Modification of Glucose Oxidase by the Covalent Attachment of a

Anal. Chem. 1997, 69, 734-742

Modification of Glucose Oxidase by the Covalent Attachment of a Tetrathiafulvalene Derivative P. N. Bartlett,* S. Booth, D. J. Caruana,† J. D. Kilburn, and C. Santamarı´a

Department of Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ, U.K.

4-[(3′-Carbohydroxypropyl)thio]-5-(methylthio)tetrathiafulvalene was synthesized and used as a mediator for the oxidation of glucose oxidase both free in solution and after covalent attachment to the enzyme through carbodiimide coupling to amine residues in the protein. The modified enzyme was characterized by isoelectric focusing gel electrophoresis and found to have a higher pI than the native enzyme. Electrochemical studies show that the singly oxidized tetrathiafulvalene derivative attached to the enzyme can act as a mediator for the direct reoxidation of the enzyme at electrode surfaces, whereas the doubly oxidized dication is not a mediator. Similar results are found for mediation by the tetrathiafulvalene derivative in solution. The application of the modified enzyme in membrane enzyme electrodes was investigated, and the response were analyzed to give kinetic information about the modified enzyme kinetics. Our studies show that the modified enzyme has good stability on storage in the absence of glucose but is less stable during continuous operation in a glucose sensor. This appears to arise from reactions between the tetrathiafulvanene groups attached to the modified enzyme and traces of hydrogen peroxide generated by the enzymatic reaction of glucose with oxygen present in the solution. The transfer of electrons between the enzyme active site and the electrode surface is most commonly the limiting factor in the fabrication of amperometric biosensors. Electrochemical mediators are frequently employed to act as a link between these two sites. Logically, the mediator molecule, generally of low molecular weight, must be positioned in between, or able to shuttle between, the active site of the enzyme and the electrode surface. In this paper we describe the covalent attachment of tetrathiafulvalene (TTF) to the peptide backbone of glucose oxidase (GOx) in order to enable the direct oxidation of the flavin prosthetic group at the enzyme active site. The stability of the resulting modified protein and the effect of the modification procedure on the protein structure are investigated. The use of TTF redox mediator groups is directly compared with results for the modification of glucose oxidase by covalent attachment of ferrocenemonocarboxylic acid. Given the range of amino acid side chains, covalent attachment is not as difficult as it may at first seem. Different amino acid residues have different reactivities, and so chemical reagents can be reasonably specific for certain amino acids. Suitably specific reactions are available for aspartic acid, glutamic acid, histidine, lysine, arginine, methionine, tryptophan, tyrosine, and cysteine. † Current address: Department of Biomedical Sciences, University of Malta, Msida, Malta, MSDO6.

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Other amino acids such as proline, valine, and alanine are less reactive. By covalently attaching a suitable mediator molecule to a specific residue, one can imagine that, provided the mediators are situated in the correct positions, the electron transfer distance is reduced into several smaller “hops” instead of a single long “jump”. Heller has referred to this as “wiring the enzyme”.1 In 1984 Hill described the covalent attachment of ferrocenemonocarboxylic acid to lysine residues of GOx using isobutyl chloroformate.2 This work demonstrated that the direct oxidation of the flavin (FAD) prosthetic group by ferrocene groups covalently attached to the protein structure was possible. However, the modification procedure involved reactive reagents that precipitated some of the enzyme. Degani and Heller subsequently described the covalent attachment of ferrocenemonocarboxylic acid to glucose oxidase and D-amino acid oxidase.3 In this work they used carbodiimide-promoted coupling of the acid to lysine residues of the protein, a technique that makes use of much milder coupling conditions. They found, by atomic absorption spectroscopy, that an average of 12 ferrocene molecules were attached to each enzyme molecule and that these attached ferrocene groups were redox active and able to mediate the reoxidation of the reduced flavin group within the enzyme. In a subsequent publication,4 the same authors showed that lysine, tyrosine, tryptophan, and histidine residues on the protein could also be used to attach other electron relays such as ferroceneacetic acid and ruthenium pentaamine. Further studies carried out by other groups have investigated the stability and kinetics of glucose oxidase modified with ferrocene derivatives. Bartlett et al.,5 using the same carbodiimide coupling procedure employed by Degani and Heller, showed that the reactivity of the ferrocene mediator was influenced by the length of the alkyl spacer arm and that this also influenced the storage stability of the ferrocene-modified enzyme at 4 °C. In a subsequent paper, Bartlett et al.6 showed that glucose oxidase modified with either ferrocenemonocarboxylic acid or the ferroceneacetic acid was unsuitable for use in an amperometric glucose sensor because of the poor stability of the ferricinum form of the mediator in neutral aqueous solution. Badia et al. investigated the enzyme activity and kinetics of glucose oxidase modified with ferrocenemonocarboxylic and -dicarboxylic acids. Using the recently published structure for the enzyme, they were able to investigate the locations of the (1) Heller, A. Acc. Chem. Res. 1990, 23, 128-134. (2) Hill, H. A. O. Eur. Pat. Appl. EPO, 125, 139 A2 (C12 Q1/68), 14, 45-46, 1984. (3) Degani, Y.; Heller, A. J. Phys. Chem. 1987, 91, 1285-1289. (4) Degani, Y.; Heller, A. J. Phys. Chem. 1988, 110, 2615-2620. (5) Bartlett, P. N.; Whitaker, R. G.; Bradford, V. Q. Talanta 1991, 38, 57-63. (6) Bartlett, P. N.; Whitaker, R. G.; Green, M. J.; Frew, J. J. Chem. Soc., Chem. Commun. 1987, 1603-1604. S0003-2700(96)00533-1 CCC: $14.00

