Available online at www.sciencedirect.com

9 (2008) 153–158www.elsevier.com/locate/microc

Microchemical Journal 8

Quartz Crystal Microbalance monitoring the real-time binding of lectin withcarbohydrate with high and low molecular mass

Mariele M. Pedroso a, Ailton M. Watanabe d, Maria Cristina Roque-Barreira c,Paulo R. Bueno b,⁎, Ronaldo C. Faria a,⁎

a Departamento de Química, Universidade Federal de São Carlos, CP 676, 13560-905, São Carlos, SP, Brazilb Instituto de Química, Departamento de Físico-Química, Universidade Estadual Paulista, CP 355, 14801-907, Araraquara, SP, Brazil

c Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. dos Bandeirantes, 3900, Ribeirão Preto 14049-900, SP, Brazild Faculdade de Filosofia, Ciências e Letras, Departamento de Física e Matemática, Universidade de São Paulo,

Av. dos Bandeirantes, 3900, Ribeirão Preto 14049-900, SP, Brazil

Received 16 October 2007; received in revised form 6 February 2008; accepted 6 February 2008Available online 13 February 2008

Abstract

A Quartz Crystal Microbalance (QCM) was used to monitor the mass changes on a quartz crystal surface containing immobilized lectins thatinteracted with carbohydrates. The strategy for lectin immobilization was developed on the basis of amultilayer system composed of Au–cystamine–glutaraldehyde–lectin. Each step of the immobilization procedure was confirmed by FTIR analysis. The system was used to study the interactions ofConcanavalin A (ConA) with maltose and Jacalin with Fetuin. The real-time binding of different concentrations of carbohydrate to the immobilizedlectin was monitored by means of QCM measurements and the data obtained allowed for the construction of Langmuir isotherm curves. Theassociation constants determined for the specific interactions analyzed here were (6.4±0.2)×104 M−1 for Jacalin–Fetuin and (4.5±0.1)×102 M−1

for ConA–maltose. These results indicate that the QCM constitutes a suitable method for the analysis of lectin–carbohydrate interactions, even whenassaying low molecular mass ligands such as disaccharides.Published by Elsevier B.V.

Keywords: Lectin; Quartz crystal microbalance; Biosensor

1. Introduction

Lectins are proteins that possess at least one non-catalyticdomain which binds reversibly to specific mono- or oligosacchar-ides [1]. Although lectins have been known since the early 19thcentury, their importance was only recognized after the 1960s,when they became extremely useful tools in biomedical researchsuch as the characterization of carbohydrates on cell surfaces orthe isolation of glycoproteins from biological fluids. Since then,numerous lectins have been isolated from many organisms,ranging from viruses and bacteria to plants and animals, playing akey role in a variety of biological processes [2].

⁎ Corresponding authors.E-mail addresses: prbueno@iq.unesp.br (P.R. Bueno),

rcfaria@power.ufscar.br (R.C. Faria).

0026-265X/$ - see front matter. Published by Elsevier B.V.doi:10.1016/j.microc.2008.02.001

Among plant lectins, Concanavalin A and Jacalin deservespecial attention. ConA, extracted from jack bean seeds(Canavalia ensiformes), exhibits great affinity for D-mannoseand its glycosides in α-anomeric form. It also reacts with terminalα-linked glucose and N-acetyl glucosamine, but with less affinity[3]. ConA is composed of four non-glycosylated 26.5 kDasubunits. There is one Ca2+ and one Mn2+ per subunit and, upondemetallization, the lectin loses its activity [4]. ConA has beenextensively used as a mitogenic stimulus for lymphocytes and forisolation and structural studies of glycoconjugates on immobilizedlectin columns, cellular or subcellular membranes in animal andbacterial cells, and also in cell separation. Jacalin is a lectin con-tained in the seeds of Artocarpus integrifolia, composed of an α-chain of 133 residues non-covalently bound to a β-chain of20 amino acids, forming an α4 β4 structure. Jacalin binds togalactose and oligosaccharides terminating with D-galactose-(β1-

154 M.M. Pedroso et al. / Microchemical Journal 89 (2008) 153–158

3)-N-acetylgalactosamine (Gal(β1-3)GalNAc) [5], a specificitythat has rendered lectin widely applicable in the isolation anddetection ofO-glycosylated proteins of mammals [6,7]. Due to itscarbohydrate-binding profile, useful biological properties havebeen reported for Jacalin [8]. Among these properties, the mostprominent is its capacity to selectively interact with human IgA1from human serum and secretions [6,9]. Most recently, Jacalin hasbeen reported as an appropriate tool to isolate O-glycosylatedIgG from bovine colostrum, which was characterized as pre-senting increased resistance to pepsin digestion and appropriatefor conferring passive immunity to calves that were not fed withnatural colostrum immediately following birth [10].

