Significance of uricase in oxidase-induced oxidative coloring reaction of p-phenylenediamine

Article abstract of DOI:10.1021/jo9605049

Uricase (urate oxidase, UOD) is found to induce the oxidative polymerization of p-phenylenediamine (PPD) effectively, which is a key reaction of color development in hair-dyeing and fur-dyeing practices. The significance of uricase is described by comparison to glucose oxidase (GOD), which also produces hydrogen peroxide as an oxidizing agent of PPD. In contrast to UOD, GOD inhibits the polymerization reaction. Spectroscopic and electrochemical study has revealed that the inhibition effect of GOD is ascribed to the glucose dehydrogenase activity, in which p-benzoquinonediimine (BQI) as the two-electron oxidized form of PPD works as an efficient electron acceptor to be reduced back to PPD, resulting in the inhibition of the subsequent polymerization of BQI. On the other hand, the UOD reaction does not compete with the polymerization of BQI owing to the lack of urate dehydrogenase activity in UOD. In addition, it has been found that UOD catalyzes the oxidation of PPD in the presence of uric acid by PPD oxidase-like and PPD peroxidase-like activities. These properties of UOD are favorable toward the oxidative generation of BQI from PPD and are responsible for the prominent ability in the oxidative coloring of PPD.

Full text of DOI:10.1021/jo9605049

5610
J . Org. Chem. 1996, 61, 5610-5616
Sign ifica n ce of Ur ica se in Oxid a se-In d u ced Oxid a tive Color in g
Rea ction of p-P h en ylen ed ia m in e
Masahiro Aoki,†,‡ Yoshio Tsujino,§ Kenji Kano,*,† and Tokuji Ikeda*,†
Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University,
Kyoto 606-01, J apan, and R & D Department, Yamahatsu Sangyo Kaisha Ltd.,
Senbonminami, Nishinari-ku, Osaka 557, J apan
Received March 14, 1996X
Uricase (urate oxidase, UOD) is found to induce the oxidative polymerization of p-phenylenediamine
(PPD) effectively, which is a key reaction of color development in hair-dyeing and fur-dyeing
practices. The significance of uricase is described by comparison to glucose oxidase (GOD), which
also produces hydrogen peroxide as an oxidizing agent of PPD. In contrast to UOD, GOD inhibits
the polymerization reaction. Spectroscopic and electrochemical study has revealed that the
inhibition effect of GOD is ascribed to the glucose dehydrogenase activity, in which p-benzoquino-
nediimine (BQI) as the two-electron oxidized form of PPD works as an efficient electron acceptor
to be reduced back to PPD, resulting in the inhibition of the subsequent polymerization of BQI.
On the other hand, the UOD reaction does not compete with the polymerization of BQI owing to
the lack of urate dehydrogenase activity in UOD. In addition, it has been found that UOD catalyzes
the oxidation of PPD in the presence of uric acid by PPD oxidase-like and PPD peroxidase-like
activities. These properties of UOD are favorable toward the oxidative generation of BQI from
PPD and are responsible for the prominent ability in the oxidative coloring of PPD.
Dyeing of hair and/or fur involves synthesis of the
a mild alternative to H2O2 in hair-dyeing practice.5,6
Screening of several oxidases for this purpose revealed
that uricase (urate oxidase, UOD), which catalyzes the
following reaction,
colored species by oxidative polymerization of monomeric
precursors.1 Commercial hair dyeing compositions usu-
ally consist of three types of reactive species: the dye
precursors (dye intermediates or developers), the color
couplers (modifiers), and the oxidizing agents. p-Phe-
nylenediamine (PPD) and hydrogen peroxide (H2O2) are
most frequently used as a precursor and an oxidizing
agent, respectively, while the color couplers may be
m-diamines, m-aminophenols, phenols, pyrazolones, or
â-diketones. The oxidizing agents trigger the coloring
development by oxidizing PPD into p-benzoquinonedi-
imine (BQI),2 which reacts with the couplers to form
several colored species and may also undergo electrophilic
attact of PPD to give a trimer where PPD is considered
to be a vital catalyst (Scheme 1).1b
In usual hair-dyeing practice, the coloring reaction is
initiated by mixing H2O2 directly with the organic
reactants. Such direct use of H2O2 at relatively high
concentrations (usually about 3%, i.e., 1 M) may damage
hair and skin due to the strong oxidizing power of H2O2.
In order to alleviate the damages, several enzyme reac-
tions were examined to induce in situ synthesis of colored
species. For example, tyrosinase was utilized to oxidize
polyhydroxy aromatic compounds directly to generate
melanin dyes.3,4 However, the methods were not satis-
factory in practice. One of the present authors (Y.T.) and
co-workers have presented an idea to utilize oxidases as
uric acid + O2 + 2H2O f allantoin + CO2 + H2O2
(1)
is superior to other oxidases examined including glucose
oxidase (GOD), pyruvate oxidase, lactate oxidase, glycerol
oxidase, and xanthine oxidase.6 The UOD-induced hair
coloring was as effective as and much milder than the
direct oxidation method using H2O2.
