ELSEVIER

Bioelectrochemistryand Bioenergetics36 (1995) 157-160

Short communication

Amperometric assay based on an apoenzyme signal amplified using

N A D H for the detection of FAD
Atsunori Hiratsuka, Mikio Kawasaki, Kiyoshi Hasebe
Division of Material Science, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo, Hokkaido 060, Japan

Received4 August1994
Keywords: FAD sensor; FMN; Riboflavin; Amperometric assay; NADH

1. Introduction
An enzyme is generally characterized by having both
a high degree of specificity and a high catalytic efficiency, and there have been many attempts to use
these enzymatic functions. For example enzymes have
been used as sensor materials and for the detection of
various substrate compounds [1,2]. However, enzymes
have multifunctional receptors, so that the enzymatic
reaction is regulated and controlled by cofactor and
allosteric modulators. It is known that the enzymes
need cofactors, such as metals or nonprotein organic
substances, in order to express their catalytic activity.
The protein portion of the apoenzyme is catalytically
inactive. It follows that, if allesterism is utilized, these
mechanisms can be applied to the detection of organic
compounds that are difficult to recognize by ordinary
methods. Several biosensors based on the apoenzyme holoenzyme reaction which can be used to detect metals have been reported [3,4]. However, this kind of
sensor has rarely been used for the determination of
organic compounds. Therefore we have developed a
method using an enzymatic reaction which includes an
organic compound as a cofactor of the enzyme. The
type of enzyme that catalyzes the reaction requires a
cofactor to participate in the electron transfer step of
the overall reaction. In addition, the cofactor should
show allosteric effects and its effects on the enzymatic
reaction should be detectable by electrochemical methods. On the basis of these requirements, we investigated the behavior of salicylate hydroxylase, which is a
cofactor in the activation of flavin adenine dinucleotide
(FAD), and developed a new amperometric assay.
FAD is a derivative of vitamin B 2 with the 7,8-dimethyl-isoalloxazine structure. Almost all the vitamin
B 2 derivatives exist as FAD in vivo. Other vitamin B 2
0302-4598/95/$09.50 1995 Elsevier ScienceS.A. All rights reserved
SSDI 0302-4598(94)01755-7

derivatives, such as riboflavin and flavin mononucleotide (FMN) are of less importance. In particular, it
is believed that trace amounts of FAD play an important role in physiological processes, for example
metabolism and normal growth. The FAD-bound enzyme is activated and converts salicylate into catechol.
Thus catechol can easily be detected by monitoring the
redox currents electrochemically [5]. We reasoned that
when FAD is removed from the enzyme active site, the
apoenzyme cannot be activated and no electrochemical
signal will be observed. However, when FAD is added
to the apoenzyme, the electrode response of catechol
will be promoted by activated enzyme. In order to
increase the electrochemical signal nicotinamide adenine dinucleotide (NADH) was used to mediate catechol reduction. This catalytic magnification of the FAD
signal may be more effective than applications which
do not use catalytic reactions. It is also expected that
this method will be suitable for the determination of
FAD if the measurement conditions are investigated in
detail.

2. Experimental
2.1. Materials

Salicylic acid was obtained from Wako Pure Chemical Industries Ltd. Pyrocatechol and riboflavin phosphate sodium salt dihydrate (FMN) were purchased
from the Kanto Chemical Co., Japan. Riboflavin and
salicylate hydroxylase ($2907) were supplied by Sigma.
Flavin adenine dinucleotide disodium salt (FAD-Na 2)
and /3-NADH were obtained from Merck AG, Germany, and the Oriental Yeast Co., Japan, respectively.

158

A. Hiratsuka et al. / Bioelectrochemistry and Bioenergetics 36 (1995) 157-160

2.2. Methods
In the absence of FAD, the apoenzyme is unable to
convert salicylate and /3-NADH to catechol and flNAD +. However, when FAD is added to the apoenzyme, it is activated and catechol is formed. Hence,
FAD can be detected indirectly by using electrochemical methods to monitor catechol production. The electrochemical measurements were performed as follows.
Appropriate amounts of purified apoenzyme,
NADH and salicylate were prepared and dissolved in
the buffer solution. Catechol was produced enzymatically from salicylate acid when FAD was added to the
solution. The signal from electroactive catechol is increased in the presence of NADH. The amount of
FAD present is estimated from electrochemical measurements of catechol.

