+
Electrical Contacting of FAD and NAD(P) Enzymes
A R T I C L E S
+
(
PQQ)-containing enzymes.9 The vast majority of redox-
integrated NAD -dependent enzyme-electrode was generated
by the two-dimensional cross-linking of an affinity complex
+
enzymes are, however, NAD(P) /NAD(P)H-dependent biocata-
lysts, and their electrical contacting reveals fundamental diffi-
culties due to the diffusional nature of the operation of these
cofactors. That is, the biocatalytic activity of these enzymes
+
generated between a PQQ-NAD monolayer and the respective
cofactor-dependent enzyme, for example, lactate dehydroge-
nase. Nonetheless, the generation of the electrically contacted
flavoenzyme-electrode, or of the NAD -dependent enzyme-
22
+
+
involves the diffusion of the NAD(P) /NAD(P)H cofactors into
the proteins, the formation of temporary complexes that enable
electron (hydride) transfer between the redox center and the
cofactor, and the subsequent diffusion of the reduced (or
electrode, required the use of the scarce amino-functionalized
+
23
FAD and NAD cofactors, respectively. This turned the
reconstitution of the enzymes or the lateral cross-linking of the
3
+
oxidized) cofactor from the protein matrixes. For the design
NAD -enzyme complexes to yield electrically contacted enzyme-
of enzyme-electrodes for bioelectronic applications, however,
it is essential to generate integrated biomaterial-electrodes that
lack diffusional components. A further difficulty accompanying
electrodes to be of limited applicability.
Recently, we reported in a preliminary communication on
the use of a boronic acid-functionalized relay monolayer as an
active interface for the ligation of native flavin adenine dinucleo-
tide, FAD, and for the surface reconstitution of the flavo-apo-
enzyme glucose oxidase on the monolayer to yield an electrically
+
NAD(P) /NAD(P)H-dependent enzymes relates to the poor
+
electrochemical properties of the NAD(P) /NAD(P)H cofactors.
The direct electrochemical oxidation of NAD(P)H or the direct
+
24
reduction of NAD(P) is kinetically unfavored leading to high
contacted enzyme-electrode. Here we wish to present the
overpotentials1 that structurally change the respective cofactors
0
detailed comprehensive study characterizing the generation of
integrated electrically contacted enzyme-electrodes of the fla-
11
and inhibit their biological function. Different redox-mediators
1
2-14
+
were employed for the catalyzed oxidation of NAD(P)H.
voenzyme glucose oxidase, the NAD -dependent enzyme lactate
+
Electroactive relays, such as o-quinones, p-quinones, phenazine,
phenoxazine, and phenothiazine derivatives, ferrocenes, and Os-
complexes, were used as catalysts for the oxidation of NAD-
dehydrogenase, and the NADP -dependent biocatalyst malate
+
dehydrogenase by the ligation of the native FAD, NAD , and
+
NADP cofactors to a relay boronic-acid monolayer associated
(
P)H. A particularly effective catalyst for the electro-oxidation
with a Au-electrode. The surface-reconstitution of the apo-
glucose oxidase on the FAD monolayer, or the two-dimensional
1
5,16
of NAD(P)H is pyrroloquinoline quinone (PQQ).
enhanced electrocatalyzed oxidation of NAD(P)H in the pres-
ence of Ca -ion was reported, and the electrocatalytic
regeneration of NADP in a PQQ-malic enzyme monolayer
assembly was achieved. Similarly, electrocatalyzed reduction
of NAD(P) was accomplished in the presence of Rh-
complexes.17
The
+
cross-linking of the complexes generated by the NAD or
2+
15
+
NADP monolayers with the respective enzyme, led to the
+
electrically contacted enzyme-electrodes.
