A novel artificial superoxide dismutase: Non-covalent conjugation of albumin with a MnIII salophen type complex

Article abstract of DOI:10.1016/j.ejmech.2010.12.023

The approach of non-covalent conjugation has recently been used and a number of metal complexes have been encaged in a protein scaffold to form functional metallo proteins. Here we investigate a novel artificial superoxide dismutase enzyme based on the non-covalent conjugation of bovine serum albumin and the manganese(III) complex of N,N′-bis(salicylidene)-3,4- diaminobenzoic acid. We report the intriguing ability of the albumin environment to increase the SOD activity of the inorganic complex. We also determined the binding constant with BSA by CD spectroscopy.

Full text of DOI:10.1016/j.ejmech.2010.12.023

European Journal of Medicinal Chemistry 46 (2011) 961e965
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
A novel artificial superoxide dismutase: Non-covalent conjugation of albumin
with a MnIII salophen type complex
*
Valentina Oliveri, Graziella Vecchio
Dipartimento di Scienze Chimiche, University of Catania, V.le A.Doria 6, 95125 Catania, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The approach of non-covalent conjugation has recently been used and a number of metal complexes
have been encaged in a protein scaffold to form functional metallo proteins. Here we investigate a novel
artificial superoxide dismutase enzyme based on the non-covalent conjugation of bovine serum albumin
and the manganese(III) complex of N,N0-bis(salicylidene)-3,4-diaminobenzoic acid. We report the
intriguing ability of the albumin environment to increase the SOD activity of the inorganic complex. We
also determined the binding constant with BSA by CD spectroscopy.
Received 28 September 2010
Received in revised form
10 December 2010
Accepted 21 December 2010
Available online 14 January 2011
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
SOD mimics
Manganese(III) complexes
BSA
CD
1. Introduction
hydrogen peroxide and molecular oxygen, and thus protect living
cells from toxic oxygen metabolites [13]. An excess of Oꢀ2 , which
Recently the synthesis of conjugated metal complexes has
become an important approach in different fields of chemistry and
biology and amongst these, in the bio-inorganic field [1].
overwhelms the amount of SOD locally available [14], is produced by
the immune system in such illnesses as inflammation or strokes. Free
radical damage has been associated with a growing number of
diseases such as rheumatoid arthritis, cancer, neurodegenerative
disorders, diabetic complications, strokes, inflammation and reper-
fusion injury [15].
The approach of non-covalent conjugation has recently been
used and metal complexes have been encagedin a protein scaffold to
form functional metallo proteins [2e12]. These artificial metallo
enzymes, also called hybrids, combine the properties of the protein
chain with the activity of the artificial prosthetic group. In some
cases the protein part determines a chiral environment, water
solubility and different substrate accessibility. About one third of all
enzymes are metallo proteins and some of the most difficult bio-
logical reactions are mediated by them. The protein scaffold is
exquisitely suited to provide a well defined second coordination
sphere. Evolution appears to have tailored the second coordination
sphere of enzymes to maximize the efficiency and the selectivity of
the catalyst. Enantioselective hybrid catalysts have been designed
for a wide range of reactions, including ester hydrolysis, hydroxyl-
ation, epoxidation, sulfoxidation, hydrogenation, transfer hydroge-
nation, and DielseAlder reactions. The role of hybrid catalysts has
been recently reported as an attractive strategy in the development
of new catalysts and for the understanding of reaction mechanisms.
Superoxide dismutases (SODs) are a family of metallo enzymes
which catalyze the dismutation of superoxide radical anions to
SOD native enzymes have shown promising anti-inflammatory
properties in both preclinical and clinical studies for the treatment
of various diseases, however there were drawbacks and several
issues associated with their use as therapeutic agents [16]. For this
reason a number of therapeutic strategies by modulating ROS
species was of particular interest [17]. In this context the design of
synthetic mimics of SOD enzymes have been studied [18,19].
Among the different families of SOD mimics, the manganese(III)
complexes of salen type ligands represent a promising new class of
compounds [6,20e23]. It has been shown that salen complexes
exert beneficial effects in several in vitro and in vivo animal models
of human diseases which involve oxidative stress ranging from
Alzheimer’s [24,25], Parkinson’s [26], Prion Disease [27] and motor
neuron disease to multiple sclerosis and excitotoxic neuronal injury
[28]. The manganese(III) salophen type complexes have been also
investigated as anticancer agents [29].
Recently, we have synthesized some SOD mimetics based on
salen derivatives conjugated with cyclodextrins [30,31]. The pres-
ence of the cyclodextrin moiety improved the solubility and the
SOD-like activity of manganese-salen moiety.
* Corresponding author. Tel.: þ39 (0) 957385064; fax: þ39 (0) 095580138.
E-mail address: gr.vecchio@unict.it (G. Vecchio).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.12.023

