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Effect of hydrogel matrix on binding kinetics of protein–protein interactions on sensor surface
Analytica Chimica Acta 456 (2002) 201–208
Effect of hydrogel matrix on binding kinetics of protein–protein interactions on sensor surface Chi-Chun Fong a , Man-Sau Wong b , Wang-Fun Fong a , Mengsu Yang a,∗ a
Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong, PR China b The Open Laboratory of Chirotechnology, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, PR China Received 3 September 2001; received in revised form 19 December 2001; accepted 10 January 2002
Abstract Surface plasmon resonance (SPR) biosensor has become a standard technology for measuring kinetics of bimolecular interactions without the need for labeling. Sensor chips coated with a carboxymethylated dextran (CMD) hydrogel matrix are commonly used for immobilizing a protein binding partner in kinetic studies. The sensor chip provides a biocompatible surface with low non-specific binding, but it also presents some problems, such as steric effect and re-binding which may bias the kinetic measurement. In the present study, the effect of hydrogel matrix on protein–protein interaction was investigated. The insulin-like growth factors (IGFs) and their binding proteins were used as the model system. Kinetic parameters obtained with either of the binding partners immobilized on the matrix were compared to evaluate the effects of the matrix on the binding kinetics. The surface capacity and sensitivity of the hydrogel-modified sensor chip (CM5) and those of a sensor chip modified with a self-assembled monolayer (SAM) were measured and the performance of both chips was discussed. © 2002 Elsevier Science B.V. All rights reserved. Keywords: SPR biosensor; Sensor chip CM5; Hydrogel matrix; Self-assembled monolayer (SAM)
1. Introduction Surface plasmon resonance (SPR) biosensor has become a standard technique for kinetic measurement of molecular interactions [1], many researches also demonstrated that the determination of binding kinetics using SPR technique are useful for the development of immunological study [2–4]. During the SPR measurement, one partner of the interaction to be studied (called a ligand) is immobilized covalently to the sensor surface and the other partner is passed over the chip in solution (called an analyte). A three-dimensional ∗ Corresponding author. Tel.: +86-852-2788-7797; fax: +86-852-2788-7406. E-mail address: [email protected] (M. Yang).
hydrogel matrix is commonly coated on the sensor chip surface to provide a favorable environment for ligand immobilization (Fig. 1A). However, steric effects on analyte binding and limits of diffusion upon dissociation of the analyte (causing re-binding of the analyte) due to the hydrogel matrix usually lead to discrepancies between SPR-measured kinetic parameters and solution data [5]. Because biomolecular interactions usually involve two proteins of different sizes, and that steric effects and diffusion obviously depend on the size of the analyte, it is necessary to understand how the choice of the immobilized ligand may change the measured kinetics of a binding interaction. While many studies have been carried out, through both experimental design and theoretical modeling, aiming at reducing the biasing effects of the
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immobilization matrix on the measured kinetics were investigated.
2. Material and methods 2.1. Equipment and materials
Fig. 1. Diagrams showing the three-dimensional structure of sensor chip CMD (A) and two-dimensional structure of self-assembled monolayer (B).
hydrogel matrix on SPR measurements [6–9], it has not been studied in detail on how the selection of the ligand molecule may affect the outcome of the kinetic measurement. In this study, we used the SPR biosensor to measure the binding kinetics of the protein–protein interaction system involving insulin-like growth factor-I (IGF-I) and their binding proteins. It is well known that insulin-like growth factor (IGF) can form complexes with at least six different IGF binding proteins (IGFBPs) in the circulation and extracellular fluids [10]. Using the SPR biosensor, the association and dissociation profiles of the interaction between IGF-I and IGFBP-1 or IGFBP-3 were obtained when either IGF-I or the IGFBP was immobilized on sensor chips coated with a carboxymethylated dextran (CMD) hydrogel or a self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (11-MUA). The kinetic parameters for each pair of interaction were determined from the profiles by using global fitting with various binding models. The effects of ligand