16
+
Experimental Section
Chemicals. Lactate dehydrogenase (LDH, EC 1.1.1.27 from rabbit
muscle, type II), malate dehydrogenase (MalD, EC 1.1.1.40 from
chicken liver), and glucose oxidase (GOx, EC 1.1.3.4 from Aspergillus
niger) were purchased from Sigma and used without further purification.
Modified NAD+ derivatives were linked to polymer ma-
trixes18 and coupled to NAD -dependent enzymes. The
coimmobilization of the polymer-bound cofactors and biocata-
lysts led to the design of bioreactors for biocatalytic transforma-
tions and to the organization of biosensor devices.21 An
+
19
7
b
Apo-glucose oxidase (apo-GOx) was prepared by a modification of
the reported method.25 The cofactors of LDH, MalD, and GOx -
2
0
+
â-nicotinamide adenine dinucleotide (NAD ), â-nicotinamide adenine
+
dinucleotide phosphate (NADP ), and flavin adenine dinucleotide
(
10) (a) Blaedel, W. J.; Jenkins, R. A. Anal. Chem. 1975, 47, 1337-1343. (b)
Samec, Z.; Elving, P. J. J. Electroanal. Chem. 1983, 144, 217-234. (c)
Burnett, J. N.; Underwood, A. L. Biochemistry 1975, 5, 2060-2066. (d)
Schmakel, C. O.; Santhanam, K. S. V.; Elving, P. J. J. Am. Chem. Soc.
(FAD), respectively - were purchased from Sigma and used without
further purification. All other chemicals, including pyrroloquinoline
quinone (PQQ), 3-aminophenylboronic acid, 2,2′-dithio-bis(ethaneam-
ine) (cystamine), dithiobis(succinimidylpropionate), 1-tetradecanethiol,
1
975, 97, 5083-5092.
(
11) Cunningham, A. J.; Underwood, A. L. Biochemistry 1976, 6, 266-271.
12) (a) Katakis, I.; Dominguez, E. Mikrochim. Acta 1997, 126, 11-32. (b)
Gorton, L.; Persson, B.; Hale, P. D.; Boguslavsky, L. I.; Karan, H. I.; Lee,
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Society: Washington, D.C., 1992; Chapter 6, pp 56-83.
(
4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt (HEPES),
tris(hydroxymethyl)aminomethane hydrochloride (Tris), 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC), glutaric dialdehyde, â-D-
(+)-glucose, lactic acid, L-malic acid, and ferrocene monocarboxylic
(13) (a) Jaegfeldt, H.; Kuwana, T.; Johansson, G. J. Am. Chem. Soc. 1983, 105,
1
1
805-1814. (b) Miller, L. L.; Valentine, J. R. J. Am. Chem. Soc. 1988,
10, 3982-3989.
acid, were purchased from Sigma and Aldrich and used as supplied.
Ultrapure water from Seralpur Pro 90 CN source was used in all
experiments.
(
14) (a) Gorton, L. J. Chem. Soc., Faraday Trans. 1 1986, 82, 1245-1258. (b)
Persson, B.; Gorton, L. J. Electroanal. Chem. 1990, 292, 115-138. (c)
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Modification of Electrodes. Au-electrodes (0.5 mm diameter Au
5
4.
2
wire, geometrical area ca. 0.29 cm , roughness factor ca. 1.4) were
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used for modifications. The Au-electrodes were cleaned by boiling in
(
16) Willner, I.; Riklin, A. Anal. Chem. 1994, 66, 1535-1539.
17) (a) Ruppert, R.; Hermann, S.; Steckhan, E. Tetrahedron Lett. 1987, 28,
2
M KOH for 1 h followed by rinsing with water. The electrodes were
(
6
583-6586. (b) Wienkamp, R.; Steckhan, E. Angew. Chem., Int. Ed. Engl.
1
982, 21, 782-783. (c) Chardonnoblat, S.; Cosnier, S.; Deronzier, A.;
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V. Biotechnol. Appl. Biochem. 1992, 15, 303-310.
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(
(
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8
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