Glucose-Grafted Gum Arabic Modified Magnetic Nanoparticles

Jun 22, 2007 - In comparison to GAMNP, the glucose-grafted GAMNP (G-GAMNP) significantly showed specific interaction with concanavalin A (Con A) with ...
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Chem. Mater. 2007, 19, 3667-3672

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Glucose-Grafted Gum Arabic Modified Magnetic Nanoparticles: Preparation and Specific Interaction with Concanavalin A Shashwat S. Banerjee and Dong-Hwang Chen* Department of Chemical Engineering, National Cheng Kung UniVersity, Tainan, Taiwan 701, Republic of China ReceiVed February 16, 2007. ReVised Manuscript ReceiVed April 13, 2007

Novel magnetic nanoparticles bearing glucose-grafted gum arabic were successfully synthesized by coupling maltose via reductive amination on gum arabic modified magnetic nanoparticles (GAMNP). By the analyses of FTIR and Raman spectra, the grafting of glucose on GAMNP was recognized. From the concentration change of maltose in solution, the amount of maltose coupled to GAMNP was estimated as 0.13 mmol/g. In comparison to GAMNP, the glucose-grafted GAMNP (G-GAMNP) significantly showed specific interaction with concanavalin A (Con A) with an association constant of about 1.26 × 109 M-1. Less particle aggregation was observed when the particle/Con A ratio was high, whereas a particle might be bound by more Con A molecules and hence led to the aggregation when the particle/ Con A ratio was low. The amount of Con A adsorbed was significantly dependent on pH, and the maximum adsorption capacity appeared around pH 7. The decrease in adsorption amount in lower and higher pH regions might be due to the release of ions from Con A and to the denaturation of Con A, respectively. In addition, it was shown that Con A molecules were bound onto G-GAMNP via the hydrogen bonds between the -OH groups of glucose and the -NH groups or oxygen atoms of asparagine in Con A.

Introduction In the past 2 decades, our understanding of molecular biology, genomics, and nanotechnology has expanded explosively. The inevitable intersection of these three disciplines has set in motion the development of an emerging research area, bionanotechnology or nanobiomedical technology, which offers exciting and abundant opportunities for discovering new processes, phenomena, and science. In particular, the advances in the synthesis and characterization of nanoscale materials allow scientists to understand and control the interactions between nanomaterials (e.g., nanowires, nanofibers, nanoparticles, nanobelts or nanoribbons, and nanotubes) and biological entities (e.g., nucleic acid, proteins, or cells) at molecular or cellular levels.1 Magnetic nanoparticles offer some attractive possibilities in biomedicine. First, they have controllable sizes ranging from a few nanometers up to tens of nanometers, which places them at dimensions that are smaller than or comparable to those of a cell (10-100 µm), a virus (20-450 nm), a protein (5-50 nm), or a gene (2 nm wide and 10-100 nm long). This means that they can “get close” to a biological entity of interest. Indeed, they can be coated with biological molecules to make them interact with or bind to a biological entity, thereby providing a controllable means of “tagging” or addressing it.2 The surfaces of these particles are generally modified through the creation of few atomic layers of organic polymer or inorganic metallic (e.g., gold) or oxide surfaces (e.g., silica or alumina), to make them biocompatible and suitable for further functionalization by the attachment of * Corresponding author. Phone: 886-6-2757575, ext.62680. Fax: 886-62344496. E-mail: [email protected].

(1) Gu, H.; Xu, K.; Xu, C.; Xu, B. Chem. Commun. 2006, 941. (2) Pankhurst, Q. A.; Connolly, J.; Jones, S. K.; Dobson, J. J. Phys. D: Appl. Phys. 2003, 36, R167.

