445

Full Paper

Electrochemical and Catalytic Properties of the Adenine

Coenzymes FAD and Coenzyme A on Pyrolytic Graphite
Electrodes
lvarez, Mar
a Jesus Lobo-Castanon, Arturo J. Miranda-Ordieres, Paulino Tunon-Blanco*
Noem
de-los-Santos-A
Departamento de Qu
mica F
sica y Anal
tica, Universidad de Oviedo, Julian Claver
a 8, 33006 Oviedo, Spain
*e-mail: ptb@fluor.quimica.uniovi.es
Received: July 15, 2004
Final version: July 30, 2004
Abstract
Redox properties of two coenzymes, flavin adenine dinucleotide (FAD) and coenzyme A (CoA) are studied on
pyrolytic graphite electrodes. Both of them exhibit an oxidation process at about 1.2 V in pH 9, associated with the
oxidation of their adenine moiety. The resulting adsorbed oxidation products have a common structure, an
electroactive quinone-imine that acts as efficient catalyst of the oxidation of NADH at low potentials. The influence
of the structure on their electrochemical features in comparison with other adenine derivatives is studied. Finally, a
modified electrode prepared with the oxidation product of CoA was used to determine NADH in a wide linear range
(1
108 2.43
105 M) with a detection limit of 3 nM.
Keywords: Electrocatalysis, Coenzyme A, FAD, NADH, Modified graphite electrodes

1. Introduction
Many metabolic enzymes require organic molecules, called
coenzymes, to carry out catalysis. Some of the most
important ones are NAD (b-nicotinamide adenine dinucleotide), FAD (flavin adenine dinucleotide) and CoA
(coenzyme A). The two first participate in electron transfer
and dehydrogenation reactions, the latter is involved in
activation and acyl-group transfers in many enzymatic
reactions. In spite of their different activities, they share
striking similarities in their structures. They have a ribonucleoside adenosine moiety, a chemically functional group
(nicotinamide, riboflavin or pantetheine respectively) and a
pyrophosphate connecting both groups (Figure 1).
We recently reported that the electrochemical oxidation
of NAD in neutral or mild alkaline media at potentials
above 1 V leads to a compound which acts as a mediator for
the oxidation of NADH [1]. This catalytic activity was
attributed to the 2,8-dioxoadenine derivative with quinoneimine structure, generated after the electrochemical oxidation of the adenine moiety in the molecule. This structure is
stabilized from nucleophilic attack by the bulk substituent in
N9 position of the adenine [2].
Coenzyme A and FAD are also adenine compounds
containing a large N9 substituent (Figure 1). Consequently,
we are interested in investigating the electrochemical
behavior of these cofactors because, in spite of the oxidation
being expected to occur in the adenine moiety, the
substituent group may strongly influence association, conformation, orientation and adsorption characteristics and
hence, the cofactors electrocatalytic properties.
Electroanalysis 2005, 17, No. 5 6

FAD has been extensively studied on several solid

electrodes such as Au [3 5], Pt [4], glassy carbon [4, 6 8],
graphite [4], and titanium oxide modified carbon fibers [9].
Nevertheless, all of these studies were focused on the redox
properties of the riboflavin moiety. On the contrary, only a
few articles concerning the electrochemistry of CoA have
been published so far. Some polarographic studies were
done [10] and CoA was determined [11, 12] to monitor the
activity of several enzymes. Finally, the electrochemical
behavior of CoA was investigated on screen-printed carbon
electrodes chemically modified with cobalt phthalocyanine
[13]. To the best of our knowledge, no electrochemical
studies dealing with the oxidation of the adenine ring of
these cofactors have been previously published, which
constitutes the goal of this work. In addition to this, both
of them are shown as precursors of a catalytic species toward
NADH oxidation.
As has been well-known for more than twenty years,
NADH can only be oxidized on solid electrodes at
appreciable rates when a great potential is applied. Furthermore, its oxidation gives rise to several products capable
of adsorbing on these surfaces and fouling them. That is why
several strategies have appeared to overcome these difficulties. Undoubtedly, the employment of electron-transfer
mediators to shift the NADH oxidation to more appropriate
and analytically useful potentials is the most popular one, as
can be seen in reviews concerning this topic [14, 15].