962
V. Oliveri, G. Vecchio / European Journal of Medicinal Chemistry 46 (2011) 961e965
Herein, we investigated a new SOD mimic based on a non-
covalent conjugation of bovine serum albumin (BSA) and a salo-
phen type manganese(III) complex. At the best of our knowledge
this is the first example where this approach was used to prepare an
artificial SOD. We report the intriguing ability of the albumin
environment to increase the SOD activity of the inorganic complex.
Particularly, we synthesized a new acid manganese(III) complex
(MnL) of N,N0-bis(salicylidene)-3,4-diaminobenzoic acid (L) (Fig. 1),
its interaction with BSA, and the SOD activity of MnL and of the
non-covalent conjugate MnLeBSA. We determined also the binding
constant with BSA by CD.
2. Results and discussion
2.1. Manganese(III) complex
Manganese complex was synthesized as reported in the litera-
ture for the similar complexes [31], at room temperature. The
UVeVis spectra were not significantly different from those of salo-
phen type complexes, in accordance with the presence of the same
coordination environment. In the ESI mass spectra, the peak at
413.1 m/z was due to the complex species [ML þ H]þ.
Fig. 2. UVeVisible spectra of BSA (ꢀ), MnL (...). Difference absorption spectrum between
MnLeBSA and MnL at the same concentration (——). [BSA] ¼ [MnL] ¼ 1.32 ꢁ10ꢀ5 M. Inset:
titration of MnL (1.65 ꢁ 10ꢀ4 M) with BSA in phosphate buffer (5 mM, pH 7.4).
CD spectra of MnLeBSA in the 190e260 nm region show the
typical CD signals due to the
the signals was slightly modified for the addition of an excess of
MnL and the -helical structure is maintained (Fig. 3). The addition
a-helical structures. The intensity of
2.2. Albumin interaction of MnL by UVeVis, fluorescence and CD
spectroscopy
a
of MnL determined a slight decrease of ellipticity. The modification
of helix content due to BSA complexation has been observed in
Albumin, being the most abundant serum protein in the blood,
its interaction is of high interest and we tested the interaction of
MnL with bovine serum albumin (BSA). The non-covalent conju-
gation of albumin to a number of drugs has been investigated
generally and has been shown to carry active molecules in the
blood [32].
different extents [35,36]. On the contrary, at
l > 300 nm a dichroic
spectrum was observed. In the circular dichroism spectra of MnL
(320e550 nm region) in the presence of BSA the band at
l
> 300 nm can be assigned to the induced CD due to MnL inter-
action with the BSA, supporting the localization of the MnL in the
pocket of the BSA.
The interaction of MnL complex with BSA was investigated by
UVeVis, CD and fluorescence spectroscopy. The MnLeBSA adduct
was formed after few minutes at room temperature. This was
verified through the CD spectroscopy.
The UVeVis spectra are reported in Fig. 2. As reported elsewhere
[33,34], we also reported the difference absorption spectrum
between BSAeMnL and MnL at the same concentration. The
UVeVis spectra of the MnL only slightly changed in the presence of
the BSA suggesting that the complexation is not assisted by coor-
dination of the metal ion by the albumin side chain. The modifi-
cation of the spectra is consistent with the interaction BSAeMnL.
The binding constant was determined for the MnLeBSA by CD
spectroscopy. The ellipticity changes were used to estimate the
binding constant and the number of binding sites using the Engel
equation as reported by Greenfield [37,38]. In this approach the
ellipticity, qi, at any total MnL concentration C0i is defined by
equation (1).
ꢀ
ꢁ
pffiffiffiffiffiffiffiffiffiffiffiffiffi
qi
¼
qmax q ꢀ q2 ꢀ s
(1)
q ¼ ð1 þ nKAC0i þ KAPÞ=2KAP and s ¼ nC0=P
0
-10
-20
-30
-40
-50
210
220
230
λ (nm)
240
250
Fig. 3. CD spectra of BSA only (6.0 ꢁ 10ꢀ6 M) (ꢀ) and in the presence of MnL
(3.0 ꢁ 10ꢀ4 M) (...).
Fig. 1. MnL.