© 1997 American Chemical Society

possible lysine modification sites with respect to the active site.7 They concluded that the rate-determining step for the oxidation of the FAD center was dependent on the location of the attached redox mediator and not on their number. They also investigated the intramolecular distance between the FAD and the ferrocene by fluorescence quenching. On the basis of their fluorescence studies, they suggested that ferrocenedicarboxylic acid and ferrocenemonocarboxylic acid are attached at different sites on the enzyme. As a result the intramolecular electron transfer rate was 103 times slower when using ferrocenedicarboxylic acid as compared to ferrocenemonocarboxylic acid. TTF and its derivatives are another group of redox molecules that have been successfully employed as redox mediators in enzyme electrochemistry. TTF itself has been used to modify electrodes by mixing the compound with silicone oil, for example, and using this to wet a graphite electrode prior to adsorption of an enzyme onto the surface of the electrode.8 Carbon paste electrodes modified with TTF derivatives have been prepared by Lee et al.9 From the results obtained with these modified electrodes, TTF derivatives show good mediating properties for a range of oxidase enzymes including glucose oxidase, lactate oxidase, and choline oxidase. Nafion-TTF-modified glassy carbon electrodes were prepared by Liu and Deng10 and used with immobilized films of glucose oxidase and bovine serum albumin cross-linked with glutaraldehyde. Studies of the mediation of glucose oxidase electrochemistry using TTF have shown that the mediator can be hydrophobically incorporated into the enzyme and that these TTF molecules are able to mediate direct oxidation of the enzyme.11 This approach has recently been applied in an enzyme switch responsive to glucose.12 However the hydrophobically incorporated TTF is slowly lost from the enzyme, presumably due to its slight solubility in aqueous solution and its enhanced solubility in the oxidized (TTF+) form and this ultimately limits the use of this approach. Finally Albery et al.13 used crystals of the organic conducting salt tetrathiafulvalenium tetracyanoquinodimethanide (TTFTCNQ) as an electrode material to carry out the oxidation of glucose oxidase. In this case, the mechanism appears to involve both soluble mediating components from the TTF-TCNQ crystals and some heterogeneous redox catalysis of the reaction.14-17 In the present work, we report the synthesis and characterization of a TTF carboxylic acid designed for the modification of redox enzymes. We present our results for the covalent modification of glucose oxidase by the TTF derivative and for the characterization of the modified enzyme both electrochemically and by IEF electrophoresis. Calibration curves for glucose (7) Badia, A.; Carlina, R.; Fernandez, A.; Battaglini, F.; Mikkelsen, S. R.; English, A. M. J. Am. Chem. Soc. 1993, 115, 7053-7060. (8) Zhao, S.; Luong, J. H. T. Biosens. Bioelectron. 1993, 8, 483-491. (9) Lee, H. S.; Liu, L. F.; Hale, P. D.; Okamoto, Y. Heteroat. Chem. 1992, 3, 303-310. (10) Liu, M. I.; Deng, J. Q. Anal. Chim. Acta 1995, 300, 65-70. (11) Bartlett, P. N.; Bradford, V. Q. J. Chem. Soc., Chem. Commun. 1990, 11351136. (12) Bartlett, P. N.; Birkin, P. R. Anal. Chem. 1993, 65, 1118-1119. (13) Albery, W. J.; Bartlett, P. N.; Craston, D. H. J. Electroanal. Chem. 1985, 194, 223-235. (14) Bartlett, P. N. J. Electroanal. Chem. 1991, 300, 175-189. (15) Kawagoe, J. L.; Niehaus, D. E.; Wightman, R. M. Anal. Chem. 1991, 63, 2961-2965. (16) Hill, B. S.; Scolari, C. A.; Wilson, G. S. J. Electroanal. Chem. 1988, 252, 125-138. (17) Zhao, S.; Korell, U.; Cuccia, L.; Lennox, R. B. J. Phys. Chem. 1992, 96, 56415652.