2.3. Apparatus
The electrochemical measurements were performed
using a BAS 100W system with a three-electrode cell
consisting of a Au working electrode, a Ag/AgC1 reference electrode and a Pt wire counter-electrode. All
potentials were measured against the Ag/AgC1 reference. The enzymatic reaction was performed at a temperature of 50 0.5C which was maintained by a
water bath.

2.4. Preparation of the apoenzyme

Various methods are available for the extraction of
FAD from the holoenzyme. For example, the Warburg-Christians' method [6], the dialysate method [7],
the ion exchange method using a TEAE-cellulose column [8] and the hydroxyapatite method [9] are well
known. We selected the Warburg-Christians' method
based on ammonium sulfate fractionation which is
commonly used for purification and proved to be a
suitable method for obtaining salicylate hydroxylase.
The apoenzyme was prepared and purified as follows.
To 1 ml of the solution containing about 10 mg of
holoenzyme was added 1 ml of a saturated ammonium
sulfate solution (pH 2.7). The precipitate was collected
by centrifugation after the mixture had been kept
overnight in refrigerator, washed with 60% saturated
ammonium sulfate solution (pH 2.7), and dissolved in 1
ml of 20 mM phosphate buffer (pH 7.5).

3. Results and discussion

3.1. Electrode response of catechol and amplification of

the signal
Cyclic voltammograms (CVs) for catechol with and
without NADH are shown in Fig. 1. The solution

--Co)

200

400

600

E / m V vs. Ag/AgCI
Fig. 1. Redox behavior of catechol containing NADH (0-20 mM) in
20 mM phosphate buffer (pH 7.5) at 50C. Scan rate, 50 mV s-1.
(a) 0 NADH; (b) 10 mM NADH; (c) 20 mM NADH.

contained 1.1 mM salicylate in 20 mM phosphate buffer

(pH 7.5) at 5&C. No oxidation peak current appeared
in the phosphate buffer solution. NADH was electrochemically inactive under these conditions. However,
in the presence of catechol, oxidation and reduction
peaks were observed at +400 mV and at + 100 mV
respectively (Fig. l(a)). When NADH was added to the
solution containing catechol, the oxidation peak current increased with increasing concentration of NADH.
However, the oxidation peak potential shifted in the
positive direction and the peak became less well defined, while the reduction peak current decreased (Figs.
l(b) and l(c)). A similar phenomenon was reported by
Tse and Kuwana [10]. The electrochemical reaction of
catechol to quinone mediated by NADH is as follows:
electrode reaction
OH
catalytic reaction
+ NADH+H+

C:o

+ 2e- +2H

OH + NAD+

Catechol can be electrochemically oxidized to oquinone, which is reduced and recycled to the dihydro
form of catechol by protons from NADH oxidization.
Thus the increased peak current is attributed to catalysis of NADH oxidation by quinone moieties. Because
of this catalytic reaction, catechol can be detected
sensitively.
Fig. 2 shows the CVs of a solution containing salicylate, NADH and apoenzyme obtained in the presence
and absence of FAD. In the absence of FAD, the
prepared apoenzyme was completely inactive and could
not generate catechol. Therefore no oxidation currents
appeared in the potential range from - 1 0 0 to + 500

A. Hiratsuka et al. / Bioelectrochemistry and Bioenergetics 36 (1995) 157-160

I ._

Table 1
Interference with the FAD signal by FMN and riboflavin
FAD
FAD + FMN
FAD + RF
Current I/A
-8.89 10-s
- 10.6x 10-s
-9.00x 10-s
Response %
100
119
101

--(a)"

0 .,--

Experimental conditions: 2.1 /zM FAD+ 1.0 mM NADH+ 1.1 mM

salicylate in 20 mM phosphate buffer (pH 7.5) at 50C. Potential,
+ 500 mV vs. Ag/AgC1.

'< -1
~

-2

-3

-4

159

--~)
I

200

400

600

E / m V vs. Ag/AgC!
Fig. 2. CVs of (a) apoenzyme and (b) FAD-bound apoenzyme obtained in 1.0 mM N A D H + 1.1 mM salicylate in 20 mM phosphate
buffer (pH 7.5) containing salicylate hydoroxylase at 50C. Scan rate,

solution containing the prepared apoenzyme, and the

oxidation current at +500 mV was monitored. No
oxidation current of catechol was observed in the absence of FAD. When FAD was added to the solution,
it was found that the oxidation currents depended on
the FAD concentration. This configuration discriminated between different concentrations of analyte in
the range 10-100 nM.