However, the significance of UOD in the hair-dyeing
practice remains unknown. If the UOD reaction worked
simply as the H2O2 supplying source, other oxidases such
as GOD could work as effectively as UOD. This question
inspired us to clarify what is happening in the oxidative
polymerization of PPD induced by UOD or other oxidases
from the viewpoint of enzyme-assisted organic synthesis.
In this work, GOD was used as a comparison to UOD.
Detailed spectroscopic and electrochemical study has
revealed that GOD reduces BQI as an efficient electron
acceptor due to its dehydrogenase activity and then
inhibits the subsequent polymerization of BQI. On the
other hand, UOD lacks such dehydrogenase activity and
does not inhibit the polymerization. Furthermore, it has
been found that UOD can function as a catalyst of the
PPD oxidation as well as the H2O2 supplying source in
the presence of uric acid to generate BQI.
* Corresponding authors.
† Kyoto University.
‡ On leave from Yamahatsu Sangyo Kaisha Ltd.
§ Yamahatsu Sangyo Kaisha Ltd.
X Abstract published in Advance ACS Abstracts, J uly 1, 1996.
(1) (a) Wall, F. E. In Cosmetics: Science and Technology, 2nd ed.;
Balsam, M. S., Sagarin, E., Eds.; Wiley: New York, 1972; Vol. 2, pp
279-343. (b) Corbett, J . F. In The Chemistry of Synthetic Dyes;
Venkataraman, K., Ed.; Academic Press: New York, 1971; Vol. 5, pp
479-495.
(2) Brown, E. In The Chemistry of the Quinonoid Compounds; Patai,
S., Rappoport, Z., Eds.; Wiley: New York, 1988; Part 2, Vol. 2, Chapter
21, p 1231.
Ma ter ia ls a n d Meth od s
Rea gen ts. Oxidases used in this study are uricase (EC
1.7.3.3, UOD) from Escherichia coli cloned with the UOD gene
(5) Tsujino, Y.; Yokoo, Y.; Sakato, K. J . Soc. Cosmet. Chem. 1991,
42, 273.
(3) Soloway, S. U.S. Patent 1966, 3251742.
(4) Fujinuma, S. J pn. Kokai Tokkyo Koho 1976, S51-47778.
(6) Tsujino, Y.; Kitayama, K.; Yokoo, Y.; Sakato, K. J . Soc. Cosmet.
Chem. J pn. 1991, 24, 220.
S0022-3263(96)00504-X CCC: $12.00 © 1996 American Chemical Society

Oxidative Coloring Reaction of p-Phenylenediamine
J . Org. Chem., Vol. 61, No. 16, 1996 5611
Sch em e 1. Mech a n ism of th e Oxid a tive P olym er iza tion of P P D a n d th e Rela ted Color in g Rea ction
of Cellulomonas flavigena7 and glucose oxidase (EC 1.1.3.4,
GOD) from Aspergillus niger. UOD was donated from Kyowa
Hakko Kogyo Co., Ltd., while GOD was purchased from Sigma.
The GOD concentrations were determined spectrophotometri-
cally using the molar extinction coefficient of 18 240 at 450
nm.8 Uric acid, glucose, and p-phenylenediamine (PPD) were
purchased from Wako and used without further purification.
All other chemicals were of reagent grade.
Ap p a r a tu s. UV-vis spectroscopy and spectrophotometry
were performed with a Shimadzu UV-2500 (PC)S spectropho-
tometer at 30 ( 2 °C. In anaerobic measurements, standard
quartz cuvettes with screw caps were used, and nitrogen gas
was passed through test solutions in the cuvettes before
measurements. Under aerated conditions, the cuvettes were
opened to the atmosphere without stirring.
All electrochemical measurements were performed with a
Bioanalytical System 100 B/W electrochemical workstation or
a Yanagimoto P-1100 potentiostat controlled with an NEC PC-
9801 RA microcomputer. Cyclic voltammetry, constant-
potential amperometry, and potential step chronoamperometry
were carried out with a three-electrode system consisting of a
glassy carbon working electrode (Bioanalytical System, i )
3.0 mm), a platinum plate auxiliary electrode, and an Ag/AgCl/
KCl(sat.) reference electrode using a conventional electro-
chemical cell at 30 ( 0.2 °C under nitrogen atmosphere. The
glassy carbon electrode was polished with 0.05 µm alumina
powder (Buehler, No. 3) before each measurement. The
alumina powder was removed by washing with water and then
by sonication in distilled water. Dioxygen concentration
measurements were carried out using an oxygen electrode
equipped with an Ag/AgCl/KCl(sat.) reference electrode (two-
electrode system, Oriental Electronics) and a 1.0-mL space-
free and air-tight electrochemical cell (Oriental Electronics)
under constant stirring. The dioxygen concentrations of test
solutions were regulated by purging air, nitrogen, oxygen gas,
or their mixture just before measurements. All spectroscopic
and electrochemical measurements were done using 0.1 M
phosphate buffer of pH 7.0, unless otherwise stated.