various bioactive molecules.3 Second, the nanoparticles are magnetic, which means that they obey Coulomb’s law, and can be manipulated by an external magnetic field gradient. The magnetic nanoparticles with suitable surface characteristics have a high potential for use in a lot of in vitro and in vivo applications. As well-documented in the literature, sugars play a critical role in the process of cell recognition.4,5 Carbohydrate-grafted polymers have been used to recognize some proteins on the basis of specific protein-saccharide interactions.5-10 Recently, diverse sugar (glucose, mannose, galactose, and lactose)-capped poly(ethylene glycol)-b-poly(D,L-lactide) block copolymers are synthesized, and their micelles of core-shell structure are obtained from water media. With the probe sugars on their surface, the micelles have a specific binding ability to target cells. Therefore, this kind of polymeric micelle system would be a cell-targeting drug carrier. However, an A-B type of block copolymer end-capped with only one sugar residue is less effective than a carbohydrategrafted polymer in mimicking the multivalent carbohydrateprotein interaction.11 Furthermore, the carbohydrate-grafted polymers are also important models for the study of the interactions between multivalent ligands and the target specific receptors. Accordingly, it is important and interesting to develop magnetic nanoparticles with the carbohydrate-grafted poly(3) Berry, C. C.; Curtis, A. S. G. J. Phys. D: Appl. Phys. 2003, 36, R198. (4) Schleepper-Scha¨fer, J.; Hu¨lsmann, D.; Djovkar, A.; Meyer, H. E.; Herbertz, L.; Kolb, H.; Kolb-Bchofen, V. Exp. Cell Res. 1986, 165, 494. (5) Dwek, R. A. Chem. ReV. 1996, 96, 683. (6) Varki, A. Glycobiology 1993, 3, 97. (7) Kiessling, L. L.; Pohl, N. L. Chem. Biol. 1996, 3, 71. (8) Roy, R. Curr. Opin. Struct. Biol. 1996, 6, 692. (9) Lee, Y.; Lee, R. Acc. Chem. Res. 1995, 28, 321. (10) Bovin, N. V.; Gabius, H. J. Chem. Soc. ReV. 1995, 24, 413.

10.1021/cm070461k CCC: $37.00 © 2007 American Chemical Society Published on Web 06/22/2007

3668 Chem. Mater., Vol. 19, No. 15, 2007 Scheme 1. Schematic Illustration of Glucose Grafting on Gum Arabic Modified Magnetic Nanoparticles (GAMNP)

mers on their surface. Recently, we have prepared gum arabic modified magnetic nanoparticles (GAMNP) as a novel magnetic nanoadsorbent by attaching gum arabic (GA) onto the surface of Fe3O4 magnetic nanoparticles (MNP) via the interaction between the carboxylic groups of GA and the surface hydroxyl groups of Fe3O4.12 Motivated by the biological application prospects of the carbohydrate-grafted GAMNP, the functionalization of GAMNP with glucose was attempted and the specific interactions with the protein concanavalin A (Con A) were investigated in this work. Experimental Section Materials. Ferric chloride, 6-hydrate was purchased from J. T. Baker (Phillipsburg, NJ). Ferrous chloride tetrahydrate was obtained from Fluka (Buchs, Switzerland). Ammonium hydroxide (29.6%) was supplied by TEDIA (Fairfield, OH). Gum arabic was purchased from Fluka (Buchs, Switzerland). Maltose and Con A were obtained from Aldrich. Bio-Rad protein assay was purchased from Bio-Rad Laboratories (Hercules, CA). The water used throughout this work was the reagent grade produced by a Milli-Q SP ultrapure water purification system from Nihon Millipore Ltd., Tokyo. All other chemicals were of analytical grade and used without further purification. Preparation of GAMNP. GAMNP were prepared according to our previous work.12 Fe3O4 MNP were prepared by coprecipitating Fe2+ and Fe3+ ions by ammonia solution and treating under hydrothermal conditions. For the binding of GA, 100 mL of solution containing 5 mg/mL of GA was mixed with 1.0 g of MNP in a stoppered bottle. The reaction mixture was sonicated for 20 min, then mixed on a vortex mixer for 5 min, and again sonicated for another 10 min. The product (GAMNP) was recovered magnetically from the reaction mixture by using a permanent magnet with a surface magnetization of 6000 G, then washed three times with 100 mL of distilled water, and finally dried in an air oven at 50 °C for 24 h and stored in a stoppered bottle for further use. Preparation of Glucose-Grafted GAMNP. The preparation of glucose-grafted GAMNP (G-GAMNP) was achieved by coupling GAMNP with maltose via reductive amination as illustrated in Scheme 1. GAMNP (25 g/L) were suspended in a phosphate buffer solution at pH 9 containing 8.8 mM maltose and 24 mM NaBH3CN. The coupling reaction was performed at ambient temperature by stirring the solution for 48 h. The product was recovered magnetically from the reaction mixture and then washed once with 0.2 M sodium phosphate (pH 9) and twice with distilled water. The amount of maltose coupled with GAMNP was estimated by analyzing the concentration change of maltose in the solution after reaction using benzidine method.13 (11) (a) DeFrees, S. A.; Gaeta, F. C. A.; Lin, U. C.; Ichikawa, Y.; Wong, C. H. J. Am. Chem. Soc. 1993, 115, 7549. (b) Matrosovich, M. N.; Mochalova, L. S.; Marinina, V. P.; Byramova, N. E.; Bovin, N. V. FEBS Lett. 1990, 272, 209. (c) Glick, G. D.; Toogood, P. L.; Wiley, D. C.; Skehel, J. J.; Knowles, J. R. J. Biol. Chem. 1991, 266, 23660. (12) Banerjee, S. S.; Chen, D. H. J. Hazard. Mater., published online, http:// dx.doi.org/10.1016/j.jhazmat.2007.01.079.