3.2. Interference

50 mV s-~.

mV. In the presence of FAD, a dramatic change in

oxidation current was observed. This change may be
due to the enzymatic formation of catechol, which is
electroactive in the potential range scanned. The enzymes bind with the FAD added to the solution containing apoenzyme to activate and catalyze the reaction
of salicylate to catechol. Therefore the apoenzyme is
converted to holoenzyme in the presence of FAD.
Hence FAD can be detect by monitoring the oxidation
currents of catechol.
Fig. 3 shows the time dependence of the oxidation
currents at various FAD contents. FAD at concentrations ranging from 0.42 to 3.36 /zM was added to the

0.2

0.0

(a)T

Almost all vitamin B 2 in living bodies is present as

FAD, and FMN and riboflavin are of less importance.
However, their effects on the electrochemical signal
were investigated. Solutions of FMN and riboflavin
were prepared in 20 mM phosphate buffer (pH 7.5)
containing 1.1 mM salicylate and 1.0 mM NADH.
Another solution was prepared in 20 mM phosphate
buffer (pH 7.5) containing 1.1 mM salicylate, 1.0 mM
N A D H and 2.1 /zM FAD. The response of electrodes
with apoenzyme, N A D H and salicylate in the solution
was monitored and compared with the oxidation current obtained with 2.1/~M FAD alone. The results are
summarized in Table 1. The enzymatic activity was not
observed when one of the structural analogs was added
to the reactant instead of FAD. Table 1 also shows the
enzymatic ability of these compounds in the presence
of 2.1 /~M FAD. The results of monitoring the oxidation currents of catechol show that no significant interference is produced by the presence of equivalent
concentrations of FMN and riboflavin.

-0.2
4. C o n c l u s i o n s

,~< -0.4
.~

-0.6
-0.8
-1.0
.1.20

(d)

I
5

,
t
I
10
15
20
Time / min

I
25

30

Fig. 3. Time dependence of catechol oxidation currents at various

FAD concentrations 1.0 mM N A D H + I . 1 mM salicylate in 20 mM
phosphate buffer (pH 7.5) at 50C; potential, + 500 mV vs. Ag/AgCI):
(a) 0 FAD; (b) 0.42 p.M FAD; (c) 1.68/~M FAD; (d) 3.36/~M FAD.

The use of allosterism, such as the a p o e n z y m e - F A D

interaction, allows the detection of organic compounds
which are difficult to sense using standard methods.
Only FAD can induce the signal. The electrode reaction of catechol can be amplified by using N A D H as a
mediator. Such that that this method can be used to
detect trace amounts of FAD, i.e. the signal sensitivity
is increased. More detailed investigations of amplification of the signal using mediators and enzyme immobilization are now in progress. It is expected that the
immobilized apoenzyme will be useful for recycling
applications.

160

A. Hiratsuka et al. /Bioelectrochemistry and Bioenergetics 36 (1995) 157-160

References
[1] R.F. Taylor, Protein Immobilization, Dekker, New York, 1991.
[2] A.P.F. Turner, I. Karube and G.S. Wilson, Biosensors, Oxford
University Press, Oxford, 1987.
[3] R.B. Thompson and E.R. Jones, Anal. Chem., 65 (1993) 730.
[4] I. Satoh, T. Kasahara and N. Goi, Sensors Actuators, B1 (1990)
499.
[5] J.E. Frew, S.W. Bayliff, P.N.B. Gibbs and M.J. Green, Anal.
Chim. Acta, 224 (1989) 39.

[6] O. Warburg and W. Christian, Biochemistry, 240 (1938) 368.

[7] H. Theorell and A.P. Nygaard, Aeta Chem. Scand., 8 (1954)
1649.
[8] K. Yagi, M. Naoi, M. Harada, K. Okarnura, H. Hidaka, T.
Ozawa and A. Kotaki, J. Biochern., 61 (1967) 580.
[9] Y. Miyake, K. Aki, S. Hashirnoto and T. Yamano, Biochirn.
Biophys. Acta, 105 (1965) 86.
[10] D.C. Tse and T. Kuwana, Anal. Chem., 50 (1978) 1315.