Oxid a se Rea ction Ra te Mea su r em en ts a n d Defin ition
of En zym e Activity. Steady state enzymatic reaction rates
of UOD and GOD were estimated by monitoring the oxygen
consumption rate using the oxygen electrode at 30 °C. The
Michaelis constant of uric acid in the UOD reaction (KS,UOD
was determined as 0.8 mM under oxygen-saturated conditions
at pH 7.0, while that of glucose in the GOD reaction (KS,GOD
)
)
was 86 mM.9 Because of the difference in the optimum pH
between UOD and GOD, one unit (U) of the enzyme activity
was defined in this work as the amount of the enzyme that
consumes 1 µmol of dissolved dioxygen (O2) per minute at pH
7.0 and at 30 °C under air-saturated conditions (0.25 mM10
as a dioxygen concentration ([O2]) in the presence of the
substrate at sufficiently high concentrations (1.3 mM uric acid
for UOD and 130 mM glucose for GOD) compared with the
corresponding Michaelis constant of the substrate. One unit
of UOD and GOD in the present definition were equivalent,
respectively, to 2.0 and 1.8 U in the unit scale estimated
according to the protocols.11
Resu lts a n d Discu ssion
Com p a r ison of UOD a n d GOD in th e Oxid a tive
Color in g Rea ction of P P D. Panel A of Figure 1 shows
spectral change of PPD associated with the UOD reaction
at pH 7.0. Upon the addition of 10 U mL-1 of UOD into
the solution containing PPD (30.8 mM) and uric acid (1.0
mM . KS,UOD), a broad absorption band increased, giving
the maximum absorption around 520 nm and a shoulder
around 430 nm. During the reaction, the practically
colorless solution of PPD changed to red-purple. Thus,
(7) Yamasaki, M.; Ishino, S.; Iwata, K.; Azuma, M.; Teshiba, S.;
Hasegawa, M.; Yamaguchi, K.; Yano, K.; Yokoo, Y.; Hashimoto, Y. J pn.
Kokai Tokkyo Koho 1994, 94, 38766.
(8) Loach, P. A. In Handbook of Biochemistry and Molecular
BiologysPhysical and Chemical Data, 3rd ed.; Fasman, G. D., Ed.;
CRC Press: Cleveland, OH, 1976; Vol. I, pp 122-130.
(9) For KS,GOD, a value of 120 mM at pH 5.6, 38 °C, was reported in:
Gibson, Q. H.; Swoboda, B. E. P.; Massay, V. J . Biol. Chem. 1964, 239,
3927.
(10) Degrand, C. J . Electroanal. Chem. 1984, 169, 259.
(11) Toyobo Enzyme Catalog 1993, p 107 and 275.

5612 J . Org. Chem., Vol. 61, No. 16, 1996
Aoki et al.
F igu r e 1. Time-dependent adsorption spectra of PPD (30.8
mM) after addition of (A) 10.0 U mL-1 of UOD in the presence
of 1.0 mM uric acid and (B) 10.0 U mL-1 of GOD in the
presence of 130 mM glucose at pH 7.0 and at 30 °C under
aerated conditions. The time interval of the spectroscopic
measurements after the addition of the enzyme was 4.0 min.
F igu r e 2. Cyclic voltammograms of 0.308 mM of PPD in the
presence of (A) 10 U mL-1 GOD and 130 mM glucose and (B)
10 U mL-1 UOD and 1.0 mM uric acid. The broken curves in
panels A and B are voltammograms in the absence of GOD
and UOD, respectively. The voltammograms were recorded
at a scan rate of 10 mV s-1 with a glassy carbon electrode in
pH 7.0 phosphate buffer at 30 °C under deaerated conditions.
the spectroscopic change is reasonably ascribed to the
oxidation of PPD with enzymatically generated H2O2,
yielding BQI and the subsequent polymerization of BQI
with PPD. The absorption bands around 430 nm and 520
nm seem to be assigned to BQI and its polymerized
product (probably the trimer in Scheme 1), respectively.12
Such spectroscopic change was not observed when either
uric acid or dissolved dioxygen was removed from the
reaction solution. Spectral changes similar to those
depicted in Figure 1A were reproduced by the direct
addition of about 3 mM of H2O2 into a PPD solution.12
These results indicate that the UOD reaction is effective
in oxidizing PPD and the subsequent coloring reaction.