electrodes based on the modified enzyme are reported, and the long-term operational stability of the modified enzyme in an amperometric membrane enzyme electrode is investigated. The results for the TTF-modified enzyme are compared to those for the ferrocenemonocarboxylic acid-modified enzyme. EXPERIMENTAL SECTION Materials. Reagent grade tetrahydrofuran (THF) was predried and distilled over sodium benzophenone ketyl prior to use. Trimethyl phosphite, dichloromethane, and petroleum ether were freshly distilled prior to use. All other solvents and chemicals were used as received. Piperazine-N,N′′-bis(2-ethanesulfonic acid) (PIPES), N-(2-(hydroxyethyl)piperazine-N′-2-ethanesulfonic acid, sodium salt (HEPES), tert-octylphenoxypolyethoxyethanol (Triton X-100), and 3-(N-morpholino)-2-hydroxypropanesulfonic acid, sodium salt (MOPSO) were obtained from Sigma. 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (DEC), ferrocene, ferrocenemonocarboxylic acid, ethylenediaminotetraacetic acid (EDTA), 4-aminoantipyrene, trichloroacetic acid, and 3,5dichloro-2-hydroxybenzene sulfonic acid (DHSA) were obtained from Aldrich. Urea, disodium hydrogen orthophosphate, hydrochloric acid, methanol, and acetic acid were purchased from Fisons. Glucose was obtained from BDH. Bis(1,3-dithiol-2-thioxo4,5-dithiolate)zinc tetraethylammonium salt (1) and 4,5-bis[(2′cyanoethyl)thio]-1,3-dithiol-2-one (3) were prepared according to reported literature methods.18-21 Glucose oxidase (GOx) from Aspergillus niger and horseradish peroxidase were gifts from MediSense Inc. IEF polyacrylamide gels, pH 4-6.5, were obtained from Pharmacia. Coomassie brilliant blue G-250 was obtained from Bio-Rad. All assay solutions were freshly prepared using water purified by a Whatman WR50 RO deionizing system followed by a Whatman Still Plus carbon filter. Instrumentation. High-field NMR experiments were performed on a Bruker AC 300 instrument. Mass spectra were obtained on a VG analytical 70-250-SE normal geometry doublefocusing mass spectrometer. Protein purification was performed on a 10 × 1.6 cm gel permeation column containing Sephadex G-15 using a three-channel peristaltic pump (Pharmacia P-3) to elute the samples through the column or on PD-10 disposable columns (Pharmacia) containing Sephadex G-25M. Electrochemical measurements were performed on an in-house-built potentiostat incorporated in a Faraday cage. IEF gel electrophoresis was carried out on a Phastsystem (Pharmacia, LKB) automated electrophoresis workstation using pH 4-6.5 acrylamide gels (Pharmacia). Absorption spectra were recorded on a HewlettPackard 8452A diode array spectrophotometer. 4,5-Bis[(2′-cyanoethyl)thio]tetrathiafulvalene (4). Vinylene trithiocarbonate (2.50 g, 18.63 mmol) and 4,5-bis[(2′cyanoethyl)thio]-1,3-dithiol-2-one (5.66 g, 18.63 mmol) were heated at 140 °C for 4 h in freshly distilled trimethyl phosphite (50 mL) under a nitrogen atmosphere, after which excess trimethyl phosphite was removed by distillation under reduced pressure. Chromatography on silica gel with dichloromethane-petroleum (18) Steimecke, G.; Sieler, H. J.; Kirmse, R.; Hoyer, E. Phosphorus Sulfur 1979, 7, 49-55. (19) Varma, K. S.; Bury, A.; Harris, N. J.; Underhill, A. E. Synthesis 1987, 837839. (20) Becher, J.; Lau, J.; Leriche, P.; Mørk, P.; Svenstrup, N. J. Chem. Soc., Chem. Commun. 1994, 2715-2716. (21) Svenstrup, N.; Hansen, T. K.; Rasmussen, K. M.; Becher, J. Synthesis 1994, 809-812.