 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

DOI: 10.1002/elan.200403180

446

lvarez et al.
N. de-los-Santos-A

Fig. 1. Structures of some adenine coenzymes.

2. Experimental

2.3. Preparation of Modified Electrodes

2.1. Reagents

The working electrode was homemade using a 3 mm

diameter pyrolytic graphite rod (Goodfellow, UK), sealed
into a polypropylene holder with epoxy resin. The electrical
contact was a brass rod. Renewal of the electrode surface
was achieved by polishing on sandpaper and washing with
purified water in an ultrasonic bath.
The electrode modification was achieved by electrochemical oxidation of CoA or FAD. Unless otherwise stated, cyclic
potential scans between  0.2 and 1.4 V were applied to the
clean graphite electrode dipped into 0.1 M phosphate
solution pH 9, until a stable background current was reached.
After that, the electrode was submerged in a phosphate 0.1 M
pH 11 solution containing CoA and the potential was held at
0.2 V under stirring for 2 minutes. Then the stirring was
stopped and the potential was cycled 20 times between  0.2
and 1.25 V at 0.05 V s1. For the preparation of oxidizedFAD-modified electrodes, the cyclic potential scans were
applied between  0.2 and 1.4 V in phosphate solutions
pH 9 containing FAD. In both cases, the corresponding
modified electrode was rinsed with water and placed in a pure
supporting electrolyte (phosphate 0.1 M pH 9). The cyclic
voltammogram of the adsorbed electron transfer mediator
was then recorded, usually between  0.2 and 0.3 V. Finally,
NADH of the appropriate concentration was added to detect
it voltammetrically or amperometrically.

Coenzyme A (sodium salt, 96%), FAD (disodium salt,

94%), and b-NADH (disodium salt, 98%), were purchased
from Sigma (Madrid, Spain) and were used as received.
Other chemicals employed were of analytical grade and all
solutions were daily prepared in purified water using a MilliQ (Millipore) system and stored at 4 8C.

2.2. Instrumentation
Voltammetric measurements were carried out with a conventional three-electrode electrochemical cell. A platinum
wire acted as counter electrode and the working electrode
was a pyrolytic graphite electrode. All the potentials were
measured and referred to the Ag j AgCl j KCl saturated
reference electrode. The cell was driven by a computercontrolled AutoLab Pgstat-10 potentiostat (EcoChemie,
The Netherlands). Static amperometric measurements were
performed with a stationary electrode dipped into a
magnetically stirred solution. A fixed potential of 0.1 V
was applied using a PAR 400 (EG&G, USA) electrochemical detector. The output was recorded on a Metrohm E586
Labograph strip chart recorder.
Electroanalysis 2005, 17, No. 5 6

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Properties of the Adenine Coenzymes

