such a rapid screen analysis of interaction between proteins and immobilized ligand using the QCM biosensor provided a promising way to discover a specific ligand for purifying protein. As the covalent coupling has been involved in the amino groups for immobilization of KM ligand, either on the sensor chip for QCM or on the silica matrix for HPAC, we could assume that rapid association in the loading solution and fast dissociation in the elution solution as shown in the measurement of QCM would be a good indicator for HPAC. Apparently, KM can be used as a novel ligand of HPAC to purify LZM. LZM, which occurs naturally in CEW, is a commercially available enzyme with many important applications.36-38 The low (35) Sauerbrey, G. Z. Phys. 1959, 155, 206-222. (36) Ghosh, R. Biochem. Eng. J. 2003, 14, 109-116. (37) Das, S.; Banerjee, S.; Dasgupta, J. Chemotherapy 1992, 38, 350-357. (38) Huang, S. L.; Huang, P. L.; Sun, Y.; Kung, H.; Blithe, D. L.; Chen, H. C. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 2678-2681.


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The frequency shift due to the association and dissociation of A on the gold electrodes of sensor chip can be calculated according to eqs 2 and 3, respectively, as previously described.23 For association,

∆F ) -

kassC∆Fmax -(kass+kdiss) (e - 1) kassC + kdiss

(2)

For dissociation,

∆F ) - ∆F0e-kdiss(t - t0) Figure 2. Typical binding and regeneration QCM sensorgram. Glycine hydrochloride solution (100 mM, pH 2.0) was used as regeneration reagent.

content of lysozyme in CEW (∼3.4%) is a real challenge for its purification. The conventional immobilized ligands used for purification of LZM are metal ion,39 dye,40 saccharide,41 and so on. Compared with these conventional ligands, KM drug ligand will be more attractive due to its high specificity, freedom from toxicity, and low cost. Regeneration and Reproducibility of QCM Sensor Chip. For repeated uses of the sensor, it is important to regenerate the sensor. Hence, the efficiencies in removing adsorbed LZM and maintaining sensor sensitivity of various regeneration reagents, such as glycine hydrochloride solution (100 mM, pH 2.0), 0.5 M NaCl solution, 10 mM HCl solution, and 10 mM NaOH solution, were compared. The mild glycine hydrochloride solution (pH 2.0) was chosen as the best one, and a typical binding and regeneration sensorgram was obtained and is shown in Figure 2. A significant decrease of frequency could be seen after 0.5 µg/µL LZM was injected in the flow cell. The frequency decreased ∼95 Hz and reached equilibrium within ∼8 min. With rinsing by the loading buffer, the frequency increased a little, indicating dissociation of adsorbed LZM, but the frequency did not return back to the initial baseline. When glycine hydrochloride solution was injected, the specifically bound LZM molecules were released and washed out in less than 20 min. The frequency recovered to the initial level. To verify reproducibility, 0.5 µg/µL LZM was chosen and determined for 5 times, respectively. The relative standard deviation (RSD) of frequency shift (∆f) was calculated as 2.6% (n ) 5), indicating that this regeneration procedure was quite reproducible. Software Based on Genetic Algorithm for Rapid Kinetic Data Analyses. Since the QCM is a continuous, real-time detector, it is possible to evaluate the interaction kinetics. The reaction between the immobilized compound (B) and the molecule in solution (A) is often assumed to follow pseudo-first-order kinetics.42 For the reversible interaction, kass

A + B {\ } AB k diss

(1)

(39) Luo, Q. Z.; Zou, H. F.; Xiao, X. Z.; Gou, Z.; Kong, L.; Mao, X. Q. J. Chromatogr., A 2001, 926, 255-264. (40) Arica, M. Y.; Yilmaz, M.; Yalcm, E.; Bayramoglu, G. J. Chromatogr., B 2004, 805, 315-323.