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ether (bp 40-60 °C) (4:1) as eluent afforded 4 (2.04 g, 5.45 mmol, 29%) as an orange-red oil: Rf (dichloromethane) 0.46; IR (CH2Cl2) 3055, 2985, 2685, 2305, 1700, 1420, 1270, 1255, 1155, 895 cm-1; 1H NMR (acetone-d , 300 MHz) δ 6.67 (2 H, s, HCdCH), 3.23 (2 6 H, t, J ) 6.6 Hz, CH2CN), 2.93 (2 H, t, J ) 6.6 Hz, SCH2); 13C NMR (acetone-d6, 75.5 MHz) δ 128.9, 120.2 (CH), 118.7 (CN), 117.9, 103.6, 32.0 (CH2CN), 19.1 (SCH2); MS-FAB m/z 374 (M+, 8), 307 (19), 289 (12), 154 (100), 136 (71), 107 (21), 89 (20), 77 (18). 4-[(2′-Cyanoethyl)thio]-5-(methylthio)tetrathiafulvalene. Cesium hydroxide monohydrate (1.07 g, 6.35 mmol) in methanol (2 mL) was added to a solution of 4 (2.50 g, 6.68 mmol) in THF (50 mL) under an argon atmosphere and stirred at room temperature for 1 h. Methyl iodide (1.04 g, 7.35 mmol) in THF (2 mL) was added in one portion, and stirring continued overnight. The solvent was evaporated under reduced pressure and the residue partitioned between dichloromethane and water. The organic phase was dried (MgSO4) and concentrated in vacuo. Chromatography on silica gel with dichloromethane-petroleum ether (bp 40-60 °C) (7:3) as eluent afforded 4-[(2′-cyanoethyl)thio]-5(methylthio)tetrathiafulvalene (2.01 g, 5.99 mmol, 90%) as an orange powder which crystallized as orange needles from dichloromethane-petroleum ether: bp 40-60 °C; mp 79-80 °C; Rf [dichloromethane-petroleum ether (bp 40-60 °C) (7:3)] 0.61; IR (CH2Cl2) 3050, 2985, 2305, 1700, 1420, 1270, 1155, 895 cm-1; 1H NMR (acetone-d6, 300 MHz) δ 6.65 (1 H, s, dCH), 6.64 (1 H, s, dCH), 3.16 (2 H, t, J ) 6.8 Hz, CH2CN), 2.87 (2 H, t, J ) 6.8 Hz, SCH2), 2.51 (3 H, s, SCH3); 13C NMR (acetone-d6, 75.5 MHz) δ 134.7, 122.2, 120.2 (CH), 120.1 (CH), 118.7 (CN), 117.1, 104.2, 31.9 (CH2CN), 18.9 (SCH2 and SCH3); MS-FAB m/z 335 (M+, 41), 289 (12), 154 (100), 136 (76), 107 (24), 89 (26), 77 (23). Anal. Calcd for C10H9NS6: C, 35.79; H, 2.70; N, 4.17; S, 57.33. Found: C, 35.88; H, 2.55; N, 4.11; S, 60.54. 4-[(3′-Carbomethoxypropyl)thio]-5-(methylthio)tetrathiafulvalene (5). Cesium hydroxide monohydrate (1.11 g, 6.59 mmol) in dry methanol (2 mL) was added to a solution of 4-[(2′cyanoethyl)thio]-5-(methylthio)tetrathiafulvalene (2.50 g, 6.68 mmol) in dry THF (50 mL) under argon and stirred at room temperature for 2 h. Methyl 4-bromobutanoate (1.19 g, 6.59 mmol) in dry THF (2 mL) was added and stirring continuously overnight. The solvent was evaporated under reduced pressure and the residue partitioned between dichloromethane and water. The organic phase was dried (MgSO4) and concentrated in vacuo. Chromatography on silica gel with ethyl acetate-petroleum ether (bp 40-60 °C) (3:7) as eluent afforded 5 (1.33 g, 3.48 mmol, 58%) as an orange oil: Rf [ethyl acetate-petroleum ether (bp 40-60 °C) (3:7)] 0.63; IR (CH2Cl2) 3050, 2985, 2305, 1735, 1435, 1420, 1260, 895 cm-1; 1H NMR (acetone-d6, 300 MHz) 6.64 (2 H, s, HCdCH), 3.62 (3 H, s, OCH3), 2.92 (2 H, t, J ) 7.0 Hz, CH2CO2Me), 2.52 (2 H, t, J ) 7.0 Hz, SCH2), 2.45 (3 H, s, SCH3), 1.92 (2 H, quintet, J ) 7.0 Hz, SCH2CH2CH2CO2Me); 13C NMR (acetoned6, 75.5 MHz) 173.3 (CO2Me), 131.7, 125.0, 120.2 (CH), 120.1 (CH), 116.6, 104.6, 51.6 (OMe), 35.6 (CH2CO2Me), 32.4 (CH2CH2CH2CO2Me), 25.5 (SCH2), 19.0 (SCH3); MS-FAB m/z 382 (M+, 71), 289 (11), 154 (100), 136 (74), 107 (23), 89 (23), 77 (23). Anal. Calcd for C12H14S6O2: C, 37.73; H, 3.69; S, 50.37. Found: C, 37.92; H, 3.65; S, 50.88. 4-[(3′-Carbohydroxypropyl)thio]-5-(methylthio)tetrathiafulvalene (6). 5 (1.50 g, 3.93 mmol) was heated at 100 °C in 10% KOH (aqueous) (25 mL) for 1 h until solution occurred. 736