Banerjee and Chen Characterization. Transmission electron microscopy (TEM) analysis was carried out using a Joel model 1200EX transmission electron microscope at an accelerating voltage of 80 kV. The sample was obtained by placing a drop of colloid solution onto a Formvarcovered copper grid and evaporating in air at room temperature. X-ray diffraction (XRD) measurement was performed on a Rigaku D/max III. V X-ray diffractometer using Cu KR radiation (λ ) 0.1542 nm). Fourier transfer infrared (FTIR) spectra were recorded on a Varian FTS-1000 FTIR spectrometer. The ζ-potentials were measured on a Malvern ZEN2600 Zetasizer Nano Z. The hydrodynamic diameter was measured by dynamic light scattering using a Malvern Autosizer 4700/PCS100 spectrometer equipped with an Ar ion laser operating at 488 nm. Thermogravimetric analysis (TGA) was done on the dried samples in air with a heating rate of 10 °C/min on a Shimadzu TA-50WSI TGA. Interaction with Con A. For the study on the interaction with Con A, 0.1 g of GAMNP or G-GAMNP was mixed with 5 mL of Con A solution (0.001-1.0 µM in 0.2 M phosphate buffer, pH 7.4) in a capped glass tube. The reaction mixture was stirred gently for 2 h at room temperature. The amount of Con A adsorbed was estimated from the concentration difference of Con A in solution before and after recognition treatment by the colorimetric method at 595 nm on a Jasco model V-570 UV-vis spectrophotometer using the Bio-Rad reagent for protein assay. The Raman spectra were measured on a Jobin Yvon/Labram Raman spectrometer.

Results and Discussion Properties of G-GAMNP. According to our previous work,12 the MNP had a mean diameter of 13.2 nm. The surface modification with GA showed no change in the spinel structure of Fe3O4, while it led to the shift of the isoelectric point from 6.78 to 3.6 and the formation of secondary particles with a mean diameter of 34.2 nm ((5.0%) due to the high molecular weight of GA. The typical TEM image is shown in Figure 1a. Furthermore, from the TGA curves of MNP and GAMNP, the amount of GA bound on the GAMNP could be estimated to be about 5.1 wt %. The typical TEM image of G-GAMNP is shown in Figure 1b. Their mean diameter was 14.1 nm ((5.0%), smaller than that for GAMNP while close to that for MNP. This revealed that the glucose grafting has resulted in the deagglomeration of secondary particles. This might be attributed to the fact that the glucose grafted onto GA changed the surface functional groups of GAMNP, leading to their deagglomeration. Figure 2 shows the FTIR spectra of GAMNP, G-GAMNP, and maltose. In the spectrum of maltose, the bands located at about 1433, 1274, 991, and 848 cm-1 were assigned to the vibrations associated with the CH2 group. The band near 1035 cm-1 was due to the mode involving deformation of the C-OH group. The modes related to the C-C-H bending and the C-O and C-C stretching were identified at about 1072, 1105, and 906 cm-1, respectively.14 In the spectrum of G-GAMNP, the intense band at 1045 cm-1 due to deformation of the C-OH group might be contributed by the combination of the band at 1035 cm-1 in the spectrum of maltose and that at 1068 cm-1 in the spectrum of GAMNP. The other two bands at 1087 and 995 cm-1 corresponded to (13) Jones, J. K. N.; Pridham, J. B. Bioch. 1954, 4288. (14) Sekkal, M.; Dincq, V.; Legrand, P.; Huvenne, J. P. J. Mol. Struct. 1995, 349, 349.