The broad application of plant lectins, especially in bio-medical research, is based on their ability to discriminate dif-ferent sugars. To optimize the use of lectins as reagents or toevaluate lectins' function in biological processes, it is of a greatvalue to establish in detail their binding properties, especially interms of the fine specificity for different carbohydrate ligands.One way to achieve this goal is to use transducer concepts tostudy their chemical interactions and sensing.

Among the modern transducer concepts, the Quartz CrystalMicrobalance (QCM) plays an important role in chemical sensing.The use of this gravimetric device as a biosensor or biomedicaldevice is growing rapidly [11–19]. The approach involved in thegravimetric devices can be also used to investigate biomolecularinteraction, mainly due to the sensitive and cost-effective detectionof chemical interactions in real time [13]. Due to its high operatingfrequencies, the QCM possesses one of the highest sensitivi-ties of all gravimetric methods. The gain of chemical specificityoccurs from a chemically active coating, which interacts withthe surrounding environment. Aspects like sensitivity, selectivity,reliability and long term stability or dynamic response are pri-marily a question of the chemical layer design [20–22]. In themostcommon approach, gravimetric devices are based on a quartzresonator that forms the frequency-determining element of anelectronic oscillator. Quartz oscillator circuits are usually designedto work at series resonant frequencies, i.e., at the maximum of thereal part of the electrical admittance or at zero phase angle, whichis almost at the same frequency for an ordinary quartz crystal os-cillator. During experiments, the changes in the resonant frequencyof the oscillator are measured and related to the analyte con-centration in the probe.

The main goal of this work is to study some features oflectin–carbohydrate interactions by using the Quartz CrystalMicrobalance as a gravimetric device and tool. The lectin–carbohydrate binding systems studied were ConA–maltose andJacalin–Fetuin.

2. Experimental

2.1. Reagents and solutions

The lectins Concanavalin A and Jacalin were used in thiswork. ConA, extracted from Canavalia ensiformes seeds, waspurchased from Sigma Chemical Co. (St. Louis, MO). Jacalin,from Artocarpus integrifolia seeds, was isolated by affinity toimmobilized D-galactose, as previously described [23].

The glycoprotein Fetuin from Sigma Chemical Co. (St. Louis,MO) was used as Jacalin ligands. Fetuin contains N- and O-glycans, most of them sialylated and having as terminaldisaccharides the following structures: Gal(β1-3)GalNAc; D-galactose-(β1-3)-N-acetylglucosamine (Gal(β1-3)GlcNAc); D-galactose-(β1-4)-N-acetylglucosamine (Gal(β1-4)GlcNAc).Maltose, a disaccharide composed of D-glucose-(α1-4)-D-glu-cose, supplied by Sigma Chemical Co. (St. Louis, MO), was usedto study the ConA binding properties. Bovine serum albumin(BSA), also from Sigma Chemical Co. (St. Louis, MO), was usedas the non-glycosylated protein to control the lectin's non-specificinteractions.

All the solutions used in the analytical procedures wereprepared with water purified by Milli-Q Millipore system,Bedford, MA (USA) with 18 MΩ. All the chemical reagentsused in the experiments were of analytical grade. Cystaminepurchased from Sigma Chemical Co. (St. Louis, MO) and a 25%v/v glutaraldehyde solution from Merck (Germany) wasemployed in the process of lectin immobilization. The ConAwas prepared and used in 0.10M phosphate buffered solution, pH7.4, with 0.15M of NaCl (PBS) containing 15.0 mM of Ca2+ andMg2+. The Jacalin was prepared and used in PBS medium.Glycine, from Sigma Chemical Co. (St. Louis, MO) was usedafter the protein immobilization to react with reminiscent terminalaldehyde of glutaraldehyde as a blocking reagent.

