Analytica Chimica Acta 435 (2001) 151–156

A new concept for the construction of an artificial dehydrogenasefor fructosylamine compounds and its application for an

amperometric fructosylamine sensor

Koji Sode∗, Yuka Takahashi, Shigenori Ohta, Wakako Tsugawa, Tomohiko YamazakiDepartment of Biotechnology, Tokyo University of Agriculture and Technology, 2-14-16 Nakamachi, Koganei, Tokyo 184-8588, Japan

Received 4 July 2000; received in revised form 6 November 2000; accepted 13 November 2000

Abstract

For the construction of an artificial dehydrogenase and an artificial dehydrogenase based sensor, the potential applicationof polyvinylimidazole (PVI) was investigated as the catalyst for the fructosylvaline (Fru-val) oxidation, a model compoundfor the glycated hemoglobin. The presence of PVI catalyzed the oxidation of Fru-val in the presence of an electron acceptor.A colorimetric determination of Fru-val was possible with the detection range from 50 �M to 10 mM utilizing PVI as thecatalyst. An amperometric sensor for Fru-val was constructed using carbon paste electrode immobilized PVI. Using thesensor system, Fru-val could be measured from 20 �M to 0.7 mM. These results indicated the further possible applicationof PVI as the catalyst for fructosylamine compounds as well as various glycated proteins including glycated hemoglobin.© 2001 Elsevier Science B.V. All rights reserved.

Keywords: Glycated hemoglobin; Fructosylamine; Fructosylamine oxidase; Polyvinylimidazole; Artificial dehydrogenase; Amperometricsensor

1. Introduction

A carbonyl sugar group combines with an aminogroup of protein, non-enzymatically, and results theformation of a labile aldimine-Schiff base, which thenundergoes the Amadori rearrangement, consequentlyforms a stable ketoamine. The products are so-calledAmadori products, fructosamine or glycated proteins(Fig. 1) [1]. Since the amount of the glycated pro-tein in serum is dependent upon both the glucoseconcentration in the serum and the lifetime of theprotein, the measurement of proportion of glycatedprotein to non-glycated protein is a good and reli-

∗ Corresponding author. Tel.: +81-42-388-7027;fax: +81-42-388-7027.E-mail address: sode@cc.tuat.ac.jp (K. Sode).

able indicator for the glycemic control in the diabeticpatients. Among the various Amadori compounds,much attention is currently being paid to glycatedhemoglobin (HbA1c) as the most important indicatorof the degree of diabetic control. Since the N-terminalresidue of �-globin is valine, fructosylvaline (Fru-val)is formed as the result of Amadori rearrangement,and the resultant hemoglobin molecules with glycated�-globin is called HbA1c. Due to the in vivo lifetimeof hemoglobin, the ratio of glycation directly referredpast blood glucose levels. Therefore, the amount ofHbA1c is a good indicator of glycemic control forover a period of 2–3 months, and is more reliablecompared with rapidly fluctuating blood glucose lev-els [2].

In order to determine the glycated proteins as wellas HbA1c levels in the blood, considerable attention

0003-2670/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S0 0 0 3 -2 6 70 (00 )01304 -0

152 K. Sode et al. / Analytica Chimica Acta 435 (2001) 151–156

Fig. 1. Proposed three-step enzymatic reaction of fructosylamine oxidase on the basis of the analogy of the monoamine oxidase catalyzingreaction [16].

has been devoted to developing selective and sensitivemethods suitable for clinical diagnostics. Ion exchangeand affinity chromatography and immuno-assay meth-ods have been developed and utilized for clinical di-agnosis [3–5]. Enzymatic determination of glycatedprotein was also proposed utilizing a known group ofenzymes, which recognizes and oxidatively degradesfructosylamine compounds, called fructosylamine ox-idases (FAOs) or Amadoriases [6–12]. Since glycatedproteins can be digested by some proteases to be gener-ated small peptide fragment containing fructosamine,the combination of FAOs and proteases is being paidmuch attention as the enzymatic method for glycatedprotein measurement [13], although further improve-ment for the selectivity and also the stability is stillrequired.

Besides, the molecular imprinting technology is thepromising area in constructing tailor made molecu-lar recognition element using synthetic polymers [14].

Although the principle and method for the preparationof tailor made molecular recognition element by MIPtechnology are now available, the development of thecatalytic center suitable for the combination with MIP,consequently being utilized as the sensor elements, isstill in progress [15].