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The aqueous phase was washed with ethyl acetate, acidified with concentrated hydrochloric acid, extracted into ethyl acetate, dried (MgSO4), and concentrated. The crude product was recrystallized from dichloromethane-petroleum ether (bp 40-60 °C) to afford 6 (1.13 g, 3.07 mmol, 78%) as orange needles: mp 84-85 °C; IR (CH2Cl2) 3420 br m, 3055, 2985, 2305, 1710, 1420, 1270, 895 cm-1; 1H NMR (acetone-d , 300 MHz) δ 6.65 (2 H, s, HCdCH), 2.94 (2 6 H, t, J ) 6.8 Hz, CH2CO2H), 2.50 (2 H, t, J ) 6.8 Hz, SCH2), 2.47 (3 H, s, SCH3), 1.92 (2 H, quintet, J ) 6.8 Hz, SCH2CH2CH2CO2H); 13C NMR (acetone-d , 75.5 MHz) 173.8 (CO H), 131.6, 122.6, 120.2 6 2 (CH), 120.1 (CH), 116.4, 104.6, 35.6 (CH2CO2H), 32.2 (CH2CH2CH2CO2H), 25.5 (SCH2), 18.9 (SCH3); MS-FAB m/z 368 (M+, 16), 307 (8), 289 (12), 154 (100), 136 (71), 107 (22), 89 (22), 77 (20). Anal. Calcd for C11H12S6O2: C, 35.91; H, 3.29; S, 52.29. Found: C, 36.03; H, 3.25; S, 54.96. Protein Modification. The enzyme was modified using the procedure developed by Degani and Heller.3,4 The mediator to GOx molar ratio was ∼230:1 for both the TTF derivative 6 and ferrocenemonocarboxylic acid. A stock solution (10 mL) of HEPES buffer (0.1 M) and urea (2 M) was prepared, and its pH adjusted to 7.0 with 2 M HCl. Triton-X100 (10 mL) and DEC (125 mg, 0.65 mmol) were added to make a 0.8 M DEC solution. This solution was cooled to 4 °C in an ice bath and then either ferrocenemonocarboxylic acid (8.7 mg, 0.038 mmol) was added to 1 mL of the solution or 6 (11.9 mg, 0.032 mmol) dissolved in 0.1 M NaOH (0.3 mL) was added to 0.9 mL of the solution. After 15 min stirring, GOx (25 mg, 0.14 µmol) was added to the mixture and stirred. The mixture was then placed in the refrigerator at 4 °C for 16 h. After reaction, the enzyme samples were purified by gel permeation chromatography on disposable columns containing Sephadex G-25 M (Pharmacia Biotech Column PD-10). Columns were first stabilized with 25 mL of buffer solution (0.1 M PIPES pH 7). The sample was then added (∼1 mL of the modification solution) and another 1.5 mL of the buffer solution was added to obtain a final volume of 2.5 mL. Once the sample had run through the column, 3.5 mL of buffer was added to elute the high molecular weight fractions (i.e., the modified enzyme). Determination of Protein Concentration and Activity. After purification, the concentration of the protein was determined by the Bio-Rad protein assay.7 A 5 mL aliquot of Bio-Rad reagent and 0.1 mL of protein solution were mixed, and the absorbance at 595 nm was recorded. The protein concentration was calculated using the absorbance-protein concentration standard curve. GOx activity was assayed using the spectrophotometric enzyme-linked assay described by Barham and Trinder.22 Electrochemical Measurements. All measurements were made using a conventional three-electrode system in a single compartment cell using a large-area Pt gauze as counter and a saturated calomel (SCE) reference electrode. Glassy carbon rotating disk electrodes (area 0.385 cm2) and associated rotator and motor controller were obtained from Oxford electrodes. The electrode was polished with alumina slurry (1 and 0.3 µm) before each experiment. All measurements were performed under continuous Ar purge to exclude oxygen from the atmosphere. Membrane Electrodes. These electrodes were prepared according to the procedure of Bartlett et al.5 A drop (10 or 20 µL) of the modified enzyme solution (0.7-1.2 mg mL-1) was applied to the glassy carbon electrode. The membrane (Sigma dialysis tubing, pretreated according to the manufacturer’s instruc(22) Barham, D.; Trinder, P. Analyst 1972, 94, 142-145.