Similar experiments were performed using GOD and
glucose instead of UOD and uric acid, respectively. In
this experiment, the enzymatic activity of GOD was
adjusted to be identical with that of UOD given in Figure
1A (10 U mL-1) at 130 mM of glucose. Therefore, the
initial rate of the H2O2 production by GOD was expected
to be identical with that in the case of UOD, though the
GOD reaction was expected to generate a larger total
amount of H2O2 than that in the case of UOD under these
experimental conditions, because of the higher concen-
tration of glucose compared with that of uric acid. In
spite of the setting of such experimental conditions, the
spectroscopic data depicted in panel B of Figure 1 shows
marked inhibition effect of GOD on the polymerization
reaction of PPD. Any increase in the absorption bands
was not observed. Rather, a small broad absorption band
around 500 nm disappeared, in which the band would
be ascribed to a very small amount of oxidized impurity
of PPD. The comparison between panels A and B in
Figure 1 clearly reveals that UOD is superior to GOD in
the PPD coloring reaction. This inhibiting effect of GOD
was observed even under anaerobic conditions when H2O2
(ca. 3 mM) was directly added into the solution containing
glucose and PPD. In the absence of glucose, however,
GOD itself did not affect the coloring reaction of PPD
induced by the direct addition of H2O2.
Differ en ce in Ca ta lytic F u n ction betw een UOD
a n d GOD. As an explanation for the inhibition effect of
GOD described above, we considered that BQI generated
by the PPD oxidation with H2O2 works as an electron
acceptor of GOD. The following cyclic voltammetric
experiments support the consideration clearly. Panel A
of Figure 2 shows effects of the GOD addition on the cyclic
voltammogram of PPD in the presence of glucose under
anaerobic conditions. PPD gave a reversible wave with
a midpoint potential of 197 mV vs Ag/AgCl/KCl(sat.) in
the absence of GOD. The redox wave is ascribed to the
two-electron oxidation into BQI and its rereduction. The
addition of GOD into the solution resulted in an appear-
ance of a well-defined catalytic oxidation wave and a
disappearance of the rereduction wave. This electro-
(12) When PPD was oxidized more strongly with H2O2 at concentra-
tions higher than 1 M, an absorption band around 415 nm increased
faster than that of 520 nm. Therefore, the former band can be assigned
to BQI. This is also supported by considering the longest π-π*
wavelength of p-benzoquinone (425 nm). The shoulder around 430 nm
in Figure 1A would be identical with the 415 nm band by considering
the overlapping of the 520 nm band.

Oxidative Coloring Reaction of p-Phenylenediamine
J . Org. Chem., Vol. 61, No. 16, 1996 5613
Ta ble 1. Kin etic P a r a m eter s of Glu cose Oxid a se a n d
Glu cose Deh yd r ogen a se Activities of GOD
chemical phenomenon is called mediated bioelectro-
catalysis,13-15 in which GOD functions as a glucose
dehydrogenase and BQI serves as an electron transfer
mediator; that is, the electrochemically generated BQI
is quickly rereduced by the substrate-reduced GOD
yielding PPD, which is again oxidized electrochemically.
Several redox compounds involving benzoquinones and
ferrocenes have been reported to work as mediators in
GOD-based bioelectrocatalysis.16-21 By considering the
resemblance in the electronic structure between BQI and
p-benzoquinone, the function of BQI seems to be reason-
able. For some other flavin-containing oxidases such as
pyruvate oxidase, xanthine oxidase, sarcosine oxidase,
and cholesterol oxidase have been revealed to exhibit
corresponding dehydrogenase activity.22-25
electron acceptor
dioxygen
BQI
kcatKM-1/106 M-1 s-1
1.3
1.4
1.1b
11.5
4.6a
0.4a
k
cat/103 s-1
KM/10-3 M-1
a
b
Apparent value (see also ref 30). A value of 0.83 mM was
reported at 38 °C and pH 5.6 in ref 9.
activity using BQI as an electron acceptor. However, BQI
is not so stable to be isolated.2 Thus, we employed an
electrochemical method, in which the reduced form of
electron acceptors can be used preferably to the oxidized
form for dehydrogenase activity measurements.26-28
At higher concentrations (140 mM) of glucose than
On the other hand, the addition of UOD did not
substantially affect the cyclic voltammogram of PPD in
the presence of uric acid (Figure 2, panel B), indicating
that BQI does not work as an electron acceptor of UOD.