The SPR biosensor instrument (BIAcoreTM X), sensor chips coated with CMD hydrogel (CM5 research grade), HBS buffer (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.05% P20, pH 7.4), and the amine coupling kit containing N-hydroxysuccinimide (NHS), N-ethyl-N -(3-diethyl-aminopropyl) carbodiimide (EDC), and ethanolamine hydrochloride were acquired from Pharmacia Biosensor (Uppsala, Sweden). 11-MUA was purchased from Aldrich (Milwaukee, WI, USA). Recombinant IGF-I was purchased from Gropep (Adelaide, Australia), recombinant human IGFBP-1 and IGFBP-3 expressed by Chinese Hamster Ovary cells were from Upstate Biotechnology Inc. (Lake Placid, NY, USA). The IGFBPs were purified by reverse phase HPLC and the purity of the IGFBPs is over 90% as demonstrated by SDS–PAGE and amino acid analysis. 2.2. Immobilization of IGF-I and IGFBPs on sensor chip surfaces The CMD surface was activated by injecting 35 l of the 0.1 M EDC/0.1 M NHS (1:1) mixture. A 35 l solution of each ligand (10 g/ml in 10 mM sodium acetate, pH 4.8) was then injected over the activated surface, followed by the injection of 35 l of 1 M ethanolamine to deactivate remaining active carboxyl groups. The sensor chip was then washed with 12 l of 100 mM HCl to remove remaining non-covalently bound ligands. All sensor chips were washed overnight before the day of experiment with HBS buffer containing 10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.05% surfactant P20, pH 7.4 to ensure a stable baseline. The immobilization procedures were carried out at 25 ◦ C and at a constant flow rate of 5 l/min HBS buffer. In another set of experiments, a SAM of 11-MUA was prepared on the gold surface of the SPR sensor chip and compared to CM5 chip for ligand immobilization and analyte binding. The commercial CM5
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chips, which is coated with CMD, was subjected to a mixture of 70% H2 SO4 and 30% H2 O2 and heated to about 90 ◦ C for 5 min to remove the CMD coating. The surface was then rinsed with deionized water, methanol and dried for 1 h at 150 ◦ C. Immediately before use, the gold chip was cleaned in argon plasma cleaner for 10 min (Model PDC 3 X G) [11]. A monolayer of MUA (100 mM in 1:1 ethanol and deionized water mixture) was formed on the gold surface by placing the chip on a MUA solution for 2 h, and rinsed with ethanol and deionized water. The integrity of the monolayer on gold surface was verified using cyclic voltammetry. Cyclic voltammergram was recorded using a potentiostat/galvanostat (Electrochemical Analysis System model 273A) in 10 mM K3 Fe(CN)6 at ambient temperature. The two-dimensional structure of SAM was shown in Fig. 1B. After drying, the chip was docked into the instrument, followed by equilibration with HBS buffer. IGF-I was immobilized on the MUA-modified surface using the same method as that on CM5 surface. In order to minimize the effect of non-specific binding on the control channel, the control surface was treated with EDC/NHS and deactivated by ethanolamine hydrochloride for several cycles in order to block the carboxylic acid groups. The binding experiments between different concentrations of IGFBP-1 and the immobilized IGF-I were carried out as described above for the CM5 surface.
effect of different immobilization partner on binding kinetics, similar sets of experiments were carried out using IGFBP-1 or IGFBP-3 as the immobilized ligand and IGF-I as analyte in solution. To verify if mass transport presents a limit on the binding experiments, the binding interaction between IGF-I and immobilized IGFBP-1 was performed using different flow rates, ranged from 4 to 20 l/min. It was observed that the binding interaction is not mass transport-limited because the kinetic parameters derived are independent of the flow rate [7].
2.3. Real-time binding measurement by the SPR biosensor
A + BAB1
Thirty-two microliters of different concentrations of either IGFBP-1 or -3 was injected over the sensor surface with immobilized IGF-I, followed by 10 min wash with HBS buffer. The sample injection period reflects the association process and the washing period reflects the dissociation process. The sensor surface was then regenerated by washing with 12 l of 100 mM HCl. All binding experiments were carried out at 25 ◦ C with a constant flow rate of 8 l/min HBS buffer. To correct for non-specific binding and bulk refractive index change, a blank channel without IGF-I was used and run simultaneously for each experiment. The sensorgram for all binding interactions were recorded in real time and were analyzed after subtracting the sensorgram from the blank channel. In order to elucidate the
A + BAB2
2.4. Kinetic analysis The sensorgrams were analyzed using BIAevaluationTM software version 3.1. Several binding models were used to globally fit the IGF–IGFBP interaction data using either non-linear regression or numerical integration methods globally. The following models have used in data fitting. 1:1 simple Langmuir binding model ka
A + BAB kd
1:1 binding with mass transport model kt
ka
kt
kd
A0 A + BAB Two-site binding model ka1
kd1 ka2
kd2
Two-site binding with mass transport model kt
A0 A kt
ka1
A + BAB1 kd1 ka2
A + BAB2 kd2
where A represents the analyte in solution, B represents the ligand immobilized on sensor surface, AB is the complexes formed by A and B, kt is the rate of
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mass transfer, ka and kd are the derived association rate constant and dissociation rate constant, respectively. To find the optimal fit for each binding interaction, different models were fitted to the data both globally (fitting all concentrations simultaneously). Both the association and dissociation phases were fitted simultaneously. The goodness of fitting for each model was judged by the reduced χ 2 values and randomness of
residue distribution. The reported kinetic constants ka and kd were derived from the simplest and the most appreciate model. The equilibrium affinity constant (KA ) was calculated based on the ratio of ka /kd . The kinetic and equilibrium constants of each pair of binding interaction were expressed as mean ± S.E.M. and estimated based on two to three independent experiments.
