evaluated from 8 independent titrations (60–80 data points per
titration). The metal-to-ligand ratios were 1 : 1 and 1 : 2, with
the ligand concentrations between 4.0 × 10−4 and 1.3 × 10−3 M.
The formation constants of the copper(II) and zinc(II) acetate
complexes (logK = 1.8 and 0.86, respectively) were taken into
account.
to generate DA560 = 0.025–0.028 min−1. The NBT reduction rate
was measured in the presence and absence of the investigated
system ([Cu2+]tot = 0–1 × 10−6 M). Control experiments, in the
presence of Cu,Zn-SOD (0–5 × 10−8 M) were also carried out.
In separate measurements, the activity of xanthine oxidase was
monitored following urate production by spectrophotometry at
298 nm, to rule out any inhibition induced by the copper(II)–
hhgh system. The SOD-like activity was then expressed by the
IC50 values (concentration that causes 50% inhibition of NBT
reduction).
Electronic absorption and CD measurements
UV-VIS spectra were measured on a Hewlett Packard 8452A
diode array spectrophotometer. The CD spectra were recorded
on a Jasco J-710 spectropolarimeter in the wavelength interval
from 300 to 800 nm. The metal ion concentration was 6 × 10−4
M
Determination of the catecholase activity
in a cell with 1 cm optical pathlength. The individual spectra
of the copper(II) complexes were calculated by the previously
mentioned computer program PSEQUAD.
The oxidation of 3,5-di-tert-butylcatechol (H2dtbc) in the pres-
ence and absence of the copper(II)–hhgh system was monitored
spectrophotometrically on a Unicam Helios a spectrophotome-
ter at 298 K by following the increase of the 3,5-di-tert-butyl-o-
benzoquinone absorption band at 400 nm (e = 1900 M−1
cm−1) in O2-saturated 86 wt% methanol–water mixture. For
the determination of the pH in this mixed solvent, the glass
electrode was calibrated by standard aqueous buffer solutions
(pH = 4.0, 7.0 and 10, Sigma) and then the actual pH was
calculated by adding 0.28 units to the pH-meter reading,
according to the method of Bates.48 The ionization constants
of water in 86 wt% methanol–water is pKw ∼ 15.6.49 The auto-
oxidation of 3,5-di-tert-butylcatechol was also determined for
each substrate concentration and pH applied, and was then
subtracted from the overall effect in order to obtain the extent
of the oxidation reaction catalyzed by the copper(II)–hhgh
complexes. The kinetic studies were carried out by the method
of initial rates, but in some cases the integral method (up to 95%
conversion) was also used. The reported data are the average
of three parallel experiments. The maximum deviation from the
main value did not exceed 10%.
EPR measurements
The EPR titrations were performed in 12 cm3 solution under
an argon atmosphere. The initial concentration of hhgh was
1.3 × 10−3 M (6.0 × 10−4 M for the spectra measured in the
neutral pH range). A Masterflex CL peristaltic pump ensured
the circulation (14 cm3 min−1) of the solution through the
capillary tube in the cavity. The EPR spectra were taken
after equilibration/circulation for 3 min at a chosen pH at
room temperature (T = 298 K) on an upgraded JEOL-JES-
FE3X spectrometer with 100 kHz field modulation, using a
manganese(II)-doped magnesium oxide powder for the calibra-
tion of the magnetic field. The series of EPR spectra were
evaluated by a recently developed two dimensional simulation
method able to adjust the formation constants of the various
species together with the magnetic parameters of the component
EPR spectra.45 Between pH 8–10, where the CuH−2L species has
the largest concentration, the fit of the EPR spectra was not
satisfactory. This indicated that additional species have to be
taken into account. Since complexes with further compositions
were supported by neither the pH-metric nor the EPR data,
structural isomers were considered in the calculation for the
complex CuH−2L. In order to justify the necessity of new species,
the improvement of the fit was analysed. We used a criterion of
normalized regression parameter Rn defined as
Acknowledgements
This work was supported by the Hungarian Scientific Research
Fund (OTKA T037385, T046953 and T043232) and by a Marie
Curie Fellowship from the European Community program
“Improving the Human Research Potential and the Socio-
Economic Knowledge Base” (contract number “HPMF-CT-
2002-01860”). T. G. thanks the Ja´nos Bolyai Foundation for
its support.