This study focuses on the development of catalyticcenter, which may work as the fructosylamine dehy-drogenase. Fig. 1 shows the proposed three-step reac-tions of fructose amine oxidase, on the basis of thepostulated mechanisms of monoamine oxidase [16].This reaction can be divided into three steps, the firststep is the formation of enzyme-substrate complex,simultaneously the oxidation of the substrate is oc-curred by forming imine compound. The next step isthe hydrolysis of imine compound. The last step is there-oxidation of the active center by the electron ac-ceptor. It is also known that spontaneous oxidation offructosylamine occurs in the alkaline condition [17].

K. Sode et al. / Analytica Chimica Acta 435 (2001) 151–156 153

Therefore, the general basis and/or nucleophilic cat-alytic center may function as the catalyst for the oxida-tive cleavage of fructosylamine. It has been reportedthat His residue has often play as the general basecatalysis active center in the oxidoreductase, such asin sarcocine oxidase [18]. Therefore, we chose imi-dazole compounds as the mimicking His residue andas a model catalytic center for the construction of anartificial fructosylamine dehydrogenase. In this study,we show the potential of polyvinylimidazole (PVI) asthe catalyst in the oxidation of Fru-val, a model com-pound for HbA1c, and its potential application for thecolorimetric determination of Fru-val and for the con-struction of an amperometric Fru-val sensor.

2. Materials and methods

2.1. Chemicals

Fru-val was synthesized according to the previousliterature [19]. 1-Vinylimidazole (Tokyo Kasei, Tokyo,Japan), phenazinemethosulphate (Kanto Chem.,Tokyo, Japan), 1-methoxyphenazinemethosulphate(m-PMS) (DOJINDO, Kumamoto, Japan) anddichlorophenol indophenol (DCIP) (Merck, Ger-many) were purchased. All other reagents were of theanalytical grade.

2.2. Synthesis of polyvinylimidazole polymer

Polyvinylimidazole was synthesized as fol-lows. In a typical preparation, 30 mmol (2.93 ml)of 1-vinylimidazole and 0.6 mmol (98.4 mg) of2,2′-azobis(2,4-dimethylvaleronitrile) as the initiatorwere mixed in a glass tube. After being degassed, thetube was sealed under Ar and heated at 45◦C as thetrigger of polymerization and incubated for 24 h atthe same temperature. Subsequently, the polymer wasmechanically ground and was sieved. A 40 �m diam-eter fractin was collected and utilized for the furtherexperiments.

2.3. Colrimetric measurement of fructosyl valine

1 mg of PVI was mixed with 200 �l 10 mM potas-sium phosphate buffer pH 7.0 containing 2 mM PMS

and 0.06 mM DCIP and 4% of Triton X-100. The ab-sorbance decrease at 600 nm by the discoloring reac-tion of DCIP caused by the addition of a sample withdifferent concentration of Fru-val was determined.

2.4. Construction of an amperometric sensorutilizing PVI

To 50 mg of carbon paste (BAS Co., Indiana, USA),20 mg of PVI was mixed and packed into the elec-trode assembly (BAS Co.). Thus, constructed carbonpaste electrode with PVI was immersed in a 10 mMpotassium phosphate buffer, pH 7.0, containing 1 mMmPMS. By applying +100 mV versus Ag/AgCl refer-ence electrode, the anodic oxidation of reduced me-diator as the result of fructosylamine oxidation wasmonitored at 50◦C.

3. Results and discussion

3.1. Characterization of PVI as the catalysis forFru-val oxidation

We first investigated the potential of imidazole com-pound as the catalyst for the oxidation of Fru-val inthe presence of electron acceptor, PMS. Fig. 2 shows

Fig. 2. Dose effect on the rate of oxidation of fructosylvaline inthe presence of 1-vinylimidazole as the catalyst. Reaction wascarried out at room temperature in 10 mM potassium phosphatebuffer (pH 7.0) with 4% Triton X-100, containing 1 mM PMS and0.06 mM DCIP and 10 mM Fru-val as the substrate.

154 K. Sode et al. / Analytica Chimica Acta 435 (2001) 151–156

the dose effect on the rate of DCIP decolorizing reac-tion mediated by PMS at pH 7.0 by the presence of1-vinylimidazole. With the increase of the amount of1-vinylimidazole, the rate of reaction also increased.We also investigated the pH and buffer concentrationdependence of the 1-vinylimidazole catalyzing reac-tion. The rate of reaction increased with increasingpH, but the rate of the reaction is also affected by thebuffer concentration. This is the typical feature of thegeneral base catalysis. Therefore, we concluded thatimidazole functions as the general base catalyst in thiscase. We then investigated the same experiment usingPVI as the catalysis (Fig. 3). With the increase of theamount of PVI, the rate of reaction also increased. Thelevel of spontaneous oxidation of Fru-val in the pres-ence of 2 mM PMS was <50% of the oxidation ratein the presence of 1 mg/ml PVI and 2 mM PMS. Therate of reaction by the monomer is higher than that byPVI. This difference is mainly due to the hydrophobicproperty of the polymer, whereas the substrate is hy-drophilic. In addition, the synthesized matrix may de-crease the rate of diffusion toward the catalytic center,imidazole group in this case. Consequently, the rate ofreaction catalyzed by PVI is slower than the reactioncatalyzed by the monomer.