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Figure 1. TEM images of GAMNP (a), G-GAMNP (b), and the Con A/G-GAMNP complexes when the G-GAMNP concentrations were 0.02 (c) and 0.004 g/mL (d) at a fixed Con A concentration of 0.1 µM.

Figure 2. FTIR spectra of GAMNP (a), G-GAMNP (b), and maltose (c).

the band at 1072 and 991 cm-1 in the spectrum of maltose, revealing the coupling of maltose with GAMNP. Since the band at 1614 cm-1 in the spectrum of GAMNP might be

due to the N-H bending, its disappearance and the appearance of some new bands at 1652, 1558, and 1508 cm-1 in the spectrum of G-GAMNP might be due to the grafting of glucose to GAMNP. To further confirm the grafting of glucose to GAMNP, the Raman spectra of GAMNP, G-GAMNP, and maltose were investigated as shown in Figure 3. The Raman spectrum of G-GAMNP (Figure 3b) shows new peaks after the coupling of maltose with GAMNP. In comparison to the spectrum of maltose, these peaks should be the signatures of the glucose on G-GAMNP. The red-shift in the wavenumber of Raman scattering might be attributed to the fact that glucose was grafted on the surface of GAMNP. The peak at 978 cm-1 could be due to the C-C stretching. The peak at 1223 cm-1 was due to C-OH stretching. The peak at 1323 cm-1 could be attributed to the C-C-H bending vibration, and the peak at 1453.74 cm-1 could be attributed to deformational vibrations involving HCH and CH2OH groups. Thus, from the analyses of FTIR and Raman spectra,

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chains of 1,6-linked galactopyranose units. Con A is a glucoside-binding lectin, so it does not get immobilized on GAMNP. This result provided a powerful evidence for the specific binding of G-GAMNP with Con A. To clarify if Con A could be adsorbed by GAMNP via the aid of the glucose dissolved in solution which might be physically adsorbed on the surface of GAMNP, 0.00250.1 µM glucose was added into the Con A solutions (0.01 µM in phosphate buffer, pH 7.4) for the adsorption experiments. It was found that no adsorption of Con A on GAMNP took place even with the addition of glucose in solution. This revealed that the G-GAMNP developed in this work are indeed valuable for the specific binding of Con A. The equilibrium constant, Kass, for the association of Con A (L) and G-GAMNP (S) (L + S ) LS) could be determined by the Scatchard plot:18 Figure 3. Raman spectra of GAMNP (a), G-GAMNP (b), and maltose (c).

Figure 4. Equilibrium isotherms for the adsorption of Con A on GAMNP (O) and G-GAMNP (]).

it could be concluded that the coupling reaction between GAMNP and maltose to yield G-GAMNP was successful. By investigating the variation of maltose concentration with time in the reaction solution, it was found that the concentration of maltose decreased from 8.8 to 5.82 mM in the first 24 h of reaction and then decreased to 5.55 mM in the next 24 h. No further decrease in concentration beyond this time was observed over a 6 day reaction period. Thus, the amount of maltose coupled onto GAMNP could be estimated to be 0.13 mmol/g. Specific Interaction with Con A. To assess the capability of G-GAMNP, Con A was chosen as a target protein because it is a well-known glucose-binding protein and exists as a homotetramer at pH > 7.0. It has four binding sites and can interact with four glucose units simultaneously.15-17 As shown in Figure 4, there was no adsorption of Con A on the GAMNP, whereas G-GAMNP exhibited a considerable adsorption capability for Con A. Obviously, the glucose residues were supposed to be responsible for the adsorption of Con A because they reacted with Con A molecules to fix them on the surface of G-GAMNP. GA is a highly branched polysaccharide which consists of D-galactopyranose units, some of which are substituted at the 6-position with side (15) Bittiger, H.; Schnebli, H. P. ConcanaValin A as a Tool; John Wiley & Sons, Ltd.: London, 1976. (16) Derevenda, Z.; Yariv, J.; Helliwell, J. R.; Kalb, A. J.; Dodson, E. J.; Papix, M. Z.; Wan, T.; Campbell, J. EMBO J. 1989, 8, 2189. (17) Mortell, K. H.; Gingras, M.; Kiessling, L. L. J. Am. Chem. Soc. 1994, 116, 12053.