Scheme 1

tions) was placed over the electrode and held in place by a silicone rubber O-ring. This method of immobilizing the enzyme at the electrode surface has the advantages that it is very easy to carry out, that the enzyme kinetics are unperturbed from those in homogeneous solution, and that suitable theory exists23 to analyze the results. It suffers from the drawback that because of the loss of some of the solution put on top of the electrode the final loading of the electrode is unknown and is not highly reproducible. RESULTS A tetrathiafulvanyl carboxylic acid 6 was prepared for covalent attachment to glucose oxidase (Scheme 1). Starting from the readily available zinc complex 1,18,19 the bis protected thione 2 was prepared following the method of Becher et al.,20,21 and converted to the corresponding oxone 3 using mercuric acetate. Trimethyl phosphite-mediated coupling of oxone 3 with vinylene trithiocarbonate gave the bis protected TTF 4 in 29% yield. Sequential monodeprotection, using cesium hydroxide in THF, and alkylation, first with methyl iodide and then with methyl 4-bromobutanoate, gave ester 5, which was hydrolyzed to the corresponding acid 6. The acid 6 was used to modify glucose oxidase by carbodiimide coupling of the acid functionality to amine residues on the protein. After reaction, the modified enzyme was obtained in purified form by gel permeation and stored in buffer at 4 °C. IEF Gel Electrophoresis. Characterization of the modified enzyme was performed by isoelectric focusing gel electrophoresis, and for comparison a sample of ferrocenemonocarboxylic acidmodified glucose oxidase was run on the same gel. In IEF gel electrophoresis, the protein is subjected to an electric field in a gel support in which a pH gradient has been set up. The protein then migrates toward and is focused at the portion of the pH gradient where the pH is equal to the isoelectric pH (or pI) of the protein, that is the pH at which, because of the balance between the ionization of acidic and basic amino acid residues in the protein,24 the protein has zero net charge. Figure 1 shows a picture of a typical gel after development with Coomassie blue to visualize the proteins. The isoelectric pH of each enzyme sample can be estimated from the relative positions on the gel for the

bands for each protein. The native (unmodified) enzyme (track 1 in Figure 1) shows a single band at pH ∼4.5 corresponding to the known pI for the enzyme.25 The two control samples (tracks 2 and 3 in Figure 1), corresponding to enzyme samples that were taken through the standard modification procedure using ferrocenemonocarboxylic acid or 6 but without adding any carbodiimide coupling reagent, are identical to the native enzyme, indicating that in the absence of the coupling reagent there is no change in the IEF behavior of the enzyme on treatment with the different mediators. The third control (track 6 in Figure 1) corresponds to an enzyme sample taken through the standard modification procedure but without adding any redox mediator. In this case, the pI of the enzyme is significantly altered and shifts to pH ∼6.1. The shift in the pI of the enzyme on treatment with carbodiimide in the absence of any mediator can be accounted for in terms of the side reactions that can occur during the modification procedure. During the modification of the enzyme with the mediator, Figure 2, the carbodiimide first reacts with the carboxylic acid groups in the mediator followed by reactions with amine residues (in this case the lysine side chain) in the protein to make an amide link.26 Glucose oxidase itself contains

(23) Albery, W. J.; Bartlett, P. N. J. Electroanal. Chem. 1985, 194, 211-222. (24) Dunn, M. J. Gel Electrophoresis: Proteins; BIOS Scientific Publishers: Oxford, U.K., 1993; pp 65-69.