Fig. 2. The overlay sensorgram of IGF-I and IGFBP-1 interactions when either IGF-I (A) or IGFBP-1 (B) was immobilized on sensor chip CM5 surface after subtracted from the control surface and the residual plot of fitting the interaction between IGFBP-1 and immobilized IGF-I using 1:1 Langmuir binding model (C).
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3. Results and discussion IGF-I and -II are known to form complexes with at least six different IGFBPs in the circulation and in the extracellular fluid. These binding proteins are important in modulating IGFs actions, facilitating storage of IGFs in the extracellular matrices, and exerting IGF-independent effects [12]. The characterization of the binding kinetics between each IGFBP and IGFs
205
will provide direct evidence to delineate the difference in the physiochemical nature of these binding proteins [4]. Earlier studies have employed the conventional competitive binding assay to characterize the relative affinities of different IGFBPs to both IGF-I and -II by using either radio labeled IGF-I or -II [13,14]. Recent advances in biosensor technology allow the characterization of binding interaction between biomolecules in real time and the determination of binding kinet-
Fig. 3. The overlay sensorgram of IGF-I and IGFBP-3 interactions when either IGF-I (A) or IGFBP-1 (B) was immobilized on sensor chip CM5 surface after subtracted from the control surface and the residual plot of fitting the interaction between IGFBP-3 and immobilized IGF-I using 1:1 Langmuir binding model (C).
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ics and affinities without radio labeling or biochemical tagging [15]. There are a number of experimental factors that users must consider when setting up a biosensor experiment for kinetic analysis [16,17]. In the present study, SPR binding experiments were carried out using purified proteins and reference cell to minimize variations caused by analyte inhomogeneity, non-specific binding, bulk refractive index changes, and matrix and instrument drift [17]. The binding interactions of IGF-I and IGFBP-1 and IGFBP-3 with either of the binding partners immobilized were measured and recorded as sensorgrams by the SPR biosensor. The binding profiles of IGF-I with IGFBP-1 and IGFBP-3 were shown in Figs. 2 and 3, respectively. Different binding models were used to fit the sensorgram as describe in Section 2, and the simple Langmuir binding model was sufficient to fit the data globally as judged by reduced χ 2 values Table 1) and randomness of residue distribution (Figs. 2C and 3C). The curve fitting was no significantly improved the reduced χ 2 values using other more complex model. The kinetic parameters for each binding pair are summarized in Table 1. Comparing to the binding profiles and the derived kinetics for different pairs of IGFBP and IGF interaction, it was obvious that the binding kinetics depend on the ligand immobilized. For the interaction with IGFBP-1 or IGFBP-3, ka values were 50- or 30-fold lower when IGF-I was immobilized than when IGFBP was immobilized; whereas kd values did not have significant change. As a result, the KA values were 80 and 16 times lower when IGF-I was immobilized, respectively. This could be due to the different sizes and molecular weights of the binding proteins and growth factors. IGFBPs are generally five to six times greater in molecular weight than IGF-I. When the smaller lig-
Fig. 4. Surface coverage of bare gold surface after stripping (A) and self-assembled monolayer presenting 11-mercaptoundecanoic acid (B) determined by cyclic voltammetry. The surface and the surface coverage was characterized by cyclic voltammetry, where a decrease in the peak current and an increase in the peak separation of a redox couple (e.g. [Fe(CN)6 ]4−/3− ) were taken as a measure of the insulating surface area.