We further examined urate dehydrogenase function of
UOD using several possible electron acceptors. Cyclic
voltammetric experiments using ferrocene instead of PPD
did not show any change in voltammograms after the
addition of UOD under conditions similar to those given
in Figure 2B, indicating that ferricinium ion does not
work as an electron acceptor of UOD. Reactivity of UOD
with 2-methyl-1,4-naphthoquinone, 2,3-dimethoxy-5-
methyl-1,4-benzoquinone, and hexacyanoferrate(III) ion
were also followed spectroscopically in the presence of
an excess amount of uric acid under anaerobic conditions.
Any enzyme reaction-related absorbance change of the
candidates did not occur upon the addition of UOD. All
these results support lack of the dehydrogenase function
in UOD.
K
S,GOD, the bioelectrocatalytic current exhibited a steady
state as shown in Figure 2A. The steady state current
measured by constant potential amperometry at 0.35 V
(is) increased with the PPD concentration ([PPD]) at least
up to 0.09 mM at the GOD concentration ([GOD]) of 8.5
× 10-7 M in deaerated phosphate buffer of pH 7.0. Under
such conditions, is is given by26-28
i ) nFA Dkcat(BQI)[GOD]/KM(BQI)[PPD] (2)
x
s
where kcat(BQI) and KM(BQI) are the catalytic constant
and the Michaelis constant of GOD for BQI: n and D
are the number of electrons and the diffusion coefficient
of PPD, respectively. F and A are the Faraday constant
and the electrode area, respectively. From the slope of
the linear is vs [PPD] plot, kcat/KM(BQI) (i.e., the rate
constant of the bimolecular reaction between GOD and
BQI) was evaluated using an experimental value of
x
nFA D estimated separately according to the Cottrell
Deh yd r ogen a se a n d Oxid a se Activities of GOD.
It is important to evaluate enzymatic kinetics of the
glucose dehydrogenase activity of GOD toward BQI and
to compare it with that of the glucose oxidase activity in
order to clarify the inhibition effect of GOD. Conven-
tional spectrophotometry might be applied to steady state
kinetic measurements of the glucose dehydrogenase
equation,29 and the results are summarized in Table 1.
Concerning electrochemical estimation of KM(BQI),
unfortunately, rigorous analytical methods have not yet
been established. In our cyclic voltammetric experiments
as shown in Figure 2A, the increased anodic current
measured from the diffusion current of PPD in the
absence of GOD at 0.3 V exhibited a Michaelis-Menten-
type dependence on [PPD] in the [PPD] range from 0.27
to 2.94 mM. Therefore, the increased current vs [PPD]
relation was analyzed in terms of the Michaelis-Menten
equation to get an apparent value of KM(BQI),30 which is
summarized in Table 1.
(13) Tarasevich, M. R. In Comprehensive Treatise of Electrochem-
istry; Srinivasan, S., Chizmadzhev, Y. A., Bockris, J . O’M., Conway,
B. E., Yeager, E., Eds.; Plenum: New York, 1985; Vol. 10, pp 231-
295.
(14) Hill, H. A. O.; Higgins, I. J . Phil. Trans. Roy. Soc. London A
1981, 302, 267.
(15) Ikeda, T.; Senda, M. In High Molecular Functional Electrodes;
Senda, M., Aizawa, M., Oyama, N., Eds. Gakkai Shuppan Center:
Tokyo, 1983, pp 131-158 (in J apanese).
On the other hand, steady state kinetic measurements
of the glucose oxidase activity of GOD was performed by
means of the oxygen electrode at a glucose concentration
of 130 mM and [GOD] ) 2.6 × 10-8 M and at pH 7.0.
(16) Kulys, J . J .; Ce´nas, N. K. Biochim. Biophys. Acta 1983, 744,
57.
(17) Ikeda, T.; Hiasa, H.; Senda, M. In Redox Chemistry and
Interfacial Behavior of Biological Molecules; Dryhurst, G., Niki, K.,
Eds.; Plenum: New York, 1988; p 193.
(18) Kulys, J .; Buck-Rasmussen, T.; Bechgaard, K.; Razumas, V.;
Kazlauskaite, J .; Marcinkeviciene, J .; Christensen, J . B.; Hansen, H.
E. J . Mol. Catal. 1994, 91, 407.
(19) Cass, A. E. G.; Davis, G.; Francis, G. D.; Hill, H. A. O.; Aston,
W. J .; Higgins, I. J .; Plotkin, E. V.; Scott, L. D. L.; Turner, A. P. F.
Anal. Chem. 1984, 56, 667.
(20) Marx-Tibbon, S.; Katz, E.; Willner, I. J . Am. Chem. Soc. 1995,
117, 9925.
(21) Gregg, B. A.; Heller, A. J . Phys. Chem. 1991, 95, 5970, 5976.
(22) Cass, A. E. G.; Davis, G.; Green, M. J .; Hill, H. A. O. J .
Electroanal. Chem. 1985, 190, 117.