[LS]/[L] ) Kass([S]0 - [LS])

(1)

where [LS] and [L] refer to the concentration of bound Con A and that of free Con A, respectively, and [S]0 refers to the total concentration of glucose residues. A plot of [LS]/ [L] versus [LS] should give a straight line with a slope of -Kass. According to the data in Figure 4, the Kass value for Con A and G-GAMNP could be estimated to be 1.26 × 109 M-1, assuming a molecular weight of 25 500 for four subunits in the Con A molecules. The Kass value was very high as compared to the association constant which was 0.8 × 103 M-1 for Con A binding to free glucose,19 revealing the glucose residues of G-GAMNP enhanced appreciably the affinity for Con A, and also the strong binding implied the multipoint interaction of glucose with Con A. Effect of G-GAMNP/Con A Ratio on Complex Morphology. The plasmon absorbance of nanoparticles is sensitive to the surrounding medium and expected to change when the particles are aggregated. The plasmon absorbance arises from the excitation of plasmons by incident light. When the separation distance between particles is comparable to or smaller than their radii, the oscillations of the plasmons from adjacent particles can become coupled, thus lowering their vibration frequency. This normally appears as absorption bands shifted to longer wavelengths. The shift is dependent on the number of particles and their spatial arrangement within an aggregate. The end result of this is a red-shift and significant broadening of the absorption bands due to the overlapping of shifted modes of vibration. The interaction of Con A and G-GAMNP was characterized spectrophotometrically for plasmon absorbance at a constant G-GAMNP concentration of 0.02 g/mL. As shown in Figure 5, the particle plasmon peak of Fe3O4 was centered at 360 nm. The Con A/G-GAMNP complex did not display any significant spectral change either in terms of shift in the plasmon absorption band or in broadening of the spectra. This indicated that the binding of Con A to G-GAMNP by complexation might not lead to their aggregation. This might be because the excess amount of particles prevented the (18) Tagawa, K; Sendai, N; Ohno, K; Kawaguchi, T; Kitano, H; Bioconjugate Chem. 1999, 10, 354. (19) Sato, K.; Imoto, Y.; Sugama, J.; Seki, S.; Inouse, H.; Odagiri, T.; Hoshi, T.; Anzai, J. Langmuir 2005, 21, 797.

Glucose-Grafted Gum Arabic Magnetic Nanoparticles

Figure 5. Absorption spectra of the Con A/G-GAMNP complexes at various Con A concentrations. The concentration of G-GAMNP was fixed at 0.02 g/mL.

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Figure 7. Effect of pH on the binding of Con A on G-GAMNP.

Scheme 2. Schematic Illustration for the Effect of G-GAMNP/Con A Ratio: (a) Less Aggregation at a Higher Particle/Con A Ratio and (b) More Aggregation at a Lower Particle/Con A Ratio

Figure 6. Hydrodynamic diameter distributions of G-GAMNP (a) and the Con A/G-GAMNP complexes when the G-GAMNP concentrations were 0.02 (b) and 0.004 g/mL (c) at a fixed Con A concentration of 0.1 µM.