V. Oliveri, G. Vecchio / European Journal of Medicinal Chemistry 46 (2011) 961e965
963
where n is the reciprocal of the number of binding sites, KA is the
binding constant and P is the BSA concentration. The equation can
be solved by estimating initial values of KA, and n and qmax and
finding the best fits using non-linear least squares curve fitting
procedures. The data are reported in Fig. 4. The fit of the data
indicated a number site of one and the KA is 3.6 ꢁ 104 M. This value
is in the range of the KA values reported for a number of molecules
interacting with albumin [39,40].
Fluorescence experiments were carried out to verify changes in
the microenvironment around the complex. The fluorescence
intensity at
l
¼ 450 nm in the MnL spectrum increased when
a solution of BSA was added to MnL (Fig. 5). These data suggest
a different microenvironment for MnL due to its insertion in the
BSA sites [35].
Site marker competitive experiments were carried out to iden-
tify the MnL binding site on BSA, using hemin (site I marker),
2,3,5-triiodobenzoic acid (TIB, site I and II marker), ibuprofen (site II
marker), thyroxine (site III marker) which specifically bind to
albumin sites [41]. In these experiments the induced CD signal of
MnLeBSA was monitored (Figs. 6 and 7). The marker probe was
added to solutions containing equimolar concentrations of MnL
and BSA. In the presence of ibuprofen and thyroxine, the induced
CD signal was not modified. When TIB was added, but an induced
CD spectrum was still observed (Fig. 6), suggesting the replacement
of MnL was not complete or the MnL changed its orientation in BSA
sites. The addition of hemin to a solution of MnLeBSA complex
changed the induced CD signal of MnLeBSA. The hemin addition
showed a weak induced effect due BSA-hemin system (Fig. 7).
These data are consistent with a competitive displacement of MnL
by hemin and TIB. On the basis of previously published data
[11,35,36,42e44] these results demonstrate that the MnL is bound
inside BSA in the site I (subdomain II A).
Fig. 5. Emission spectra of MnL (2.45 ꢁ 10ꢀ6 M) in the presence of various concen-
trations of BSA, 0.0 (ꢀ), 1.22 ꢁ 10ꢀ6
(
...), 2.45 ꢁ 10ꢀ6 M (——).
complexes did not interfere with the reaction of the xanthine/
xanthine oxidase system. Inhibition of NBT (250 M) reduction by
m
the complex, when plotted as V0/Vcatꢀ1 against the [complex],
yielded a straight line with a slope kcat/(kdetector[NBT]). V0 is the
uninhibited reduction rate of the detector molecule and Vcat is the
reduction rate of the detector molecule inhibited by the complex
(Fig. 8). kdetector is the constant for the reaction between the Oꢀ2 and
the detector molecule NBT and it is 5.88 ꢁ 104 Mꢀ1 sꢀ1. The slope of
the line, obtained by the best fit of the experimental data, allowed
the calculation of the kcat value. The I50 value (the concentration
which causes the 50% inhibition of NBT reduction) of MnL is 1.3
( ꢂ 0.2) ꢁ 10ꢀ6 M and its kcat is 1.2( ꢂ 0.1) ꢁ 107 Mꢀ1 sꢀ1. The results
are very similar to those in the literature for similar complexes [47].
In the presence of albumin, the MnL behavior is interesting. The
albumin did not show any interference with the assay and did not
destroy the complex. The I50 value of the MnLeBSA adduct is 1.9
( ꢂ 0.3) ꢁ 10ꢀ7 M and kcat is 7.7( ꢂ 0.9) ꢁ 107 Mꢀ1 sꢀ1. These values
are significantly better than that of the simple MnL in keeping with
the ability of albumin to bind the manganese(III) complex. We also
determined the rate constant for Mn2þ in the presence of BSA in
order to discount the possibility of MnL decomposition assisted by
2.3. SOD activity
Rate constants for the reactions of MnL and the MnLeBSA
adduct with the superoxide anion, were determined by competi-
tion kinetics using NBT as the detector molecule, as reported
elsewhere [45]. We verified that the cytochrome c interfered with
the assay as reported for other manganese complexes [46]. For this
reason we determined the I50 values using only the NBT assay.
Possible interferences with the xanthine/xanthine oxidase reaction
of the test compounds were examined by following the rate of urate
production at 295 nm, in the absence of the target molecule. The
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0
2.00x10-4
4.00x10-4
6.00x10-4
Fig. 6. CD spectra in phosphate buffer 5 mM (EtOH 2%) of MnLeBSA (9.0 ꢁ 10ꢀ5 M) in
the presence of TIB. (1) TIB 0 M, (2) TIB 7.0 ꢁ 10ꢀ5 M, (3) TIB 1.4 ꢁ 10ꢀ4 M, (4) TIB
(2.1 ꢁ 10ꢀ4 M), (5) TIB 2.8 ꢁ 10ꢀ4 M, (6) TIB (9 ꢁ 10ꢀ5 M) and BSA only (9 ꢁ 10ꢀ5 M).
[complex] (M)
Fig. 4. Ellipticity at 350 nm. BSA is 9.4 ꢁ 10ꢀ5 M (phosphate buffer 5 mM, pH 7.4).