447

3. Results and Discussion

3.1. Redox Properties of CoA
Previous studies with several adenine derivatives, systematically showed that the adsorption of these molecules was
stronger on pyrolytic graphite electrodes (PGE) than on
carbon paste ones. In fact, for adenosine 5-diphosphate
(ADP), the initial surface coverage resulted to be four times
higher and the loss of adsorbed material after 46 min of
continuous cycling was 10% lower using graphite electrodes.
For this reason this material was selected as electrode
surface.
On PG electrodes, CoA undergoes a sharp oxidation
process with a peak potential of 1.206 Vat pH 9 (Figure 2,
peak Ia). This value is very close to that obtained for other
molecules containing an adenine moiety [2]. It is wellknown that the oxidation of N9 substituted adenine compounds takes place at more positive potentials than adenine
free base [1, 2, 16]. Therefore, this oxidation process can be
attributed to the oxidation of the adenine ring that gives rise
to a 2,8-dioxoadenine derivative. The current intensity of
this process decreases in successive scans. Only when the
scan is reversed upon 1.2 V a reduction peak (IIc) is
observed on the first and subsequent backward scans at
potentials near 0 V at pH 9. On the second forward scan a
new oxidation peak (IIa) appears forming a quasi-reversible
redox system with the reduction one. The current intensity
of this process increases with the number of cycles meaning
that an accumulation of the oxidation product occurs on the
electrode surface. The only difference with other adenine
nucleotides is the appearance of two additional processes
IIIc and IVa (Figure 2 inset). The former diminishes with the
number of cycles and finally disappears, the latter at lower
potentials than IIa, is irreversible and disappears in the
second scan. The redox behavior of FAD within this
potential window was analogous to CoA. For this molecule,
neither peak IIIc nor peak IVa were observed.
A well-defined quasi-reversible redox system could be
observed when the corresponding modified electrode with
either CoA or FAD is transferred into a fresh solution
without soluble cofactor (Figure 3, dashed line). The formal
potential measured at pH 9 was found to be 0.012  0.003
and 0.009  0.003 V for CoA and FAD respectively, which
suggests that a common electroactive specie is formed
during the electro-oxidation of these molecules. The peak
separation is 7 mV at 5 mV s1 suggesting facile charge
transfer. The symmetrical profile of the peaks and the small
peak separation indicate the adsorption nature of the
process that was confirmed when a linear plot was obtained
between the current intensity and the scan rate (I/A 3

105 v/(V s1)  4.9
106, r 0.9993, 25  v  500 mV s1
for CoA), as expected for a confined species on the
electrode surface. The strength of this adsorption was
evaluated under continuous cycling. Figure 4 shows the
fractional coverage as a function of the number of cycles for
CoA. During first cycles, the decay is sharper and afterwards
the rate of decay slowed down. This behavior is similar to
Electroanalysis 2005, 17, No. 5 6

Fig. 2. Cyclic voltammograms obtained with a PGE in a 0.1 M

phosphate solution pH 9.0 containing CoA 0.1 mM. Dotted line:
first potential scan; dashed line: second scan; solid line: 10th scan.
Scan rate: 50 mV s1.

Fig. 3. Cyclic voltammograms obtained in pH 9 with a modified

PGE from 0.1 mM CoA solution in 0.1 M phosphate pH 11.
Dashed line: pure supporting electrolyte. Solid line: in the
presence of 0.1 mM of NADH.

other adsorbed mediators and indicates that in the early

times the weakest bound material is lost. However, after 100
cycles 78% of the initial coverage still remains. In comparison with other mediators, the adsorption can be considered
quite strong. Some o-quinones lose more than 50% of the
coverage [17, 18] and 3,4-dihydroxibenzaldehyde (3,4DHB) and 2,5-DHB maintain 60 and 85% respectively
after 30 min of continuous cycling [19]. The decay for nitrofluorenones derivatives stabilizes after several hundred of
cycles and seems to be more stable when adsorb on glassy
(> 90%) than on gold (> 75%) in the presence of Ca2 [20].
 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

448

Fig. 4. Normalized surface coverage of a PGE modified with

oxidation product of CoA as a function of the number of cycles.

The oxidation of the coenzyme A was carried out in

solutions of different pH (from 7 to 11). It was found that the
formal potential of the process II measured in pH 9 was
independent on the pH of the solution in which the previous
oxidation step was performed. Therefore, the oxidation of
this molecule yields only one oxidized species in the whole
pH range tested. In addition to this, the E8 is in accordance
with those obtained for other oxidized adenine derivatives
[2], suggesting that the electroactive species adsorbed on the
graphite electrode is the 2,8-dioxoadenine derivative containing a quinone-imine structure. Redox processes of these
groups involve protons, so we anticipate a dependence of the
formal potential on the pH. To verify this, the modified
electrode with oxidized CoA was immersed in solutions of
pH ranging from 7 to 11 and the formal potential was
calculated. The slope of the E8-pH plot (E8/V 0.507
0.055 pH) was very close to the theoretical value expected
for an electrode reaction involving the same number of e as
H. The width peak at half height is 56 mV at 5 mV s1 and
pH 9. The expected value for a 2 e process is 45 mV (90/n).
The difference found between experimental and theoretical
values can be explained in terms of electrostatic repulsion
due to the negative nature of the molecule [21]. CoA has not
only three negatively charged phosphate groups but also it
has a thiol group whose pKa is 9.6 [10]. Consequently, the
thiol group is partially deprotonated at pH 9. It was
observed that the peak width is smaller at pH 8 and 7,
where the thiol group is protonated, and it increases in more
alkaline media.