These results indicated that PVI functioned as thecatalyst in the oxidation of Fru-val in the presence ofelectron acceptor.

Fig. 3. Dose effect on the rate of oxidation of fructosylvaline inthe presence of PVI as the catalyst. Reaction was carried out atthe same condition described in Fig. 2, except that using 2 mMPMS instead of 1 mM.

Fig. 4. Correlation between fructosylvaline concentration and therate of oxidation in the presence of 5 mg/ml PVI as the catalyst(�) or in the absence (�). Reaction was carried out at roomtemperature in 10 mM potassium phosphate buffer (pH 7.0) with4% Triton X-100, containing 2 mM PMS and 0.06 mM DCIP,Fru-val as the substrate. The reaction was carried out at the roomtemperature.

Since 1-vinylimidazole nor PVI could utilize oxy-gen as the electron acceptor to proceed this reactionand did not evolve hydrogen peroxide, this reactionwas categorized as the mimic of the Fru-val dehydro-genase.

Fig. 4 shows the correlation between Fru-val con-centration and the rate of reaction in the presenceor absence of PVI. In the presence of 5 mg/ml PVI,a good linear correlation was observed from 50 �Mto10 mM Fru-val. Most popular clinical method forfructosamine is based on the reduction of nitrobluete-trazolium chloride (NBT) to a dye under alkaline con-ditions [17]. However, this method requires precisebuffer for the assay which may result in numerousinterference of measurements. In order to meet the re-quirement of much reliable method for fructosamineassay, enzymatic methods have been developed uti-lizing FAO. We have also reported the isolation andcharacterization of FAO, and its application for colori-metric determination of Fru-val [20]. At the moment,none of the enzyme can utilize intact glycated proteinas the substrate. Therefore, glycated protein shouldbe digested by adequate protease prior to the enzymeassay. However, in such system, protease should beinactivated before the assay, and also the removal of

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denaturant is essential in order to proceed FAO cat-alyzing reaction. In addition, considering that thepreparation of FAO requires the specific medium con-taining Fru-val as the nitrogen source and costly, andalso that the yield was very low, the further develop-ment of PVI catalyst with substrate specificity is beingexpected.

3.2. An amperometric sensor for Fru-val utilizing PVI

We then constructed an amperometric sensor forthe measurement of Fru-val utilizing PVI as a mim-icking system for enzyme sensor utilizing FAO. Themeasurement was based on the re-oxidation of thereduced mPMS, which was resulted by the oxidationof Fru-val. By the injection of the sample containingFru-val, the steady state current increased and reachedto another steady state within 3 min. Fig. 5 shows thecorrelation between sensor signal and Fru-val concen-tration. With the increase of Fru-val concentration,the current increase also increased, and a good linearcorrelation was observed in the range that we exam-ined (20 �M to 0.7 mM). The reproducibility of thesensor signals was examined by the four consecutiveinjection of the sample, and obtained within 10%errors. The bare carbon paste electrode also re-sponded with the increase of Fru-val concentration,however, the level was <15% of the electrode im-

Fig. 5. Calibration curve for amperometric fructosylamine sensoremploying PVI as the catalyst. The measurements were carried outin 10 mM potassium phosphate buffer pH 7.0, containing 1 mMm-PMS, at 50◦C.

mobilizing PVI as the catalyst. The localization ofcatalysis on the surface of electrode resulted in thehigh sensitivity, which was usually observed in theenzyme electrode. The detection limit of this sensorwas about 20 �M, which is enough sensitivity for thedetection of HbA1c derived Fru-val. Therefore, usingPVI as the catalysis, we have constructed the fructo-sylamine sensor. The reproducibility of the sensor rotat this moment is within 10%. The reproducibility isdependent upon the manufacturing process in mixingcarbon paste and PVI. The potential instability ofthe system may be the electron acceptor used in thisstudy, m-PMS. Since the catalysis, PVI itself is stableand can be operated at higher temperature and also inthe presence of denaturant, or even in the presence ofprotease, the direct measurement of glycated protein,such as HbA1c or glycated albumin, will be achieved.Our goal of this research is to construct an artificial en-zyme with substrate specificity based on the catalyticcenter shown in this study. The use of MIP technologywill enable to introduce the specificity for the PVIcatalysis.

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