and (IGF-I) was immobilized on the hydrogel surface, the immobilization matrix might pose steric hindrance for the larger IGFBP molecules to bind with the immobilized IGF molecule. As a result, association rate was decreased. On the other hand, the diffusion of smaller molecule (IGF-I) is fast, which could enhance the association rate when the larger IGFBP molecules were immobilized. Over the last decades, there were many studies of SAM as an alternative immobilization surface for various binding ligand [18–20]. In our study, the CMD hydrogel matrix was stripped off to generate a bare gold surface and self assembly of the MUA onto the gold surface was also achieved for further study. As shown in Fig. 4, the formation of the MUA monolayer completely blocked the contact of
Table 1 Kinetic parameters of the binding between IGF-I and IGFBP-1 or IGFBP-3 on hydrogel matrix (sensor chip CM5) and on self-assembled monolayer (SAM) Interaction matrix
Analyte (binding partner)
Ligand (immobilized)
Association rate constant (×105 M−1 s−1 )
CM5 CM5 CM5 CM5 SAM
IGFBP-1 IGF-I IGFBP-3 IGF-I IGFBP-1
IGF-I IGFBP-1 IGF-I IGFBP-3 IGF-I
0.16 7.83 0.73 21.6 3.15
± ± ± ± ±
0.01 0.06 0.02 0.56 0.17
Dissociation rate constant (×10−4 s−1 ) 8.12 4.81 4.04 7.26 15.9
± ± ± ± ±
0.12 0.17 0.75 0.19 0.44
Equilibrium constant (KA ) (×108 M−1 ) 0.20 16.3 1.81 29.8 1.98
± ± ± ± ±
0.01 0.59 0.34 1.09 0.12
χ2
1.22 1.51 0.96 0.87 0.36
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Fig. 5. The sensorgram of interaction between IGFBP-1 and IGF-I immobilized on a self-assembled monolayer (A) and the residual plot of fitting the interaction using 1:1 Langmuir binding model (B).
K3 Fe(CN)6 with the gold surface. IGF-I was subsequently immobilized through the same method as that for immobilization on CMD surface. The IGF-I immobilization densities on both SAM and CMD surfaces were controlled to similar (0.25 and 0.31 ng/mm2 , respectively). This created a similar surface binding capacity for the comparison between SAM and CMD surfaces. The binding sensorgram of IGFBP-1 and IGF-I immobilized on SAM were recorded (Fig. 5A). The binding profile was also fitted by the same model used for the interaction carried out on CMD surface, e.g. simple Langmuir binding model. The derived kinetics were listed in Table 1, and the residual plot distribution was shown in Fig. 5B. We found that the ka and kd values were 16- and 2-fold higher and the equilibrium affinity was about 8-fold higher on SAM surface than those on the CMD surface. This clearly indicated that some of the steric effects and limits on analyte
diffusion presented by the CMD hydrogel matrix could be reduced by the SAM monolayer.
4. Conclusion CMD surfaces are commonly used for kinetic study of macromolecular interaction. But the CMD hydrogel matrix may bias the kinetic measurement due to steric effect. In the present study, we performed a very careful experimental design and data fitting to characterize the binding kinetic of IGF-I and IGFBP-1 and IGFBP-3 interactions. As the result, different immobilization partner could differ the binding profiles and kinetic parameter, and SAM monolayer could reduce some of the steric effects presented by the CMD hydrogel matrix. Our study also demonstrated that SPR-derived kinetic rate constants couldn’t be considered as absolutely “true” values. However, it
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is a powerful technique to perform comparative analysis, analyte screening under the same experimental condition. Acknowledgements The study was supported by the Research Grant Council, Hong Kong (CityU Project No. 9040388) and Area of Strategic Development Grant A008 from the Hong Kong Polytechnic University. References [1] R.L. Rich, D.G. Myszka, Curr. Opin. Biotechnol. 11 (2000) 54–61. [2] F. Deckert, F. Legay, J. Pharm. Biomed. Anal. 23 (2000) 403–412. [3] S. Sonezaki, S. Yagi, E. Ogawa, A. Kondo, J. Immunol. Methods 238 (2000) 99–106. [4] M.S. Wong, C.C. Fong, M. Yang, Biochim. Biophys. Acta 1432 (1999) 293–301. [5] S.R. Haseley, P. Talaga, J.P. Kamerling, J.F. Vliegenthart, Anal. Biochem. 15 (1999) 203–210.