3.2. Electrocatalysis of NADH

The catalytic electrochemical oxidation of NADH by the
oxidized-FAD- and oxidized-CoA-modified electrodes was
explored by cyclic voltammetry. When the oxidized FADPGE electrode was placed into a 0.1 M phosphate solution
pH 9 containing NADH 0.1 mM, an increase in the oxidation current at about 0 V was observed (data not shown).
This was accompanied by a small decrease in the oxidizedElectroanalysis 2005, 17, No. 5 6

lvarez et al.
N. de-los-Santos-A

FAD reduction peak. The direct oxidation of NADH was

observed at about 0.3 V. This is characteristic of an electrocatalytic effect. The riboflavin moiety of the molecule is also
expected to be active as electrocatalyst for NADH oxidation. However, because of the low potential of this redox
system, the reaction between NADH and FAD in solution
or even immobilized on solid electrodes is slow. Gorton et al.
proposed to tune the E8 to more positive values in order to
accelerate the reaction rate taking into account the relationship found between logarithm of the second order rate
constant and the formal potential of the mediator [22]. Even
though no good electrocatalytic properties towards NADH
could be observed because its E8 was still too negative, a
positive shift of 200 mV (E8  0.250 vs SCE) was achieved
using TiO2 modified carbon fibers [9]. Karyakin et al. [8]
proposed the oxidation of FAD on glassy carbon electrodes
at extreme positive potential in aqueous acidic medium as a
new way of obtaining a NADH electrocatalyst from FAD.
They attribute the catalytic activity to a redox-polymer
generated after oxidation of the riboflavin moiety in FAD
[8]. However we have demonstrated that the catalytic
activity observed by these authors is in fact due to surfacequinone groups generated during the oxidation process until
2 2.5 V [23]. We found that, after electro-oxidation of its
adenine moiety, FAD gives rise to a mediator for NADH
oxidation more effective than that arisen from the riboflavin
one. The catalytic current obtained with this mediator was
about 6.76 mA cm2 for 0.1 mM NADH.
An identical experiment was carried out with a modified
electrode with the oxidation product of CoA, which resulted
to be a much more efficient catalyst for the oxidation of
NADH (Figure 3). In this case, a great enhancement of the
oxidation current occurs at potential near the E8 of the
redox process of the mediator and the concomitant decrease
in the reduction current was more clearly observed. Additionally, only a small oxidation current could be measured at
potential at which the non-mediated NADH oxidation takes
place (typically 332 mV in pH 9 on PGE).
The oxidation of graphite electrodes gives rise to surface
quinones that are able to catalyze the NADH oxidation. In
order to investigate the possible contribution of these
surface-quinone groups to the observed catalytic effect, a
bare PGE was subjected to the same previous oxidative step
but in the absence of cofactors. Only the uncatalyzed
oxidation of NADH appeared after transferring this electrode to pH 9 solution. Thus, the contribution of surface
quinones can be ruled out.
The catalytic efficiency can be evaluated by measuring the
catalytic current to redox process current ratio [24]. Its value
was found to be 8.2 and 4.7 for CoA and FAD, respectively,
using 0.1 mM NADH indicating that the former is a better
catalyst. This different behavior could be explained in terms
of the linear and flexible side-chain of CoA that provides
better interaction between the NADH and the CoA-based
mediator when compared with the bulky riboflavin group in
FAD.
In any case, the electrocatalytic current can be useful to
measure NADH at a more appropriate potential than with a
 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Properties of the Adenine Coenzymes