cross-linking between particles and Con A. Similar findings have been reported for the binding of Con A to dextran on surface.20 The dynamic light scattering method was used to further substantiate the above explanation. The hydrodynamic diameters of G-GAMNP and the Con A/G-GAMNP complexes were measured as shown in Figure 6. The hydrodynamic diameter of G-GAMNP was 26.8 nm. When the concentration of G-GAMNP was fixed at 0.02 g/mL, the hydrodynamic diameters of the Con A/G-GAMNP complexes for all Con A concentrations (0.001-1.0 µM) were found to be 28-30 nm, confirming the above explanation that each Con A was bound separately on the particle under this condition. However, when the concentration of GGAMNP was fixed at 0.004 g/mL, the hydrodynamic diameters of the Con A/G-GAMNP complexes for all Con A concentrations (0.001-1.0 µM) increased to be 50-65 nm. This revealed that when the G-GAMNP/Con A ratio was less a particle might be bound by more Con A molecules and, hence, led to the aggregation. On the other hand, at higher G-GAMNP/Con A ratios, the excess amount of particles prevented the cross-linking between particles and Con A, and so the particles were bound separately on the Con A. The TEM images for the Con A/G-GAMNP complexes at different G-GAMNP/Con A ratios are shown in Figure 1, parts c and d. In comparison with Figure 1b, it was found that the TEM image of the Con A/G-GAMNP complex at a high G-GAMNP/Con A ratio (Figure 1c) was similar to (20) Zhang, J.; Roll, D.; Geddes, C. D.; Lakowicz, J. R. J. Phys. Chem. B 2004, 108, 12210.

that of G-GAMNP before interaction with Con A (Figure 1b). However, at a low G-GAMNP/Con A ratio, particle aggregation was significantly observed (Figure 1d). This provided further evidence for the above suggestion. Accordingly, the effect of G-GAMNP/Con A ratio on the complex morphology could be illustrated by Scheme 2. Effect of pH on Con A Binding. Figure 7 shows the effect of pH on the binding of Con A on G-GAMNP. It was found that the amount of Con A adsorbed was significantly dependent on pH and the maximum adsorption capacity appeared around pH 7. Farina and Wilkins21 reported that the exposure of Con A to an acid environment caused release of ions (manganese and calcium) from Con A which is required for carbohydrate binding. The Ca2+ and Mn2+ are situated 4.25 Å apart and are in close proximity (9-13 Å) to the carbohydrate binding site, they help to position the amino acids that form contacts with the carbohydrates. Large changes have been observed in the crystallographic structure of Con A upon demetalization of the lectin, which results in the loss of its carbohydrate binding ability. They are apparently initiated mainly by the removal of calcium ion, which causes the destruction of both the Ca2+ and the carbohydrate combining sites. Thus, the lower pH might (21) Farina, R. D.; Wilkins, R. G. Biochim. Biophys. Acta 1980, 631, 428.

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Figure 8. Raman spectra of Con A (a), G-GAMNP (b), and the Con A/GGAMNP complexes at pH 7.4 (c), pH 5.0 (d), and pH 9.0 (e).

result in the decrease in the amount of Con A adsorbed on G-GAMNP. On the other hand, the decrease in the adsorbed Con A above pH 7 might be mainly due to the denaturation of Con A and the lowering of mass transfer rate caused by increase of viscosity of the solution.22 Since the pI value of Con A was 4.5-5.5 and that of GAMNP was 3.6, it was believed that the electrostatic interactions between the protein and G-GAMNP played a minimal role on the binding of Con A. This is why G-GAMNP had specific interaction with Con A. Binding Mechanism of Con A on G-GAMNP. Figure 8 shows the Raman spectra of Con A and the Con A/GGAMNP complexes at various pH values. In the spectra of Con A (Figure 8a) the amide I band showed a strong frequency and occurred at 1683 cm-1. The amide II band occurred at 1565 cm-1. The amide III band occurred at 1240 cm-1. The remaining band at 1466 cm-1, which appeared in the protein spectra, was neither an amide band nor an aromatic amino acid band. Its assignment is uncertain at present. The amide I band consisted of amide carbonyl Cd O stretching, with smaller contributions of C-N stretching and N-H bending. The amide II and III bands involved significant C-N stretching, N-H bending, and C-C stretching.23-29 The Raman spectra of the Con A/G-GAMNP complex at pH 7.4 showed three major peaks with high intensity as compared to G-GAMNP at 1447, 1553, and 1670 cm-1. On the basis of these observations some specific conclusions could be drawn. The 1677 and 1457 cm-1 frequencies measured in Con A were shifted in the Con A/G-GAMNP complex as given above. This indicated that the binding of Con A to G-GAMNP was by the help of -NH groups which (22) Hoshino, K; Taniguchi, M; Kitao, T; Morohashi, S; Sasakura, T; Biotechnol. Bioeng. 1998, 60, 568. (23) Song, S.; Asher, S. A. J. Am. Chem. Soc. 1989, 111, 4295. (24) Mirkin, N. G.; Krimm, S. J. Mol. Struct. 1996, 377, 219. (25) Chen, X. G.; Asher, S. A.; Scheitzer-Stenner, R.; Mirkin, N. G.; Krimm, S. J. Am. Chem. Soc. 1995, 117, 2884. (26) Chen, X. G.; Scheitzer-Stenner, R.; Asher, S. A.; Mirkin, N. G.; Krimm, S. J. Phys. Chem. 1995, 99, 3074. (27) Wang, Y.; Purrello, R.; Georgiou, S.; Spiro, T. G. J. Am. Chem. Soc. 1991, 113, 6359. (28) Wang, Y.; Purrello, R.; Jordan, T.; Spiro, T. G. J. Am. Chem. Soc. 1991, 113, 6368. (29) Krimm, S.; Bandekar, J. AdV. Protein Chem. 1986, 38, 181.