[6] N.J. de Mol, E. Plomp, M.J. Fischer, R. Ruijtenbeek, Anal. Biochem. 279 (2000) 61–70. [7] L.H. Christensen, Anal. Biochem. 249 (1997) 153–164. [8] R.L. Glaser, Anal. Biochem. 213 (1993) 152–161. [9] P. Schuck, Biophys. J. 70 (1996) 1230–1249. [10] J.I. Jones, D.R. Clemmons, Endocr. Rev. 16 (1995) 3–34. [11] E. Kalb, S. Frey, L.K. Tamm, Biochim. Biophys. Acta 1103 (1992) 307–316. [12] S. Rajaram, D.J. Baylink, S. Mohan, Endocr. Rev. 18 (1997) 804–831. [13] Y. Oh, H.L. Muller, D.-Y. Lee, P.J. Fielder, R.G. Rosenfeld, Endocrinology 132 (1993) 1337–1344. [14] D.R. Clemmons, M.L. Dehoff, W.H. Busby, M.L. Bayne, M.A. Cascieri, Endocrinology 131 (1992) 890–895. [15] U. Jonsson, L. Fagerstam, B. Ivarsson, B. Johnsson, R. Karlsson, K. Lundh, S. Lofas, B. Persson, H. Roos, I. Ronnberg, Biotechniques 11 (1991) 620. [16] R.L. Rich, D.G. Myszka, Curr. Opin. Biotechnol. 11 (2000) 54–61. [17] D.G. Myszka, Curr. Opin. Biotechnol. 8 (1997) 50–57. [18] J. Lahiri, L. Isaacs, J. Tien, G.M. Whitesides, Anal. Chem. 71 (1999) 777–790. [19] J. Rao, L. Yan, J. Lahiri, G.M. Whitesides, R.M. Weis, H.S. Warren, Chem. Biol. 6 (1999) 353–359. [20] N. Horan, L. Yan, H. Isobe, G.M. Whitesides, D. Kahne, PNAS 96 (1999) 11782–11786.
Effect of hydrogel matrix on binding kinetics of protein–protein interactions on sensor surface Chi-Chun Fong a , Man-Sau Wong b , Wang-Fun Fong a , Mengsu Yang a,∗ a
Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong, PR China b The Open Laboratory of Chirotechnology, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, PR China Received 3 September 2001; received in revised form 19 December 2001; accepted 10 January 2002
Abstract Surface plasmon resonance (SPR) biosensor has become a standard technology for measuring kinetics of bimolecular interactions without the need for labeling. Sensor chips coated with a carboxymethylated dextran (CMD) hydrogel matrix are commonly used for immobilizing a protein binding partner in kinetic studies. The sensor chip provides a biocompatible surface with low non-specific binding, but it also presents some problems, such as steric effect and re-binding which may bias the kinetic measurement. In the present study, the effect of hydrogel matrix on protein–protein interaction was investigated. The insulin-like growth factors (IGFs) and their binding proteins were used as the model system. Kinetic parameters obtained with either of the binding partners immobilized on the matrix were compared to evaluate the effects of the matrix on the binding kinetics. The surface capacity and sensitivity of the hydrogel-modified sensor chip (CM5) and those of a sensor chip modified with a self-assembled monolayer (SAM) were measured and the performance of both chips was discussed. © 2002 Elsevier Science B.V. All rights reserved. Keywords: SPR biosensor; Sensor chip CM5; Hydrogel matrix; Self-assembled monolayer (SAM)
1. Introduction Surface plasmon resonance (SPR) biosensor has become a standard technique for kinetic measurement of molecular interactions [1], many researches also demonstrated that the determination of binding kinetics using SPR technique are useful for the development of immunological study [2–4]. During the SPR measurement, one partner of the interaction to be studied (called a ligand) is immobilized covalently to the sensor surface and the other partner is passed over the chip in solution (called an analyte). A three-dimensional ∗ Corresponding author. Tel.: +86-852-2788-7797; fax: +86-852-2788-7406. E-mail address: [email protected] (M. Yang).
hydrogel matrix is commonly coated on the sensor chip surface to provide a favorable environment for ligand immobilization (Fig. 1A). However, steric effects on analyte binding and limits of diffusion upon dissociation of the analyte (causing re-binding of the analyte) due to the hydrogel matrix usually lead to discrepancies between SPR-measured kinetic parameters and solution data [5]. Because biomolecular interactions usually involve two proteins of different sizes, and that steric effects and diffusion obviously depend on the size of the analyte, it is necessary to understand how the choice of the immobilized ligand may change the measured kinetics of a binding interaction. While many studies have been carried out, through both experimental design and theoretical modeling, aiming at reducing the biasing effects of the
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immobilization matrix on the measured kinetics were investigated.