bare graphite electrode, especially with the electrode

modified by the oxidation product of CoA, for which a
reduction of 300 mV in the overvoltage needed for NADH
oxidation is achieved. For all of these reasons, this modified
electrode was employed in further experiments.
The catalytic current for NADH linearly increases with
the square root of the scan rate even at submillimolar
concentrations, suggesting a diffusion-controlled reaction.
(Icatalytic/A 1.6
105 v1/2/(V s1)1/2  8.8
107, r 0.9993,
n 25, 5 v  700 mV s1). Similar experiments performed
with PGE modified by the oxidation product of s-adenosylmethionine (SAMe) showed that the diffusion control
was only achieved at concentrations above 1 mM. Below
this value, the control gradually changed from pure diffusion
to pure adsorption found at 10 mM [25]. This discrepancy
might be explained taking into account that SAMe does not
contain pyrophosphate group and it has a positive charge in
the methionine moiety while CoA is a polyanion, which
prevents the adsorption of NADH that is also negatively
charged.

3.3. Optimization of Modification and Detection

Conditions
As mentioned above, the oxidation product of CoA is
capable of catalyzing the NADH oxidation at low potentials
with high efficiency and sensitivity. These results encouraged us to develop a modified PGE to be employed as a
transduction tool for the determination of NADH. In order
to optimize the procedure of electrode modification, several
parameters influencing the quantity of molecules adsorbed
on the electrode were studied.
As was mentioned before, the same species was generated
in all media assayed, however, the efficiency of this process
was quite different depending on the pH of generation. In
contrast with other adenine nucleosides and nucleotides the
highest amount of catalyst was obtained from solutions of
pH 11. All the adenine derivatives exhibited higher mediator surface coverage (G) between pH 8 and pH 10. In this
case, a sharp increase in G and in catalytic current was
observed when the oxidation of CoA was performed at
pH 11. Therefore, this solution was selected for further
investigations.
Preliminary experiments demonstrated that CoA could
be preconcentrated on graphite electrodes. Thus, a preconcentration step was added before electro-oxidation and
factors that control this step were evaluated. Variation of the
electrode potential, and thus, the charge on the surface, can
create a different environment, which affect the orientation,
conformation and the way of adsorption of CoA, and
eventually, it will influence the effectiveness of the oxidation
and/or the accessibility of the mediator in the subsequent
interaction with NADH. The best results were obtained
using a conditioning potential of 0.2 V for 2 min at pH 11.
Longer times did not provide higher surface coverages.
The scan rate and the number of cycles used during the
oxidation step control the electrolysis time. Both paramElectroanalysis 2005, 17, No. 5 6

449
eters were evaluated and the highest G was achieved using 20
cycles at 50 mV s1. It is worth noting the behavior with
respect to scan rate. The amount of catalyst sharply
decreases when the scan rate increases. This trend was also
observed for other adenine nucleotides (AMP, ADP and
ATP) and was just the opposite for adenine nucleosides
(SAMe [25], cladribine [26] and fludarabine [27]), suggesting the relevant contribution of phosphate groups to the
stabilization of the catalytic species. N9-substituent in
adenine moiety was previously shown to be an essential
requirement to observe any catalytic effect attributed to
oxidized adenine derivatives. It is remarkable to note that
polynucleotides (dA20 [28] and polyA [29]), which present
phosphate groups, behave as nucleosides. The formation of
other non-catalytic products preferentially to the catalytic
species at low scan rates or the destruction of the catalyst by
other radical species as well as their polymeric nature might
justify their behavior.
The need for reaching the oxidation potential of CoA to
generate the catalyst was apparent again when the upper
potential of the sweep was varied. The G increases up to
1.25 V. A plateau was found using more positive potentials,
therefore this value was chosen as final potential in the cyclic
voltammetry procedure to prepare the modified electrode.
Finally, the concentration of CoA in the oxidation step
was optimized and resulted to be 0.1 mM. At higher
concentrations, similar surface coverages were obtained
likely because not all the adsorbed material is electroactive
or cannot be easily oxidized. In addition to this, no better
catalytic current were obtained using higher concentrations
of cofactor. Figure 5 shows the electrocatalytic current as a
function of the surface coverage. As can be seen, the
catalytic current increases with increasing surface coverages
reaching a plateau for coverages above 8.2
1011 mol
cm2. This value is lower than that found for adenine
nucleotides (5
1010 mol cm2) [2], which is in good
agreement with the smaller size of these derivatives in
comparison with CoA. This implies that the monolayer is
reached with lower G.