(25) Swoboda, B. E. P.; Massey, V. J. Biol. Chem. 1965, 240, 2209-2215. (26) Imoto, T.; Yamada, H. In Protein Function; Creighton, T. E., Ed.; Oxford University Press: Oxford, U.K., 1989; pp 247-277.

Figure 1. IEF poly(acrylamide) pH 4.0-6.5 electrophoresis gel for the modified enzyme and control samples. All samples were purified by gel permeation chromatography before IEF analysis and prepared in 0.05 M, pH 7.0 buffer solution. Samples: (1) native GOx; (2) GOx mixed with ferrocenemonocarboxylic acid but with no coupling reagent; (3) GOx mixed with 6 but with no coupling reagent; (4) GOx modified with ferrocenemonocarboxylic acid; (5) GOx modified with 6; (6) GOx treated with coupling reagent in the absence of a mediator.

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Figure 2. Scheme for the covalent modification of a protein using carbodiimide coupling reagent showing the various possible side reactions. See text for discussion.

a number of carboxylic acid groups (for example, from aspartate and glutamate residues) which can also react with the carbodiimide to form a reactive species that can undergo subsequent hydrolysis (route I in Figure 2), react with an amine group in the same or a different enzyme molecule (route II in Figure 2), or undergo an intramolecular reaction to produce a stable N-acylurea species attached to the enzyme (route III in Figure 2).27 These four possible reactions have different effects on the overall charge on the protein and its pI. The hydrolysis reaction (I) leaves the acid group, and thus the charge and pI, unchanged. The intermolecular reaction (II) will have an effect on the charge on the protein, but the extent of this is difficult to determine. The major effect of the intermolecular reaction, if it occurs, is to form oligomers of the protein. However, these are expected to be removed from the sample during the gel chromatography purification step following modification and therefore will not show up in the IEF electrophoresis. The formation of the N-acylurea (III) effectively removes a negative charge on the protein and replaces it with a species that may be ionized to give a positive charge. Consequently, the overall charge on the enzyme will be less negative and this will result in a higher pI. This is consistent with the results observed when the enzyme was incubated in the modification solution containing only carbodiimide. Finally, turning to the two modified enzyme samples which have been treated with both the coupling reagent and either ferrocenemonocarboxylic acid or 6 (tracks 4 and 5 in Figure 1), these both have a pI greater than the native enzyme and show bands at ∼5.2. Both are clearly different from both the native enzyme and the controls. These results indicate that the modi(27) Jones, J. The Chemical Synthesis of Peptides; Oxford Science Publications, Oxford University Press: Oxford, U.K., 1991; pp 51-54.

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fication procedure leads to the covalent modification of the enzyme. For both modified enzyme samples, the bands are broad when compared with those of the native enzyme, indicating a spread of pI values. This is consistent with nonspecific chemical modification of the protein.24 Bearing in mind that the TTF derivatives are attached to the lysine residues of GOx, we would expect the protein to become more negatively charged on modification because positively charged protonated lysine residues are exchanged for uncharged mediator groups and this would have the effect of lowering the pI of the enzyme. However, our results show that the pI shifts in the opposite direction upon modification for both the TTF derivative and ferrocene-modified GOx samples. Similar shifts were observed in other work on ferrocene-modified GOx.5 We believe that this is because when the mediator and carbodiimide are present in the modification solution formation of the N-acylurea derivative (reaction III) still takes place in parallel with the reaction of the mediator with lysine residues within the protein (reaction IV). The net effect of this is that the pI still increases but not by so large an amount as in the absence of the mediator. This is consistent with all of our IEF electrophoresis data. UV-Visible Spectroscopy. A sample of the TTF-modified GOx was studied by UV-visible spectroscopy, both with and without glucose present in the solution, and the spectra were compared to those obtained for the native enzyme under identical conditions. The resulting spectra are shown in Figure 3. In the absence of glucose, corresponding to the oxidized (FAD) form of the enzyme, the native GOx has two well-defined adsorption maxima at 380 and 465 nm. These are characteristic of the oxidized form of the flavin prosthetic group.25 On addition of glucose, and in the absence of oxygen, the enzyme is reduced