2.2. Apparatus

QCM experiments were carried out using 10 MHz AT-cutquartz crystals (Elchema, 16 mm in diameter) with gold electrodes(5 mm in diameter) on both sides. Prior to the experiments, theelectrode surface was cleaned with a solution of 7:3 H2SO4

(conc.): H2O2 30% v/v. After the rinsing, the crystal was placed ina homemade polytetrafluoroethylene cell with one face in contactwith the air and the other with the solution. The frequencyvariations of the quartz crystals weremonitored using a homemadeoscillator circuit coupled to a Hewlett–Packard frequency countermodel HP 53131A driven by a computer for data acquisition.

The FTIR measurements were carried out with a Bruckermodel Equinox 55 spectrometer with 30Spec accessory fordiffuse reflectance.

2.3. Protein immobilization methodology

The methodology employed for lectin immobilization wasbased on multilayer assembling on the gold surface of thepiezoelectric transducer quartz crystal. The cleaned piezo-electric quartz crystal was incubated for 12 h in an aqueoussolution with 15 mM cystamine. The crystal was then washedwith water and was incubated for 30 min in 2.5% v/vglutaraldehyde solution. Subsequently, the crystal was washedwith PBS to remove residual glutaraldehyde molecules. Thecrystal was finally exposed to the proteins solutions (1.0 mg/mlof Jacalin or ConA) for 1 h and then rinsed with PBS. To blockreminiscent aldehyde groups the lectin-modified crystal surfacewas exposed to glycine solution at 4% w/v for 30 min andwashed with PBS.

Fig. 2. QCM frequency variation monitored during the addition of differentconcentrations of maltose to a quartz crystal modified with ConA. Eachresponse was a medium of four measurements obtained with different crystalswith lectin immobilized.

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3. Results

The multilayer immobilization system was studied by FTIRduring each step of the immobilization process to confirmthe formation of the multilayer covalently binding system.Accordingly, Fig. 1 presents the FTIR spectra for all the immo-bilization steps: cystamine monolayer (Fig. 1a), cystamine–glutaraldehyde bilayer (Fig. 1b) and cystamine–glutaralde-hyde–ConA (Fig. 1c). The FTIR measurement was taken usinga cleaned quartz crystal as reference. Fig. 1a depicts two mainbands in the FTIR spectrum, at 1530 and 1690 cm−1, whichwere attributed to primary amine N–H of cystamine molecule.Fig. 1b shows the formation of a new band at around 1010 cm−1,which can be attributed to C–N present between the cystamineand glutaraldehyde. Finally, the ConA binding to the bilayer wasconfirmed by FTIR analysis, which provided a band at around1595 cm−1, as indicated in Fig. 1c, and which was attributed tothe carbonyl group of the protein. It should be noted that theFTIR analysis was in complete agreement with the QCManalysis and confirmed the expected binding of the molecules. Asimilar strategy of cystamine–glutaraldehyde bilayer was usedto immobilize Jacalin.

ConA is one of the most studied lectins [24] and its mainspecific feature is to distinguish between α and β-glycosideconfigurations, possessing a higher affinity for α-glycosides(e.g., mannose and glucose) [25]. Since the sugar bindingproperty of ConA requires Ca2+ and Mn2+, all the measure-ments of its established interactions were conducted in PBScontaining Ca2+ and Mn2+. Jacalin is a lectin with high affinityfor D-galactose, especially when presented as the disaccharideGal(β1-3)GalNAc, as found in the O-glycans attached to thehinge region of human IgA1, independently of the presence ofterminal sialic acid [6,7]. The variation of oscillation frequencyin real-time was constructed by adding different amounts ofcarbohydrate to the cystamine–glutaraldehyde–lectin multi-layer transducer system and by monitoring the frequencyvariation of this system as a function of time. Fig. 2 shows thereal-time plots obtained for the ConA–maltose. The figureshows the means and standard deviations for four independentdeterminations for ConA–maltose interaction. In general,

Fig. 1. a) FTIR spectra of cystamine monolayer, b) cystamine–glutaraldehydebilayer, and c) cystamine–glutaraldehyde–ConA.

lectins present a high affinity for polysaccharides, possessingan association constant sometimes three orders of magnitudehigher than the monosaccharide association constant [24]. Thisfact justifies the negative results obtained during the evalua-tion of mannose and/or glucose binding process to ConA. Incontrast, the ConA to maltose binding process was successfullyevaluated. This outcome was probably due to the higher asso-ciation constant of these systems in comparison to the inter-action association constant of ConAwith glucose and mannose.Furthermore, the higher molecular mass of maltose, in compari-son to mannose and glucose, likely favor the evaluation of thislast kind of interaction.