Fig. 5. Variation of the catalytic current measured for a 0.1 mM

NADH solution with the surface coverage of a PGE modified by
oxidation product of CoA.
 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

 lvarez et al.
N. de-los-Santos-A

450
In general, optimal conditions of catalyst generation may
differ from the best conditions for NADH determination.
Transferring the modified electrode to other solution allows
us to optimize separately both steps. The pH of the detection
solution was varied from 7 to 12. All the adenine-based
mediators previously assayed showed higher catalytic
currents in more acidic media, although high catalytic
activity remains in alkaline media in contrast to most
mediators studied so far. In this case, following a similar
trend, the highest catalytic currents were obtained at pH 7
and 8. However, in order to achieve a greater reduction of
the overvoltage, pH 9 was chosen because of the decrease in
the analytical signal can be overcome by the benefits of
measuring at lower potentials (better selectivity).

The stability was evaluated carrying out calibration

experiments after different periods of time and keeping
the modified electrode in phosphate 0.1 M pH 9 when not in
use. Although the adsorbed mediator is quite stable, the
addition of NADH induces a rapid loss of sensitivity sharper
in the early times. It could be due to molecules leaching from
the electrode surface or electrode fouling by oxidation
products of NADH. After 8 hours the sensitivity is still 80%
of the initial. The electrode can be used for 3 days and during
the fourth day, an increase in the response time up to 96 s
was observed. Nonetheless, the electrode modification is
very simple and rapid thus, the device can be easily prepared
daily.

4. Conclusions
3.4. Amperometric Measurement of NADH
The catalytic oxidation of NADH by oxidized CoA
modified PGE was carried out amperometrically in stirred
solutions using an applied potential of 0.1 V to assure that all
the mediator is quickly oxidized on the electrode surface. A
typical amperogram is displayed in Figure 6. A linear
dynamic range of more than 3 orders of magnitude was
found, between 1
108 and 2.43
105 M (I/A 0.0264
[NADH]/M 7
109, r 0.999, n 15). The detection
limit was 3 nM, one of the lowest reported up to date and
very similar to those reached with other adenine nucleotides. The response is very fast (8.5 s) and reproducible
(4.2%, n 5). The reproducibility was evaluated from the
slope of five calibration plots performed with different
modified electrodes.

In spite of having different metabolic roles and participating

in different reactions, the three coenzymes NAD, CoA and
FAD share some structural similarities and we have now
demonstrated that this confers them very similar electrochemical properties such as acting as mediators for NADH
oxidation after being oxidized at potentials above 1.2 V in
pH 9.
The increase of the levels of NADH in cells seems to be
promising to discriminate between normal and carcinogenic
cells. In this context, the use of these molecules as precursors
of an adsorbed mediator to prepare modified electrodes
constitutes an alternative to solve this analytical problem
due to their sensitivity and easy preparation. Additionally, it
could be used to determine either more than 300 substrates
of the NAD/NADH-dependent dehydrogenases or the
enzyme activity of these dehydrogenases.

5. Acknowledgements
Authors thank FICYT for financial support (Project N0 PB lvarez also thanks
EXP01 28) and Noem
de los Santos A
Ministerio de Educacion, Cultura y Deportes (Spain) for
FPU grant.

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Fig. 6. Amperometric response to NADH of a PGE modified

with the oxidation product of CoA in a magnetically stirred
solution (phosphate 0.1 M pH 9) at 0.1 V.
Electroanalysis 2005, 17, No. 5 6

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 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim