J . Am. Chem. Soc. 15'94,116, 3617-3618 I
Electrostatic Control of the Electron Transfer Enabling Binding of Recombinant Glucose Oxidase and Redox Polyelectrolytes
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Ioanis Katakis. Ling Ye, and Adam Heller'
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Deparimeni of Chemical Engineering The Uniuersiiy of Texas ai Ausfin Ausfin. Texas 78712-1062
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Received Ociober 29, 1993 Controlling the formation of adducts between polyelectrolytes and enzymes isofinterest inenzymeimmobilization'"and protein purification.'.' Controlling the formation of adducts between redox polyelectrolytesand redox enzymes is of specific relevance to electrochemical. e.&. amperometric, biosensors. In these, electrons may flow via a 'wiring" redox polymer from reaction centers of the enzyme to an electrode. "Wiring" of an enzyme enables the measurement of its turnover rate as an electrical current.6 We report on controlling adduct formation and 'wiring" through controlledvariation of the isoelectric point of recombinant glucose oxidase rGOx, (&o-glucose, oxygen oxidoreductase EC 1.1.3.4). Electrostatic interaction between polyanionic or polycationic rGOx (if,., rGOx with net negative or positive charge) and similarly or oppositely charged redox polyelectrolytes in aqueous solutions controls the formation of complexes and the flow of electrons from the substrate-reduced enzyme to the redox polyelectrolyte. The currents increase when the electrostatic interaction is attractive and diminish when it is repulsive. Recombinant, yeast-derived rGOx,'.8 in which the amount of peripheral oligosaccharide is 5 times higher than that in wildtype Aspergillus niger-derived glucose oxidase, was used. Part of theoligosaccharide was periodate-oxidizedgJ0to polyaldehyde. By controlling the 104- concentration. reaction time, and temperature. 20-60 (spectrophotometrically assayed") aldehyde functions were formed in each enzyme subunit. The polyaldehyde was reacted with pentaethylene hexamine at a high hexamine/ enzyme ratio so as to avoid cross-linking. The resulting polySchiff base was reduced with NaBH, to the polyamine-modified enzyme. Between 120 and 360 secondary and primary amine functions were thus introduced into the periphery of each rGOx ( I ) (a) BCi~J.;khcllcnbcrgcr.A.;Larh.J.; Fisher. I. Rlmhlm. Rlophys. (b) Colovchcnko.N. P.; Kslaeva. 1. A,; Akmenko. V. K. Rlokhlmlyo 1992.57.1031. ( c ) Decher.G.;Esslcr, F.;Hong.J. D.; Lowack, K.;Schmitt.J.;Lvw. Y.Polym. Pmpr.. Am. Chcm.Sm.Dlu.Polym. Chcm.
Acto 1980.61 2.451.
1993. 34, 745.
( 2 ) (a) Furwaki. S.; h i . N. Rlotrchnol. Rlorng. 1983. 25, 2209. (h) Leain%V.; Ruckenmein. E. Collold Polym. Scl. 1988, 266. 1187. (3) (a) Roth. C. M.; hnhoff, A. M. Imnpvlr 1993.9.962. (b) Wehtje, E.; Adlercrcutz. P.; Mattiasson. E. Rlotrchnol. Rlorng. 1993.41. 171. (4) (a) Dautrcnbcrg. H.; Kcelz, 1.;Linow. K.-1.; Philipp. E.; Rother, 0. Polym. Prepr..Am. Chcm. Soc. DIU.Polym. Chcm. 1991.32,594. (b) Eucll, S. A,; Middieton. J. C.; McConnick. C. L. Polym. Preppr.. Am. Chrm. Sm. Dlu.Polym. Chcm. 1991.32.573. (c) Kokufuta. E.;Shimi~u.H.;Nskamura. I.Macromolcculrs1981.14.1178. (d)Kokufuta.E.;Takahashi.K.Polymsr
1990.31. 1177. ( 5 ) (a) Park. 1.M.; Muhobcrac.B. B.;Dubin.P.L.;Xia. I. Moc"olecu1e~ 1992. 25. 290. (h) Ganzler. K.; GrNc. K. S.; Cohcn. A. S.; Karger. E. L.; Guttman. A.; Cookc, N . C. AMI. Chcm. 1992.64.2665. (c) Nguycn. T, Q. Makromol. Chem. 1986. 187,2567. (d) Luck". P. F.; Ansarifar. M. A. 8,. Polym. J . 1990. 22, 233. (6) Helier. A. J. Phys. Chem. 1992. 96, 3579. (7) Dc Baeuciier. A,; Vasavada. A,; Dohct. P.; Ha-Thi. V.; De Beukelaer, M.; Erpicum. T.; De Clcrck, L.; Hanotier, J.; Roscnbcrg. S. RlolTchnology 1991. 9, 559. (8) Frederick,K. R.;Tung,J.; Emorick. R.S.;Ma*arz, F. R.;Chamberlain. S. H.; Vaaavada,A.; Roscnbcrg.S.;Chahb=arly.S.;Schopteptcr.L. M.; Masaey, V. 3.8101. Chrm. 1990, 365. 3793, (9) Yasuda. Y.;Takahashi, N.; Murachi. T.Bloehrmlstry 1971.10,2624. (IO) Nakamum. S . ; Hayashi. S.; Koga. K. Rlochlm. Rlophyr. Acta 1976, 445, 294. ( I I ) S a w i c ~E.; , Hauaer.T. R.; Stanley. T.W.; Elbm. W. Awl. Chcm. 1961, 33. 93.
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Figure 1. Agarme gel iaocleclric focusingof chemically altered enzymes and their redoxpolyelcctrolytccomplcxcs. Thecathode isat the topand the anode at the bottom. The pH profile is indicated a1 the right. The arrow indicate thesampleapplication point. Lanes 1.17 IEFstandards. Lanes 2-6: 8 pg of ffiOx, ffiOx-8.5. ffiOx-9, ffiOx-7. and ffiOx-6, respcclively. Lane 7: 5 pg of POr-SA. Lane 8: 5 #g of POs-EA. Lanes 9-1 I : complexes of --EA with 75.77,80 wt 40 IGOX. respectively. Lanes 12-14 complexes of POs-SA with 62.44, and 29 wt 40 ffiOx-9, respcclively. Lanes I S and 16: complexes of POs-EA with 44 and I 5 wt 96 ffiOx9. respectively. .4
FI-
Wr.EA POs.SA 2. slNc1UrCa and compitions of the redox polyelectrolytes.
molecule. while approximately one-half (36 units m g ' ) of the initial specificactivity was retained. The isoelectric points of the derivatized enzymes ranged from 3 for unmodified rGOx to 9 for the most heavily modified enzyme. Isoelectric focusing (IEF) N n S for some of these enzymes are shown in Figure I , lanes 2-6. By reacting 70 kDa poly(viny1pyridine)complexed with [Os(bpy),Cl~]"l"' with bromocthylamine, the water-soluble redox polycation POs-EA (Figure 2, left) was formed.~2-~~ and by reacting it with bromosuccinicacid, thewater soluble polyanionic zwitterion POs-SA (Figure 2, right) was prepared. The IEF NnS of these polymers are shown in Figure I , lanes 7 and 8. POs-EA. being a polycation at any pH. does not focus when migrating to the cathode. POs-SA focuses at pH 4.8. where protonation of free pyridine rings of the poly(viny1pyridine) backbone balances the exccss negative charge associated with the succinate functions. Complexing of enzymes and polyelectrolytes was observed through the IEF migration patterns'' of Figure 1. lanes 9-16. When a complex was not formed, e&, between native rGOx and POs-SA,bothofwhicharepolyanionsatpH 7, thetwocomponents migrated and focused independently at their respective isoelectric points of 3 and 4.8. However, when a complex was formed, the (12) Grim. E. A.: Hcllcr. A. J. Phyr. Chrm. 1991, 95, J976. (I31 Katakis. 1.; Hcllcr. A. AMI. Chrm. 1992. 64. 1008. (141 Y e . L : Hammerle. M.; Olrthoorn. A. J , Schuhmann. W.; Schmidt. H.-L.. Dum. J : Hiller. A. AM^ Chrm. 1993. 63. 238. (IS) Kataks, 1.; Davidson. L.; Hciler. A,. unpublished rcsulla
OOO2-7863/94/1516-361'7$04.50/0 0 1994 American Chemical Society
3618 J. Am. Chem. SOC.,Vol. 116, No. 8, 1994
Communications to the Editor Table 1. Relationship between the Current, Normalized for Bound
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t
t
0.8
0.6 0.4
ffiox
ffiOx-9
Enzyme Activity, and the Enzyme Binding Capacity of the Redox Polyelectrolyte
enzyme
mlymer
relative enzyme activity. A
ffi0x ffi0x ffi0~-9 ffi0~-9
POs-EA POs-SA POs-EA POs-SA
1.0f0.15 0.4 f 0.15 9 . 2 f 0.3 4.0f0.3
activity current, normalized I (nA) current, IIA 327 0.89 17.81 53.14
327
2 2 13
capacity (%) 75