To evaluate the interaction of immobilized Jacalin withglycans attached to a protein chain, the glycoprotein Fetuin wasemployed as analyte, as showed in the Fig. 3a. The figureshows the means and standard deviations for four indepen-dent determinations for Jacalin–Fetuin interaction and three

Fig. 3. QCM frequency variation monitored during the addition of differentconcentrations of: (●) Fetuin and (○) BSA to a quartz crystal modified withJacalin. Each response was a medium of four measurements obtained withdifferent crystals with lectin immobilized.

Fig. 5. Langmuir isotherm plots for ConA binding with maltose, calculated bythe QCM study (Fig. 3).

156 M.M. Pedroso et al. / Microchemical Journal 89 (2008) 153–158

independent determinations for Jacalin–BSA interaction. Thevariation of oscillation frequency in the real-time curve wasobtained by adding progressively higher amounts of Fetuin to thecystamine–glutaraldehyde–Jacalin multilayer system (Fig. 3a).To confirm the specificity of the Jacalin binding to the glycans andnot to the peptide chain of Fetuin, an unglycosylated protein, i.e.,bovine serum albumin, was used as analyte. As Fig. 3b indicates,no significant frequency variation was detected.

To confirm that the variation in the transducer frequencycorresponds to the specific binding of lectin–carbohydrate pairrather than to other non-specific types of interaction, such ascarbohydrate–Au or carbohydrate–glutaraldehyde, a cleanedquartz crystal was first plunged into a maltose solution. In thelast situation, no variations in transducer frequency were noted.The same evaluation was carried out for the quartz crystal withan immobilized cystamine–glutaraldehyde bilayer and, again,no variation in the transducer frequency was detected. Hence, avariation in frequency observed when a cystamine–glutaralde-hyde–lectin multilayer transducer is exposed to a carbohydratemedium was attributed to the specific lectin–carbohydrate inter-action [14–19,26–28]. The variation in the oscillating frequencyfor ConA–maltose system, observed in the Fig. 2, was about100 Hz and for Jacalin–Fetuin was about 200 Hz. The frequencyvariation for maltose addition is higher compared to the Fetuinand this higher value can be explained by taking into account thedifference in molecular weight (i.e., 342.3 g mol−1 for maltoseand 48.4 kDa for Fetuin). The observed results indicate thatmaltose interacted not only specifically, i.e. by specific bindingwith ConA, but there are also other chemical protein environ-ments where likely electrostatic interaction must occur, i.e.physical interactions by means of electrostatic-like forces. Toconfirm the specificity of ConA–maltose interaction, three addi-tions of maltose were carried out to the crystal with ConAimmobilized using a PBS without the presence of Ca2+ and Mn2+

ions in solution. There was no variation in the oscillation fre-quency of quartz crystal indicating that the ConA loses its activity[4]. The results are reinforced by the fact that the associationconstant obtained for ConA–maltose interaction is lower than that

Fig. 4. Langmuir isotherm plots for Jacalin binding with Fetuin calculated by theQCM study (Fig. 2).

obtained for Jacalin–Fetuin interaction, as discussed below in thefollowing paragraph.

The association constant (Ka) of the lectin–carbohydratebinding was calculated by an appropriate Langmuir adsorptionmodel [28–31], using the curves shown in Figs. 2 and 3. The Ka

for Jacalin–Fetuin was calculated to be (6.4±0.2)×104 M−1, aconstant derived from theLangmuir isotherm represented in Fig. 4.For the ConA–maltose binding systems, the mean value of theassociation constant was (4.5±0.1)×102 M−1, according to thedata shown in Fig. 5. The association constant values obtainedherewere of the same magnitude as those found in the literature, usingdifferent methodologies, i.e., titration microcalorimetry [24],affinity chromatography [32], fluorescence analysis [33] andsurface plasmon resonance (SPR) [29]. This finding allows us toconclude that the Quartz CrystalMicrobalance is a useful method-ology to study protein–carbohydrate interactions, even for lowmolecular mass molecules.