(23) Kulys, J .; Wang, L.; Daugvilaite, N. Anal. Chim. Acta 1992,
265, 15.
(24) Miki, K.; Kinoshita, H.; Yamamoto, Y.; Taniguchi, N.; Ikeda,
T. Denki Kagaku 1995, 63, 1121.
(25) Nakase, H.; Kano, K.; Ikeda, T. Presented at the 41st annual
meeting of Polarography and Electroanalytical Chemistry, Nov 27-
28, 1995, Yokohama, J apan. Abstract: Rev. Polarogr. (Kyoto) 1995,
41, 91.
(26) Save´ant, J . M.; Vianello, E. Electrochim. Acta 1965, 10, 905.
(27) Coury, J r., L. A.; Oliver, B. N.; Egekeze, J . O.; Sonsnoff, C. S.;
Brumfield, J . C.; Buck, R. P.; Murray, R. W. Anal. Chem. 1990, 62,
452.
(28) Ogino, Y.; Takagi, K.; Kano, K.; Ikeda, T. J . Electroanal. Chem.
1995, 396, 517.
(29) For the Cottrell equation, see, for example: Bard A. J .; Faulkner
L. R. In Electrochemical Methods; Wiley: New York, 1980; 1980; p
142. In our case, chronoamperometric measurements were done with
x
a potential step from 0 to 0.35 V. The resulting i t values were
practically constant at least within a time (t) range from 1 to 12 s.
(30) Our recent study using digital simulation suggests that depen-
dence of the increased current in mediated bioelectrocatalysis on the
mediator concentration follows the Michaelis-Menten-type equation in
the presence of an excess amount of substrate. However, the apparent
value of KM as well as kcat is a function of not only the corresponding
real value but also time and others, and then the real KM could not be
distinguished from the apparent one. However, the real KM(BQI) value
seems to be smaller than that estimated here, because the latter
involves the depletion effect of glucose near the electrode surface.

5614 J . Org. Chem., Vol. 61, No. 16, 1996
Aoki et al.
Sch em e 2. Mech a n ism of th e In h ibition of th e Oxid a tive P olym er iza tion of P P D by GOD
F igu r e 3. Dependence of the bimolecular rate constant
between GOD and quinones (kcat/KM) on the formal redox
potential of the quinones at pH 7.0 (E°′pH 7): (1) BQI (this
work), (2) 2-bromo-1,4-benzoquinone, (3) tetrabromo-1,4-ben-
zoquinone, (4) 1,4-benzoquinone, (5) 2-methyl-1,4-benzoquino-
ne, (6) 2,3-dichloro-1,4-naphthoquinone, (7) 2-methyl-5-methoxy-
1,4-benzoquinone, (8) 1,4-naphthoquinone. The kcat/KM values
(at 25 °C) for compounds 2-8 were taken from ref 16, except
for 4b, which were taken from ref 18. The redox potentials
are referred to the standard hydrogen electrode (SHE).
F igu r e 4. Oxidative polymerization rate of PPD (ν) as a
function of (b) the H2O2 concentration in the absence of UOD
and uric acid and (9) the UOD concentration in the presence
of 1.0 mM uric acid without addition of H2O2, at pH 7.0 and
at 30 °C under aerated conditions. The ν value was defined
as an increase in the absorbance at 520 nm in a given period
(see Figure 1A), because the absorbance coefficient of the
polymerized product was unknown. The reaction at [H2O2] )
0 (or [UOD] ) 0) is related to the auto-oxidation by dissolved
dioxygen.
The dioxygen concentration dependence of the reaction
rates followed the Michaelis-Menten equation. Table 1
summarizes the kinetic parameters thus evaluated.
Surprisingly, the kcat/KM(BQI) value is ca. 9 times
larger than that of dioxygen (kcat/KM(O2)) under the
present conditions. This implies that BQI is reduced
much faster than dioxygen when BQI and dioxygen
coexist at identical concentrations less than the corre-
sponding KM values. For quinone-mediated and GOD-
catalyzed systems, the bimolecular reaction rate con-
stants between quinones and GOD have been found to
satisfy a linear free energy relation.16-18 Interestingly,
the present value of kcat/KM(BQI) falls on the relation as
shown in Figure 3. Thus, the large value of kcat/KM(BQI)
seems to originate from the high redox potential of the
BQI/PPD redox couple. On the other hand, although
some ambiguity exists in the estimation of KM(BQI),30 the
value is smaller than that of dioxygen (KM(O2)), indicating
stronger affinity of BQI to GOD compared with O2. This
strong glucose dehydrogenase activity of GOD toward
BQI is responsible for the inhibition effect of GOD in the
PPD polymerization. GOD produces H2O2 with consump-
tion of dissolved dioxygen, of which the concentration is
initially smaller than KM(O2) under aerated conditions.