2. Material and methods 2.1. Equipment and materials
Fig. 1. Diagrams showing the three-dimensional structure of sensor chip CMD (A) and two-dimensional structure of self-assembled monolayer (B).
hydrogel matrix on SPR measurements [6–9], it has not been studied in detail on how the selection of the ligand molecule may affect the outcome of the kinetic measurement. In this study, we used the SPR biosensor to measure the binding kinetics of the protein–protein interaction system involving insulin-like growth factor-I (IGF-I) and their binding proteins. It is well known that insulin-like growth factor (IGF) can form complexes with at least six different IGF binding proteins (IGFBPs) in the circulation and extracellular fluids [10]. Using the SPR biosensor, the association and dissociation profiles of the interaction between IGF-I and IGFBP-1 or IGFBP-3 were obtained when either IGF-I or the IGFBP was immobilized on sensor chips coated with a carboxymethylated dextran (CMD) hydrogel or a self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (11-MUA). The kinetic parameters for each pair of interaction were determined from the profiles by using global fitting with various binding models. The effects of ligand
The SPR biosensor instrument (BIAcoreTM X), sensor chips coated with CMD hydrogel (CM5 research grade), HBS buffer (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.05% P20, pH 7.4), and the amine coupling kit containing N-hydroxysuccinimide (NHS), N-ethyl-N -(3-diethyl-aminopropyl) carbodiimide (EDC), and ethanolamine hydrochloride were acquired from Pharmacia Biosensor (Uppsala, Sweden). 11-MUA was purchased from Aldrich (Milwaukee, WI, USA). Recombinant IGF-I was purchased from Gropep (Adelaide, Australia), recombinant human IGFBP-1 and IGFBP-3 expressed by Chinese Hamster Ovary cells were from Upstate Biotechnology Inc. (Lake Placid, NY, USA). The IGFBPs were purified by reverse phase HPLC and the purity of the IGFBPs is over 90% as demonstrated by SDS–PAGE and amino acid analysis. 2.2. Immobilization of IGF-I and IGFBPs on sensor chip surfaces The CMD surface was activated by injecting 35 l of the 0.1 M EDC/0.1 M NHS (1:1) mixture. A 35 l solution of each ligand (10 g/ml in 10 mM sodium acetate, pH 4.8) was then injected over the activated surface, followed by the injection of 35 l of 1 M ethanolamine to deactivate remaining active carboxyl groups. The sensor chip was then washed with 12 l of 100 mM HCl to remove remaining non-covalently bound ligands. All sensor chips were washed overnight before the day of experiment with HBS buffer containing 10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.05% surfactant P20, pH 7.4 to ensure a stable baseline. The immobilization procedures were carried out at 25 ◦ C and at a constant flow rate of 5 l/min HBS buffer. In another set of experiments, a SAM of 11-MUA was prepared on the gold surface of the SPR sensor chip and compared to CM5 chip for ligand immobilization and analyte binding. The commercial CM5
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chips, which is coated with CMD, was subjected to a mixture of 70% H2 SO4 and 30% H2 O2 and heated to about 90 ◦ C for 5 min to remove the CMD coating. The surface was then rinsed with deionized water, methanol and dried for 1 h at 150 ◦ C. Immediately before use, the gold chip was cleaned in argon plasma cleaner for 10 min (Model PDC 3 X G) [11]. A monolayer of MUA (100 mM in 1:1 ethanol and deionized water mixture) was formed on the gold surface by placing the chip on a MUA solution for 2 h, and rinsed with ethanol and deionized water. The integrity of the monolayer on gold surface was verified using cyclic voltammetry. Cyclic voltammergram was recorded using a potentiostat/galvanostat (Electrochemical Analysis System model 273A) in 10 mM K3 Fe(CN)6 at ambient temperature. The two-dimensional structure of SAM was shown in Fig. 1B. After drying, the chip was docked into the instrument, followed by equilibration with HBS buffer. IGF-I was immobilized on the MUA-modified surface using the same method as that on CM5 surface. In order to minimize the effect of non-specific binding on the control channel, the control surface was treated with EDC/NHS and deactivated by ethanolamine hydrochloride for several cycles in order to block the carboxylic acid groups. The binding experiments between different concentrations of IGFBP-1 and the immobilized IGF-I were carried out as described above for the CM5 surface.
effect of different immobilization partner on binding kinetics, similar sets of experiments were carried out using IGFBP-1 or IGFBP-3 as the immobilized ligand and IGF-I as analyte in solution. To verify if mass transport presents a limit on the binding experiments, the binding interaction between IGF-I and immobilized IGFBP-1 was performed using different flow rates, ranged from 4 to 20 l/min. It was observed that the binding interaction is not mass transport-limited because the kinetic parameters derived are independent of the flow rate [7].