Fig. 6. Monitoring of QCM frequency variation of ConA immobilized duringthree series of additions of different maltose concentrations. (□) First, (●)second and (π) third series of maltose addition. After each addition the crystalwas washing with HCl/glycine solution and PBS.

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The ConA–maltose interaction was mainly evaluated bymeans of a procedure consisting of three additions in series offixed maltose concentration to the same quartz crystalcontained immobilized ConA (Fig. 6). After each series ofmaltose addition the system was exposed to a 1 mM solutionof HCl/glycine (pH 2.8). The main purpose of the lastprocedure was to remove the maltose binding to the ConA.The first and second curves, Fig. 6a and b, shows similarresponses indicating that the number of ligand-binding sitesdetected in the crystal surface does not change significantlybetween the first and second maltose series addition. However,in the third series of maltose addition, after the treatment withHCl/glycine, a significant decrease in the oscillating frequencywas observed (Fig. 6c). This was attributed to the fact that thethird HCL/glycine treatment was sufficiently aggressive toinduce the partial denaturation of the protein immobilized andconsequently the numbers of ligand-binding sites wasdecreased. At this moment, it is very important to stress thatthe association constant obtained for the three series ofmaltose addition was (5.6±0.8) ×102 M− 1, (3.5±1.2)×102 M−1 and (3.2±1.2)×102 M−1. As the values are all ofthe same magnitude it is possible to infer that in spite of thefact that the response of third treatment with HCL/glycineshowed a different value, the system shows similar associationconstants. This suggests that the third treatment with HCL/glycine produces only a variation in the number of ligand-binding sites, but the mechanism of interaction remains thesame. This also explains why it has not observed any sig-nificant mass variation between first and second treatmentwith HCL/glycine. Furthermore, it is possible to infer,considering ConA is composed of four subunits not covalentlybound (forming a tetramer), that with the third aggressivetreatment likely few subunits may be denatured (and/orremoved), exposing new subunits to the environment. There-fore, at the end of the third treatment, the number of ligand-binding sites are not maintained, i.e. it is decreased. In otherwords, considering that the functionalized crystal containsapproximately a monolayer of proteins exposed to theenvironment, likely during the third HCl/glycine treatment,the remained subunit that are covalently binding to the crystalsurface, by means of cystamine and glutaraldehyde bilayer,must suffer partial denaturation. As a result, the amount ofligand-binding sites decrease and consequently the response inoscillating frequency also decreases, as can be observed fromanalysis of Fig. 6c. The fact that there was no significantvariation observed in the affinity constant corroborates theprevious analysis, showing also that the binding mechanism isnot changed.

4. Conclusion

Lectin–carbohydrate interactions were evaluated using theQCM approach. This electrogravimetric methodology accu-rately characterized specific interactions between ConA–maltose and Jacalin–Fetuin. The strategy for protein immobi-lization used in the present study was adequate. The QCMmethodology provides an easy way for real-time monitoring of

binding to simple or complex carbohydrates, as well forcalculating the association constant involved in this kind ofinteraction. The values of the association constants calculated inthis study are coherent with those reported in the literature,which were obtained by employing several different systems tostudy molecular interactions. Moreover, the association con-stant values obtained for ConA–maltose and Jacalin–Fetuinsystems are similar to those previously reported for similar pairsof interacting molecules. The present study demonstrates theability of a Quartz Crystal Microbalance to detect and evaluatethe lectin–carbohydrate interactions, even when assaying lowmolecular mass ligands such as disaccharides.

Acknowledgements

The authors acknowledge the financial support of theBrazilian research funding agencies FAPESP, CNPq andCAPES. The authors are also indebted to FAPESP (São PauloState Research Funding Agency) for funding projects Nos. 02/06693-3, 06/05746-7 and 05/02998-2. We are indebted toSandra Maria de Oliveira Thomaz for the careful purification ofJacalin.

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