The generated H2O2 oxidizes PPD to yield BQI. With a
decrease in the O2 concentration and then with an
increase in the BQI concentration, GOD becomes liable
to switch the function from glucose oxidase to glucose
dehydrogenase. Thus, BQI is reduced back to PPD,
resulting in the inhibition of the subsequent polymeri-
zation (or coloring) of BQI (Scheme 2). Disappearance
of the small peak around 500 nm (probably due to
oxidized impurity of PPD) upon the GOD addition (Figure
1B) would be also attributable to the dehydrogenase
reaction of GOD. In other words, the polymerization and
the glucose dehydrogenase reaction compete with each
other toward BQI.
Com p a r ison of UOD-In d u ced Oxid a tion a n d Di-
r ect H2O2 Oxid a tion . We further investigated the
effectiveness of the UOD-induced oxidative polymeriza-
tion of PPD as compared with the conventional oxidative
polymerization by the direct addition of H2O2. Figure 4
shows the dependence of [UOD] and [H2O2] on the
oxidative polymerization rate (ν). In the direct oxidation
with H2O2, the reaction rate increased parabolically with
[H2O2]. On the other hand, when the UOD reaction was
utilized for the oxidative polymerization, the rate in-
creased linearly with [UOD]. The UOD-induced oxida-
tion using 40 U mL-1 of UOD is as effective as the direct
oxidation with ca. 10 mM of H2O2 under the present
conditions. This is very interesting from the stoichio-

Oxidative Coloring Reaction of p-Phenylenediamine
J . Org. Chem., Vol. 61, No. 16, 1996 5615
F igu r e 5. Effect of PPD (30.8 mM) on the time-dependent dioxygen concentration change caused by 0.3 U mL-1 of UOD in the
presence of 0.1 mM uric acid at pH 7.0 and 30 °C in a space-free and air-tight electrochemical cell. The oxygen concentrations
were expressed by the output current measured using an oxygen electrode. The cell was aerated before the experiments. The
inset shows the result under identical conditions except for the UOD concentration, which was 0.03 U mL-1
.
metric point of view. During the experiments, the
spectroscopic cuvettes were opened to the atmosphere
without stirring. However, the dioxygen depletion oc-
curred in the cuvette due to the larger amount of uric
acid (1 mM) compared to the dissolved dioxygen (0.25
mM). This is evidenced by the experimental result that
the reaction rate increased up to at least about two times
by periodic stirring to supply dioxygen into the cuvette
during the kinetic measurements. Therefore, the total
amount of H2O2 produced by the UOD reaction would be
ca. 0.25 mM, or at most 1 mM even when dioxygen could
be supplied sufficiently. The direct oxidation with H2O2
at such low concentrations cannot oxidize PPD so ef-
fectively as the UOD-induced oxidation, as judged from
Figure 4. Therefore, some other functions to enhance the
PPD oxidation would be expected in UOD.
UOD-urate-O2 ternary complex is reduced with DDT
yielding UOD, uric acid, hydroxy radical, and the oxidized
form of DDT.31
We have further found that UOD accelerates the PPD
oxidation with H2O2 under deaerated conditions in the
presence of uric acid, as shown in panel A of Figure 6.
Uric acid is essential to the acceleration of the PPD
oxidation. This means that UOD exhibits PPD peroxi-
dase-like activity in the presence of uric acid, that is, that
UOD catalyzes the oxidation of PPD by H2O2 to produce
BQI in the presence of uric acid. The dependence of the
PPD peroxidase-like reaction rate on [H2O2] depicted in
panel B of Figure 6 follows the Michaelis-Menten-type
kinetics with a Michaelis constant around 2-4 mM.
Such peroxidase-like activity of UOD was also effective
for the oxidation of 4-methylcatechol: under anaerobic
conditions in the presence of uric acid, 4-methylcatechol
solution exhibited a new adsorption band with the
maximum at 390 nm, which is almost the same as that
of the 4-methyl-1,2-benzoquinone in aqueous solution
(401 nm)32 or that generated from 4-methylcatechol using
horseradish peroxidase in ethyl acetate saturated with
aqueous phosphate buffer (382 nm).33 These results
suggest that the UOD-urate complex is responsible for
the PPD peroxidase-like activity and probably for the
PPD oxidase-like activity also. The expression of such
alternative function of the enzyme-substrate complex is
not unique for UOD. For example, nucleoside oxidase
from Pseudomonas maltophilia LB-86 is reported to show
laccase-like activity only in the presence of the substrate
(nucleoside) together with the inherent nucleoside oxi-
dase activity.34,35
En h a n ced Oxid a tion of P P D by UOD. In order to
understand the enhanced oxidation of PPD by UOD, we
have selected a working hypothesis that UOD exhibits
PPD oxidase-like and/or PPD peroxidase-like function-
(s) to oxidizePPD catalytically. Figure 5 shows a time-
course of the dioxygen consumption during the UOD
reaction in the absence and presence of PPD (30.8 mM)
at 0.1 mM of uric acid and 0.25 mM of O2 (air saturation).