2.3. Real-time binding measurement by the SPR biosensor
A + BAB1
Thirty-two microliters of different concentrations of either IGFBP-1 or -3 was injected over the sensor surface with immobilized IGF-I, followed by 10 min wash with HBS buffer. The sample injection period reflects the association process and the washing period reflects the dissociation process. The sensor surface was then regenerated by washing with 12 l of 100 mM HCl. All binding experiments were carried out at 25 ◦ C with a constant flow rate of 8 l/min HBS buffer. To correct for non-specific binding and bulk refractive index change, a blank channel without IGF-I was used and run simultaneously for each experiment. The sensorgram for all binding interactions were recorded in real time and were analyzed after subtracting the sensorgram from the blank channel. In order to elucidate the
A + BAB2
2.4. Kinetic analysis The sensorgrams were analyzed using BIAevaluationTM software version 3.1. Several binding models were used to globally fit the IGF–IGFBP interaction data using either non-linear regression or numerical integration methods globally. The following models have used in data fitting. 1:1 simple Langmuir binding model ka
A + BAB kd
1:1 binding with mass transport model kt
ka
kt
kd
A0 A + BAB Two-site binding model ka1
kd1 ka2
kd2
Two-site binding with mass transport model kt
A0 A kt
ka1
A + BAB1 kd1 ka2
A + BAB2 kd2
where A represents the analyte in solution, B represents the ligand immobilized on sensor surface, AB is the complexes formed by A and B, kt is the rate of
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mass transfer, ka and kd are the derived association rate constant and dissociation rate constant, respectively. To find the optimal fit for each binding interaction, different models were fitted to the data both globally (fitting all concentrations simultaneously). Both the association and dissociation phases were fitted simultaneously. The goodness of fitting for each model was judged by the reduced χ 2 values and randomness of
residue distribution. The reported kinetic constants ka and kd were derived from the simplest and the most appreciate model. The equilibrium affinity constant (KA ) was calculated based on the ratio of ka /kd . The kinetic and equilibrium constants of each pair of binding interaction were expressed as mean ± S.E.M. and estimated based on two to three independent experiments.
Fig. 2. The overlay sensorgram of IGF-I and IGFBP-1 interactions when either IGF-I (A) or IGFBP-1 (B) was immobilized on sensor chip CM5 surface after subtracted from the control surface and the residual plot of fitting the interaction between IGFBP-1 and immobilized IGF-I using 1:1 Langmuir binding model (C).
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3. Results and discussion IGF-I and -II are known to form complexes with at least six different IGFBPs in the circulation and in the extracellular fluid. These binding proteins are important in modulating IGFs actions, facilitating storage of IGFs in the extracellular matrices, and exerting IGF-independent effects [12]. The characterization of the binding kinetics between each IGFBP and IGFs
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will provide direct evidence to delineate the difference in the physiochemical nature of these binding proteins [4]. Earlier studies have employed the conventional competitive binding assay to characterize the relative affinities of different IGFBPs to both IGF-I and -II by using either radio labeled IGF-I or -II [13,14]. Recent advances in biosensor technology allow the characterization of binding interaction between biomolecules in real time and the determination of binding kinet-
Fig. 3. The overlay sensorgram of IGF-I and IGFBP-3 interactions when either IGF-I (A) or IGFBP-1 (B) was immobilized on sensor chip CM5 surface after subtracted from the control surface and the residual plot of fitting the interaction between IGFBP-3 and immobilized IGF-I using 1:1 Langmuir binding model (C).