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(3)

where ∆F is frequency change, C is the concentration of the free A, which holds constant in a continuously flowing solution, ∆Fmax is the frequency change after a complete saturation of the surface of the crystal with A, t is time, t0 is time at start of reaction, and F0 is frequency at t0. Based on the above equations, the kinetic analysis software based on GA was developed to calculate the kinetic constants expediently.24 Using to this special software, experimental sensorgram data could be fitted to eq 2 for the association rate constant (kass) and to eq 3 for the dissociation rate constant (kdiss). The magnitude of the χ2 and comparison between calculated and experimental curves are used to evaluate the quality of the fitted data. The equilibrium constant KD for LZM can be obtained as a ratio

KD ) kdiss/kass

(4)

The sensorgram data in Figure 2 were chosen as an example and analyzed rapidly using our kinetic analysis software to obtain the binding constants. The Mr ) 14 300 was used for the molecule of LZM. The curves fit to frequency change data for the determination of kass and kdiss are shown in Figure 3. It could be seen from Figure 3 that the quality of the fitted data in the progress of both adsorption and desorption was good when comparing the calculated curves to the experimental one. Results of the kinetic analyses of the interaction between immobilized KM and LZM are shown in Table 1 (group 1). The magnitudes of χ2 were 0.87 and 1.8 for adsorption and desorption progress, respectively, which indicated that precise quantitative information on the kinetics of binding could be obtained expediently. The same kinetic analysis method was applied to determine the affinity binding constants of the other five groups of sensorgram data obtained from the above reproducibility experiment (the concentrations of injected LZM were all same, 0.5 µg/µL). Results of the kinetic analyses are summarized in Table 1. From Table 1, KD value for LZM was obtained as KD ) (3.73 ( 0.085) × 10-5 M. The RSD of KD was 2.3%, which implied that each sensorgram could provide enough information on the kinetic constant with acceptable precision. In view of the lower KD value, LZM showed a relatively high affinity to immobilized KM. Furthermore, the value of KD is in the range of 10-4-10-8 M, (41) Ruckenstein, E.; Zeng, X. Biotechnol. Bioeng. 1997, 56, 610-617. (42) Eddowes, M. J. Biosensors 1987, 3, 1-15.

Scheme 2. Procedure for Preparing KM Immobilized Affinity Packing and Blank Packing

Figure 3. Curve fits (solid lines) to frequency change data (dotted line) for the determination of kass and kdiss. (a) Frequency modulation (in Hz) due to specific adsorption of LZM (0.5 µg/µL) to the quartz crystal coated with KM; (b) frequency modulation (in Hz) due to specific desorption of LZM from the quartz crystal coated with KM. Table 1. Kinetic and Affinity Data from the QCM Analysis of the Interaction between LZM and Imobilized KM Ligand

1 2 3 4 5 6

concn (µg/µL)

kass (M-1 s-1)

χ2a

kdiss (× 103 s-1)

χ2b

KD (× 10-5 M)

0.5 0.5 0.5 0.5 0.5 0.5

122 116 130 125 128 133

0.87 0.82 0.89 0.98 0.94 1.02

4.63 4.32 4.75 4.81 4.78 4.84

1.8 1.7 2.0 2.2 1.9 2.3

3.80 3.72 3.65 3.85 3.73 3.63

which is suitable for an affinity ligand.43 Information obtained on these kinetic constants provided further evidence to assess whether KM is suitable for use as an affinity ligand in the HPAC purification process. HPAC Purification Using the Selected KM Ligand. The preparation procedure of the affinity packing using the selected KM ligand and its potential application for the separation and purification of LZM were discussed in detail. Salt-Induced Immobilization of KM on Epoxy-Activated Silica Matrix of HPAC. It has been proved that salt-induced immobilization of small ligands onto an epoxy-activated stationary phase is (43) Mohr, P.; Pommerening K. Affinity Chromatography: Practical and Theoretical Aspects; Marcel Dekker: New York, 1985.