As shown in Figure 5, the total dioxygen consumption
as well as the initial dioxygen consumption rate (see the
inset) were increased by the addition of PPD. Such PPD-
dependent acceleration of the dioxygen consumption did
not occur in the absence of uric acid. This means that
UOD exhibits PPD oxidase-like activity in the presence
of uric acid, that is, that UOD catalyzes the oxidation of
PPD by dioxygen to produce BQI in the presence of uric
acid. Because the PPD oxidase-like reaction proceeds
parallel to the inherent urate oxidase reaction, detailed
enzymatic kinetics of the PPD oxidase-like reaction could
not be evaluated. Uchiyama et al. have reported a
similar property for other UOD (origin: not specified)
using dithiothreitol (DDT) as a reductant and explained
the phenomena by the proposed mechanism that the
(31) Uchiyama, S.; Shimizu, H.; Hasebe, Y. Anal. Chem. 1994, 66,
1873.
(32) Horner, L.; Burger, T. Liebigs. Ann. Chem. 1967, 708, 105.
(33) Yang, L.; Murray, R. W. Anal. Chem. 1994, 66, 2710.
(34) Isono, Y.; Hoshino, M. Agric. Biol. Chem. 1989, 53, 2197.
(35) Ikeda, T.; Hashimoto, Y.; Senda, M.; Isono, Y. Electroanalysis
1991, 3, 891.

5616 J . Org. Chem., Vol. 61, No. 16, 1996
Aoki et al.
F igu r e 6. Panel A: Effect of the UOD concentration on the H2O2 concentration-dependent oxidative polymerization rate of PPD
(ν) in the presence of 1.0 mM uric acid at pH 7.0 and at 30 °C under deaerated conditions. The UOD concentration/U mL-1: (O)
0, (b) 23.3, (2) 45.8, ([) 88.7. The ν values were evaluated as described in the caption of Figure 4. Panel B: UOD-accelerated
oxidative polymerization rate of PPD (νUOD) as a function of the H2O2 concentration. The symbols represent the UOD concentration
given in panel A. The νUOD values were evaluated by subtracting ν in the absence of UOD (O in panel A) from the observed ν in
the presence of UOD (b, 2, [ in panel A). The Michaelis constant of H2O2 were evaluated as (b) 2.6 mM, (2) 2.1 mM, and ([)
4.2 mM on the basis of the Michaelis-Menten equation.
Sch em e 3. P r op osed Mech a n ism of UOD-In d u ced
Oxid a tive P olym er iza tion of P P D
for the novel UOD-induced oxidative polymerization of
PPD. The detailed mechanism of the expression of the
PPD oxidase-like activity and the PPD peroxidase-like
activity remains to be solved, and the study is in progress.
Recently, high cell density cultivation and high recom-
binant UOD production of E. coli cloned with cDNA of
UOD has been established for the large-scale commercial
production of UOD.7,36 Therefore, the UOD is available
in high purity and inexpensively. In addition, this
enzyme is very stable.37 Therefore, the UOD-induced
PPD oxidation method can be subjected to practical hair-
dyeing purposes. In this method, uric acid with low
solubility can be formulated as suspension and be
continuously supplied into the reaction phase with a
decrease in the concentration. As a result, H2O2 will be
continuously generated at relatively low concentrations.
This situation is also very convenient in practice in order
to minimize the toxic effect of H2O2. In this UOD-induced
method, the reaction is triggered simply by exposure to
air. Then, all organic reactants and UOD can be formu-
lated together. This is another advantage to develop one-
pack-type products for hair-coloring.
Con clu sion
The oxidation of PPD induced by UOD can be il-
lustrated as shown in Scheme 3. Under aerobic condi-
tions, UOD oxidizes PPD as well as uric acid, yielding
BQI and H2O2 (as well as allantoin). The generated H2O2
also oxidizes PPD to yield BQI. The PPD oxidation with
H2O2 is catalyzed by the UOD-urate complex. Because
of the lack of urate dehydrogenase activity in UOD, BQI
can undergo the subsequent polymerization effectively.
The latter property as well as the expression of the PPD
oxidase-like and PPD peroxidase-like activity is essential
Ack n ow led gm en t. The authors would like to ex-
press their thanks to Kyowa Hakko Co. Ltd., J apan, for
a kind gift of uricase.
J O9605049
(36) Nakagawa, S.; Oda, H.; Anazawa, H. Biosci. Biotech. Biochem.
1995, 59, 2263.
(37) Under storage at 40 °C, over 90% of the UOD activity was
retained after 6 months.