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were the binding sites and to some extent by the oxygen atoms and hydroxyl groups of the protein via hydrogen bonds. The appearance of the intense peak at 1553 cm-1 might be due to the hydrogen bonding between Con A and G-GAMNP. Hydrogen bonding definitely alters the electronic structure somewhat as predicted by the theoretical calculations of Nishimoto et al.30 and substantiated by the 13C NMR observations of van Schagen and Muller.31 So from the above results it could be assumed that the mechanism of Con A binding specifically to glucose was due to the oxygen atoms of asparagine in Con A, which accepted hydrogen bonds from the 6-OH and 4-OH of glucose, and to the -NH groups of asparagine in Con A, which also donated a hydrogen bond to the 4-OH of glucose.32 In the case of the Raman spectra of the Con A/G-GAMNP complexes at pH 5.0 and 9.0 no high-intensity peaks at the above-mentioned regions were observed. The results confirmed our observations that the binding of Con A was not favorable at low and high pH regions. Conclusions Novel glucose-grafted gum arabic modified magnetic nanoparticles (G-GAMNP) have been prepared by coupling maltose via reductive amination on gum arabic modified magnetic nanoparticles (GAMNP). The grafting of glucose on GAMNP was recognized by the analyses of FTIR and Raman spectra, and the amount of maltose coupled to GAMNP was estimated to be 0.13 mmol/g. The G-GAMNP showed specific affinity to Con A. From the equilibrium isotherm, the association constant of Con A and G-GAMNP could be estimated to be about 1.26 × 109 M-1 by the Scatchard plot. At a higher particle/Con A ratio, less particle aggregation was observed. However, at a lower particle/Con A ratio, a particle might be bound by more Con A molecules and, hence, led to the aggregation. With increasing the pH, the adsorption amount of Con A increased first and then decreased after reaching a maximum around pH 7. The decrease in adsorption amount at lower pH values might be due to the release of ions from Con A and that at higher pH values might be resulted by the denaturation of Con A. The amount of Con A adsorbed was significantly dependent on pH, and the maximum adsorption capacity appeared around pH 7. The decrease in adsorption amount in lower and higher pH regions might be due to the release of ions from Con A and to the denaturation of Con A, respectively. The electrostatic interactions between the protein and G-GAMNP played a minimal role on the binding of Con A. In addition, the binding of Con A on G-GAMNP was by the help of -NH groups and oxygen atoms of asparagine in Con A, which might form hydrogen bonds with the -OH groups of glucose. Such a glucose-grafted material may find applications in biomedicine. Acknowledgment. We are grateful to the National Science Council (Contract No. NSC 94-2214-E006-006) of the Republic of China for their support of this research. CM070461K (30) Nishimoto, K.; Watanabe, Y.; Yagi, K. Biochim. Biophys. 1978, 26, 34. (31) Van Schagen, C. G; Muller, F. Eur. J. Biochem. 1981, 120, 33. (32) Sharon, N.; Lis, H. Chem. ReV. 1998, 98, 637.