an effective route.44-46 The increased coupling reactivity can be explained as a result of a salt-induced hydrophobic interaction between the affinity ligand and the surface of the stationary phase. The increase in concentration of the affinity ligand near the reactive epoxy groups leads to an increase in the rate of reaction between the nucleophilic groups such as amino and hydroxyl groups on the affinity ligand and the epoxide.46 In our experiment, efficient immobilization of the KM ligand onto epoxy-activated silica was also achieved in the presence of a high concentration of potassium phosphate (immobilization procedure illustrated in Scheme 2). The effect of potassium phosphate concentrations on the immobilization of KM was estimated by a series of measurements. Reactions were carried out with 100 mg of activated silica and 2 mg of KM, in different salt concentrations (0-3 M potassium phosphate) at pH 7.9. After a reaction time of 48 h, the mixtures were filtered and washed out. Unreacted KM was estimated from the absorbance at 205 nm of the wash filtrate. It was found that the extent of immobilization of KM was sensitive to the concentration of salt in the reaction. High salt concentration of potassium phosphate was clearly beneficial in the coupling of KM to epoxyactivated silica support. The optimum salt concentration was chosen as 2.5 M, and at this concentration, 96% of KM could be immobilized on the stationary phase. Specific Interaction of LZM with KM Immobilized Affinity Column. To study the specificity of the interaction of immobilized KM with LZM, other proteins were also injected into KM immobilized affinity column and eluted under the elution conditions studied as shown in Figure 4. It can be clearly seen from Figure 4 that these proteins have not been retained on the column. The low nonspecific binding with other proteins will be very useful for purification of LZM from a complicated sample using the KM immobilized affinity column. It is also interesting to mention that the above interaction screening results by HPAC are quite in accordance with the one by the QCM biosensor, as the same amino groups of the KM ligand were used for the immobilization either on the silica matrix for HPAC or on the sensor chip of QCM. The influence of pH on the interaction of LZM with immobilized KM was investigated. The optimum pH was chosen as 7.0 with maximal adsorption of LZM. The flow rate also exhibited (44) Bauer-Arnaz, K.; Naplitano, E. W.; Roberts, D. N.; Montali, J. A.; Hughes, B. R.; Schmidt, D. E., Jr. J. Chromatogr., A 1998, 803, 73-82. (45) Wheatley, J. B.; Lyttle, M. H.; Hocker, M. D.; Schmidt, D. E., Jr. J. Chromatogr., A 1996, 726, 77-90. (46) Wheatley, J. B.; Schmidt, D. E., Jr. J. Chromatogr., A 1999, 849, 1-12.


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Figure 4. Comparison of interaction of KM immobilized affinity column with various proteins (protein concentration 0.5 µg/µL). Table 2. Properties of KM Immobilized Silica Matrix KM bound on silica (mg/g of beads) binding capacity of LZM (mg/g beads) protein recovery of LZM (%)

Figure 5. Chromatographic behaviors of LZM (0.5 µg/µL) on blank column (a) and KM immobilized affinity column (b).

significant influence on the effective adsorption of LZM. The optimum flow rate was chosen as 0.5 mL/min, under which maximal adsorption could be achieved. Under the chosen conditions, the affinity column packed with KM immobilized silica beads showed specific adsorption for LZM, while there was no retention of LZM on the blank column at the same conditions as shown in Figure 5. The blank matrix without KM ligand showed no interaction with LZM. The results obtained confirmed that KM was indeed a valuable specific ligand for purification of LZM. Properties of KM Immobilized Silica Matrix. The amount of KM immobilized on silica was determined as described.26,47 A suitable amount of KM immobilized silica was well suspended in a mixture of water, ethanol, and 1-butanol in a volume ratio of 20:30:50 by ultrasonication. The absorbance of the solution at 205 nm was measured against hydrolyzed epoxy-activated silica as the reference. The standard curve can be made by quantitative additions of KM to the hydrolyzed silica and the absorbance measured under the same conditions. (47) Wu, X. J.; Liu, G. Q. Biomed. Chromatogr. 1996, 10, 228-232.

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25.8 4.0 99.4

Binding capacity determination was carried out by overloading the KM immobilized affinity column and by measuring the amount of adsorbed LZM after elution with 50 mM potassium phosphate buffer (pH 7.0) containing 0.5 M NaCl. The quantity of LZM in the eluant was determined spectrophotometrically at 280 nm by the standard curve method. Protein recovery of the affinity column was also determined. The properties of KM immobilized silica matrix are all summarized in Table 2. For the enzyme, the capacity was ∼4 mg of protein/g of the KM bound silica. Analytical HPAC for Determination of the Affinity Constant between LZM and Immobilized KM. To understand quantitatively the interaction of KM matrix and LZM, an experimental approach using analytical HPAC could be used.48-50 Frontal analysis was performed by continuously applying various concentrations of LZM to the KM immobilized affinity column at a flow rate of 0.5 mL/min. Solutions of different concentrations, [P]0, of mobile LZM were passed through the column and monitored by UV absorbance (λ ) 280 nm) until a plateau of maximum absorbance was observed (Figure 6). At this time, saturation was achieved and the eluate had the same concentration of mobile LZM as the initial applied solution. The variation of elution volume V h was plotted according to the equation51

KD 1 1 ) + [P]0 M M V h - V0 T T

(5)

where V h (mL) is elution volume at which the affinity matrix is (48) Yang, J.; Hage, D. S. J. Chromatogr., A 1996, 725, 273-285. (49) Hage, D. S. J. Chromatogr., B 2002, 768, 3-30. (50) Chaiken, I. M. J. Chromatogr. 1986, 376, 11-32.