Figure 3. UV-visible spectra of native and TTF-modified GOx in the absence and presence of glucose. In both cases the absorption bands at 380 and 465 nm associated with oxidized flavin disappear when the flavin within the enzyme is reduced in the presence of glucose.

and the flavin converted to the fully reduced (FADH2) form and the two adsorption maxima characteristic of the FAD disappear. In comparison, the spectra of the TTF-modified enzyme show similar behavior with bands at 380 and 465 nm that disappear on addition of glucose, indicating the presence of the active flavin center in the modified enzyme. In addition, there is a strong absorption at shorter wavelengths with a shoulder at 320 nm and tailing into the visible region which is associated with the TTF groups attached to the enzyme. Electrochemical Characterization. The TTF derivative, 6, is an effective mediator for oxidation of GOx in homogeneous solution. Figure 4a shows cyclic voltammograms for 6 in solution with glucose oxidase both with and without glucose present. In the absence of glucose, 6 exhibits two one-electron redox processes corresponding to the TTF/TTF+ and TTF+/TTF2+ couples at 0.14 and 0.41 V. Analysis of the cyclic voltammetry data indicates that the TTF dication is unstable under these conditions and undergoes an irreversible reaction on the voltammetric timescale. Upon addition of glucose, there is a significant increase in the current corresponding to oxidation to the TTF radical cation form of 6 arising from the catalytic regeneration of the reduced form of the mediator by the enzyme. This catalytic current decreases significantly upon oxidation of the TTF derivative to its dication state above 0.3 V as a result of the poor stability of the TTF dication in this solution. This decrease in the catalytic current cannot be attributed to fouling of the electrode because the catalytic response is recovered on the return cycle. Similar results have been reported by Zhao and Luong28 for mediation of GOx oxidation by the water-soluble TTF-2-hydroxypropyl-βcyclodextrin complex, although in contradistinction, Yu et al.29 have reported efficient mediation of GOx oxidation by both oxidation states of TTF. Turning to the modified enzyme, Figure 4b shows the corresponding voltammograms for the TTF-modified enzyme with and without glucose present in the solution. In the absence of added glucose, the voltammetry corresponding to oxidation and reduction of the TTF species attached to the enzyme is clearly seen at (28) Zhao, S.; Luong, J. H. T. Anal. Chim. Acta 1993, 282, 319-327. (29) Yu, T.; Liu, H.; Deng, J.; Liu, Y. J. Appl. Polym. Sci. 1995, 58, 973-980.

Figure 4. Cyclic voltammograms recorded at a glassy carbon (area 0.385 cm2) electrode at 5 mV s-1 in the absence of glucose and in the presence of 50 mmol dm-3 glucose: (a) for 0.54 mmol dm-3 6 in 0.1 mol dm-3 pH 7 PIPES buffer containing 2.5 mg mL-1 GOx; (b) 0.09 mg mL-1 TTF-modified GOx.

around +0.19 and +0.42 V vs SCE. These potentials are close to those found for 6 in homogeneous solution, indicating that the TTF couple is not significantly perturbed by attachment to the enzyme. On addition of glucose, a significant catalytic response is observed at ∼+0.2 V at potentials corresponding to the oxidation of the attached TTF groups to the TTF+ form. Again the catalytic response decreases upon oxidation of the TTF groups to the TTF2+ state, indicating that the TTF dication attached to the enzyme is unstable on the voltammetric time scale. On the reverse sweep, the mediation recovers as the TTF+ state is reformed, indicating that the reduction in the catalytic current does not arise from poisoning of the electrode. Calibration Curve for Glucose. Electrochemical titrations of TTF-modified GOx with glucose were performed at a rotating membrane enzyme electrode. The electrocatalytic current at a constant applied potential of +0.35 V was measured after the addition of aliquots of glucose. The electrode was rotated at 9 Hz, and before the first injection of glucose, the background current was allowed to stabilize for 2.5 h. Between consecutive additions of glucose the current reached a constant value within 3 min. Figure 5 shows a plot of the limiting currents measured for each glucose addition as a function of the resulting glucose concentration. At low glucose concentrations (