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ics and affinities without radio labeling or biochemical tagging [15]. There are a number of experimental factors that users must consider when setting up a biosensor experiment for kinetic analysis [16,17]. In the present study, SPR binding experiments were carried out using purified proteins and reference cell to minimize variations caused by analyte inhomogeneity, non-specific binding, bulk refractive index changes, and matrix and instrument drift [17]. The binding interactions of IGF-I and IGFBP-1 and IGFBP-3 with either of the binding partners immobilized were measured and recorded as sensorgrams by the SPR biosensor. The binding profiles of IGF-I with IGFBP-1 and IGFBP-3 were shown in Figs. 2 and 3, respectively. Different binding models were used to fit the sensorgram as describe in Section 2, and the simple Langmuir binding model was sufficient to fit the data globally as judged by reduced χ 2 values Table 1) and randomness of residue distribution (Figs. 2C and 3C). The curve fitting was no significantly improved the reduced χ 2 values using other more complex model. The kinetic parameters for each binding pair are summarized in Table 1. Comparing to the binding profiles and the derived kinetics for different pairs of IGFBP and IGF interaction, it was obvious that the binding kinetics depend on the ligand immobilized. For the interaction with IGFBP-1 or IGFBP-3, ka values were 50- or 30-fold lower when IGF-I was immobilized than when IGFBP was immobilized; whereas kd values did not have significant change. As a result, the KA values were 80 and 16 times lower when IGF-I was immobilized, respectively. This could be due to the different sizes and molecular weights of the binding proteins and growth factors. IGFBPs are generally five to six times greater in molecular weight than IGF-I. When the smaller lig-
Fig. 4. Surface coverage of bare gold surface after stripping (A) and self-assembled monolayer presenting 11-mercaptoundecanoic acid (B) determined by cyclic voltammetry. The surface and the surface coverage was characterized by cyclic voltammetry, where a decrease in the peak current and an increase in the peak separation of a redox couple (e.g. [Fe(CN)6 ]4−/3− ) were taken as a measure of the insulating surface area.
and (IGF-I) was immobilized on the hydrogel surface, the immobilization matrix might pose steric hindrance for the larger IGFBP molecules to bind with the immobilized IGF molecule. As a result, association rate was decreased. On the other hand, the diffusion of smaller molecule (IGF-I) is fast, which could enhance the association rate when the larger IGFBP molecules were immobilized. Over the last decades, there were many studies of SAM as an alternative immobilization surface for various binding ligand [18–20]. In our study, the CMD hydrogel matrix was stripped off to generate a bare gold surface and self assembly of the MUA onto the gold surface was also achieved for further study. As shown in Fig. 4, the formation of the MUA monolayer completely blocked the contact of
Table 1 Kinetic parameters of the binding between IGF-I and IGFBP-1 or IGFBP-3 on hydrogel matrix (sensor chip CM5) and on self-assembled monolayer (SAM) Interaction matrix
Analyte (binding partner)
Ligand (immobilized)
Association rate constant (×105 M−1 s−1 )
CM5 CM5 CM5 CM5 SAM
IGFBP-1 IGF-I IGFBP-3 IGF-I IGFBP-1
IGF-I IGFBP-1 IGF-I IGFBP-3 IGF-I
0.16 7.83 0.73 21.6 3.15
± ± ± ± ±
0.01 0.06 0.02 0.56 0.17
Dissociation rate constant (×10−4 s−1 ) 8.12 4.81 4.04 7.26 15.9
± ± ± ± ±
0.12 0.17 0.75 0.19 0.44
Equilibrium constant (KA ) (×108 M−1 ) 0.20 16.3 1.81 29.8 1.98
± ± ± ± ±
0.01 0.59 0.34 1.09 0.12
χ2
1.22 1.51 0.96 0.87 0.36
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Fig. 5. The sensorgram of interaction between IGFBP-1 and IGF-I immobilized on a self-assembled monolayer (A) and the residual plot of fitting the interaction using 1:1 Langmuir binding model (B).
K3 Fe(CN)6 with the gold surface. IGF-I was subsequently immobilized through the same method as that for immobilization on CMD surface. The IGF-I immobilization densities on both SAM and CMD surfaces were controlled to similar (0.25 and 0.31 ng/mm2 , respectively). This created a similar surface binding capacity for the comparison between SAM and CMD surfaces. The binding sensorgram of IGFBP-1 and IGF-I immobilized on SAM were recorded (Fig. 5A). The binding profile was also fitted by the same model used for the interaction carried out on CMD surface, e.g. simple Langmuir binding model. The derived kinetics were listed in Table 1, and the residual plot distribution was shown in Fig. 5B. We found that the ka and kd values were 16- and 2-fold higher and the equilibrium affinity was about 8-fold higher on SAM surface than those on the CMD surface. This clearly indicated that some of the steric effects and limits on analyte
diffusion presented by the CMD hydrogel matrix could be reduced by the SAM monolayer.
4. Conclusion CMD surfaces are commonly used for kinetic study of macromolecular interaction. But the CMD hydrogel matrix may bias the kinetic measurement due to steric effect. In the present study, we performed a very careful experimental design and data fitting to characterize the binding kinetic of IGF-I and IGFBP-1 and IGFBP-3 interactions. As the result, different immobilization partner could differ the binding profiles and kinetic parameter, and SAM monolayer could reduce some of the steric effects presented by the CMD hydrogel matrix. Our study also demonstrated that SPR-derived kinetic rate constants couldn’t be considered as absolutely “true” values. However, it
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