Figure 6. Analytical HPAC experiment performed with LZM on the KM immobilized affinity column. The varying concentrations of LZM were as follow: (a) 4.31 × 10-6, (b) 8.62 × 10-6, (c) 1.72 × 10-5, and (d) 2.58 × 10-5 M.

Figure 7. Chromatogram of CEW on KM immobilized affinity column. Chromatographic conditions: CEW injected, 100 µL; flow rate, 0.5 mL/min; detection, UV 280 nm; buffer A, 50 mM potassium phosphate buffer containing 100 mM NaCl, pH 7.0; buffer B, 50 mM potassium phosphate buffer containing 1 M NaCl, pH 7.0; eluted condition, 0-35 min buffer A, 35-60 min 10% buffer B, 60-80 min buffer B, and 80-90 min buffer A.

half-saturated, V0 (mL) is void volume of the column, KD (M) is the dissociation constant of complex of the immobilized KM and LZM, MT (mol/g of silica) is the total amount of immobilized KM, and [P]0 (M) is the initial concentration of mobile LZM. From a plot of 1/(V h - V0) versus [P]0 (shown in the inset of Figure 6), KD can be determined as a ratio KD ) intercept/slope ) 3.58 × 10-5 M. The correlation coefficient (r) was equal to 0.995. The KD value obtained by analytical HPAC is in good agreement with the one obtained by the QCM biosensor. However, the QCM biosensor offers several advantages over conventional analytical HPAC to calculate the binding constants: the QCM biosensor requires low amounts of reagents without any special preparation, allows measurements in real time with high precision and rapidity, and provides conveniently further quantitative (51) Shai, Y.; Flashner, M.; Chaiken, I. M. Biochemistry 1987, 26, 669-675.

Figure 8. SDS-PAGE analysis of fractions obtained from the purification of LZM from CEW. Lanes: (a) molecular mass markers; (b) standard LZM; (c) CEW; (d) LZM purified from the KM immobilized affinity column.

information on the kinetic rate constants (kass and kdiss) other than equilibrium dissociation constants (KD). Separation and Characterization of Purified LZM. A rapid and efficient isolation of LZM from CEW solution was achieved with the KM immobilized affinity column and the profile of CEW proteins eluted from the column was shown in Figure 7. The central fraction of the elution peak was collected, dialyzed, concentrated, and then analyzed by SDS-PAGE. The electropherogram in Figure 8 has shown that eluted fractions have high purity since only one band was detected. Densitometric scanning of the gel lane containing the purified fraction indicated that the band accounted for 93% of the total protein. Enzyme activity of purified LZM and standard LZM at the same concentration (0.1 µg/µL) was measured and compared. The calculated specific activity values were 71 326 and 72 450 units/mg for purified LZM and standard LZM, respectively. From the results it can be concluded that nonspecific binding of proteins in CEW on the KM immobilized affinity column can be negligible during the process of affinity separation and higher enzyme activity remains after elution in our experimental conditions. Analytical Chemistry, Vol. 77, No. 13, July 1, 2005

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Reproducibility of the HPAC method was also investigated. RSD of elution peak areas were 1.8 (n ) 6) and 3.5% (n ) 3) for standard LZM solution (50-µg injection) and CEW solution (100µL injection), respectively. These results confirmed that our HPAC method had good reproducibility. The use of KM as a novel affinity ligand will make LZM purification much more convenient and affordable, opening up new avenues in the study and application of this important enzyme. CONCLUSIONS This is the first demonstration of a new screening strategy for novel affinity ligand selection using the QCM biosensor. Using this screening strategy, an aminoglycoside antibiotic drug, KM, was discovered for the first time to be a novel specific ligand for the purification of LZM. The KM immobilized affinity column proved to be quite useful for the separation and purification of LZM directly from CEW. The rapid screen analysis of interaction

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between immobilized drug ligand and various receptor proteins in solution by the QCM biosensor provides a promising strategy to discovery specific ligand for purifying some valuable proteins and its application will be widening soon. ACKNOWLEDGMENT We thank Prof. Yuanhua Shao and Dr. Ronghua Yang of Peking University for helpful discussions. We are also grateful to the National Natural Science Foundation of China (20405001 and 20335010) and Postdoctoral Science Foundation of China (2004035245) for financial support of this work.

Received for review January 6, 2005. Accepted April 19, 2005. AC050026E

Selection of Ligands for Affinity Chromatography Using Quartz Crystal

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