1 − Rn = N(1 − R)
which indicates wether the fit is improving faster than the N
number of adjusted EPR parameters, when a new species is
included in the calculation. For the accepted equilibrium model
including 7 species and 52 EPR parameters, the regression
parameters R and Rn were found to be 0.99804 and 0.8981,
respectively. Omitting one of the isomers of CuH−2L, the regres-
sion parameters decreased considerably (45 EPR parameters,
Rn = 0.8619). If Cu2L2 or CuH−3L(b) was considered in addition
to the accepted complexes the Rn parameter also decreased. On
the other hand, assuming two isomers with the composition
of CuH−1L both regression parameters increased (59 EPR
parameters, R = 0.99828 and Rn = 0.8985). However, the
increase of the Rn value is within the experimental error (0.04%),
therefore the presence of a further isomer is not justified. Further
details of the method and the evaluation procedure have been
described previously.45,46
References
1 I. So´va´go´, in Biocoordination Chemistry, ch.: Metal complexes
of peptides and their derivatives, ed. K. Burger, Ellis Horwood,
Chichester, 1990.
2 H. Kozlowski, W. Bal, M. Dyba and T. Kowalik-Jankowska, Coord.
Chem. Rev., 1999, 184, 319–346.
3 C. Harford and B. Sarkar, Acc. Chem. Res., 1997, 30, 123–130, and
references therein.
4 E. C. Long, Acc. Chem. Res., 1999, 32, 827–836, and references
therein.
5 E. Farkas, I. So´va´go´, T. Kiss and A. Gergely, J. Chem. Soc., Dalton
Trans., 1984, 611–614.
6 T. Gajda, B. Henry, A. Aubry and J.-J. Delpuech, Inorg. Chem., 1996,
35, 586–593.
7 J. H. Viles, E. Cohen, S. B. Prusiner, D. B. Goodin, P. E. Wright and
H. J. Dyson, Proc. Natl. Acad. Sci. USA, 1999, 96, 2042–2047.
8 E. Aronoff-Spencer, C. S. Burns, N. I. Avdievich, G. J. Gerfen, J.
Peisach, W. E. Antholine, H. L. Ball, F. E. Cohen, S. B. Prusiner and
G. L. Millhauser, Biochemistry, 2000, 39, 13760–13771.
9 D. Valensin, F. M. Mancini, M. Luczkowski, A. Janicka, K.
Wisniewska, E. Gaggelli, G. Valensin, L. Lankiewicz and H.
Kozlowski, Dalton Trans., 2004, 16–22.
10 S. Lehmann, Curr. Opin. Chem. Biol., 2002, 6, 187–192.
11 L. B. Corson, J. J. Strain, V. C. Culotta and D. W. Cleveland, Proc.
Natl. Acad. Sci. USA, 1998, 95, 6361–6366.
12 C. S. Burns, E. Aronoff-Spencer, C. M. Dunham, P. Lario, N. I.
Avdievich, W. E. Antholine, M. M. Olmstead, A. Vrielink, G. J.
Determination of the superoxide dismutase activity
The SOD-like activity was studied at 298 K using the indirect
method of Nitroblue Tetrazolium (NBT) reduction.47 The
superoxide anion was generated in situ by the xanthine/xanthine
oxidase reaction, and detected spectrophotometrically by mon-
itoring the reduction of NBT at 560 nm. The reactions were
carried out in a phosphate buffer (5 × 10−2 M), containing
NBT (5 × 10−5 M) and xanthine (1 × 10−4 M). The reaction was
initiated by adding an appropriate amount of xanthine oxidase
D a l t o n T r a n s . , 2 0 0 5 , 3 1 8 7 – 3 1 9 4
3 1 9 3