M. R. L. Stratford et al. / Bioorg. Med. Chem. 20 (2012) 4364–4370
4365
ScanIt (version 1.0) software (Ó van Baten & Baur, 2002). Kinetic
-
N
Cu2+
N
Cu2+
O
O2
N
analysis was conducted using the Origin software (version 7,
OriginLab Corp., Northampton, MA, USA). Simulations were per-
formed using an in-house computer model. Spectral changes were
examined using the kinetic mode of the UV–vis Chemstation
A0801(66) software (Agilent Technologies, Hannover, Germany).
The oxygen uptake was found to fit first-order kinetics from
which the total oxygen utilisation (Ut) and the inactivation rate
constant (k2) were obtained in accordance with the equation:
N
N
O
-
N
N = histidine ligand
oxy-tyrosinase
catechol:
a monooxygenase
substrate
hydroquinone: not
a monooxygenase
substrate
HO
HO
HO
OH
H
N
N
N
H
N
O
-
O
-
N
N
N
N
N
Cu2+
Cu2+
Cu2+
Cu2+
k
-
Ut ¼ 1 E0ð1 ꢀ expðꢀk2tÞÞ
O
N
-
N
O
N
-
-
O
k2
O
H
O
where E0 = the initial enzyme concentration and k1 = the catecho-
lase rate constant, as described by Land and co-workers.8 For large
t the solution is Ut = (k1/k2) E0. Analysis of the oximetry data for
first-order exponential decay (using Origin software) gave values
for Ut and k2. Values of k1 were estimated from the best fit of the
first-order equation to the oximetry data using an in-house com-
puter model.
O
H
N
H
N
Cu2+
O
-
N
N
N
N
Cu2+
-
-
O
O
H
O
2.1.2. Hplc/mass spectrometry
Incubations were made at room temperature using 2 mL speci-
men tubes containing millimolar substrate(s) in deionised water to
which 50 units of tyrosinase were added. Control incubations were
made without tyrosinase.
quinone
reductive Cu0
elimination
elimination
H+
N
H2O
N
H2O
N
Cu1+
N
Cu2+
Separations were carried out using a Waters 2695 separations
module (Waters, Elstree, UK) with an Atlantis dC18, 150 ꢁ
3.0 mm hplc column (Waters) maintained at 35 °C. The hplc elu-
ents were: A, 10 mM formic acid; B, acetonitrile, with gradients
of 5–70% B (catechol), 2–70% B (DOPA) and 20–85% B (4-ethylphe-
nol) over 5 min. The flow rate was 0.5 mL minꢀ1, which was split
N
N
N
N
N
Cu1+
Cu0
N
N
N
O-
O
deoxy-tyrosinase
O
+
O
O
l
l minꢀ1
H
after diode-array detection to give a flow rate of ꢂ200
O
inactivated tyrosinase
to the mass spectrometer. The eluent was monitored using a
Waters 2996 diode-array detector (215–450 nm, 2.4 nm resolu-
tion) and a Waters Micromass ZQ mass detector (mass range
115–500 Da) in negative ion mode using electrospray ionisation,
and using Waters Empower 2 software. The mass detector em-
ployed the following conditions: capillary voltage, 2.1 kV; cone
voltage, 20 V; source temperature, 120 °C; desolvation tempera-
ture, 425 °C; desolvation gas flow, 450 L hꢀ1; cone gas flow,
Scheme 1. Catechol is a monooxygenase tyrosinase substrate and hydroquinone is
not.
the case of dopa) which we interpret as evidence that 1,4-benzo-
quinone is formed by redox exchange of hydroquinone with the
enzyme-generated ortho-quinone.
100 L hꢀ1
.
Oxygen uptake (Fig. 2) showed increased oxygen utilisation in
all cases compared to the controls without hydroquinone. In the
case of catechol this was partly accounted for by a decrease in
the inactivation rate (k2) derived from the first-order kinetic
parameters and partly due to an increase in oxidation rate (k1).
In the case of dopa, as the formation of 1,4-benzoquinone de-
creased there was evidence of increased formation of dihydroxyin-
dole 4b, although indolequinone 4c formation (k = 473 nm) was
little affected (Fig. 1).
In the case of 4-ethylphenol no inhibitory effect on the kinetics
of oxygen utilisation in the presence of equimolar hydroquinone
was observed and the formation of 1,4-benzoquinone was associ-
ated with a shortening of the ‘lag-period’.9 The half life of the
lag-period was 7.55 0.22 min in the presence of hydroquinone
in comparison with 8.43 0.25 min for the control.
Calculation of the effect of hydroquinone on the oxygen stoichi-
ometry showed that, in the case of catecholic substrates (catechol
and dopa), the control value was close to two moles product per
mole oxygen and 1:1 for the monohydric phenol substrate, 4-eth-
ylphenol, as expected (Fig. 3, open circles).
In the case of catechol oxidation in the presence of hydroqui-
none there was a marked discrepancy after about 600 nmol prod-
uct had formed with increased oxygen utilisation without obvious
formation of product, and with a final 1:1 stoichiometry, implying
the generation of a monohydric phenol substrate. This was the
It should be noted that under these conditions all phenolic
derivatives showed ions at Mꢀ1 whereas ortho-quinone deriva-
tives lacking any acidic OH substituents showed ions at M+1,
where M is molecular weight.
3. Results
Under the conditions employed in these experiments the autox-
idation of hydroquinone was negligible and we confirmed that
hydroquinone is not a primary substrate for tyrosinase, there
being: (i) no spectrometric evidence of loss of substrate or product
formation; (ii) no oxygen utilisation; and (iii) no evidence of prod-
ucts by hplc/mass spectrometry. However, in the presence of tyros-
inase substrates we detected the formation of 1,4-benzoquinone
and we compared the effect of equimolar hydroquinone on the
tyrosinase-catalysed oxidation of three substrates, namely cate-
chol, L-3,4-dihydroxyphenylalanine (dopa), and 4-ethylphenol. In
all cases 1,4-benzoquinone was generated as shown by the strong
absorbance at 246 nm in the final spectra of the products in com-
parison with the controls incubated in the absence of hydroqui-
none (Fig. 1).
The formation of 1,4-benzoquinone was at the expense of the
initial ortho-quinone product of the primary substrates as shown
by the diminution of absorbance at 400 nm (and at 304 nm in
Stratford, Michael R.L.