964
V. Oliveri, G. Vecchio / European Journal of Medicinal Chemistry 46 (2011) 961e965
been recently reported [11]. The presence of an anionic ligand as L at
physiological pH, and then of a neutral complex as MnL could
probably determine the greater interaction of the complex with BSA.
The microenvironment of the protein could increase the SOD
activity of the MnL. This microenvironment around the complex
should be more hydrophobic than that of water in a similar way to
the cyclodextrin cavity [50]. Because no significant change was
observed in the UVeVis spectra, the metal coordination of side
chain can be excluded.
3. Conclusion
The non-covalent approach for designing functional metallo
enzymes was used for the first time to prepare an artificial SOD
enzyme. The protein environment has a positive effect on the SOD
activity of the inorganic complex and the MnLeBSA adduct shows
a significantly increased SOD activity.
On the basis of the interest for the SOD mimetics based on
inorganic compounds, this result suggests a new strategy in the field
of the SOD mimetic systems and adds to fascinating perspectives on
the artificial metallo enzyme for red biotechnology applications.
Fig. 7. CD spectra in phosphate buffer 5 mM of MnLeBSA (9.0 ꢁ 10ꢀ5 M) in the
presence of hemin (cuvette 0.5 cm). (1) Hemin 0 M, (2) hemin 2.64 ꢁ 10ꢀ5 M, (3)
hemin 3.92 ꢁ 10ꢀ5 M, (4) hemin 2.64 ꢁ 10ꢀ5 M and BSA only (9.0 ꢁ 10ꢀ5 M).
BSA. As reported elsewhere [48] Mn2þ shows SOD activity (I50 ¼ 3.0
( ꢂ 0.2) ꢁ 10ꢀ7 M and its kcat is 4.9( ꢂ 0.1) ꢁ 107 Mꢀ1 sꢀ1) but the
presence of BSA does not modify significantly the I50 value (2.9
( ꢂ 0.2) ꢁ 10ꢀ7 M).
4. Experimental
4.1. Materials
Furthermore, we tested if the adduct BSAeMnL was not
destroyed in the presence of superoxide anions. This was demon-
strated by a series of CD experiments in the presence of system
xanthine/xanthine oxidase that generated superoxide anions in
situ. Xanthine and/or xanthine oxidase did not affect the induced
CD signal of MnLeBSA adduct.
Commercially available reagents were used directly, unless
otherwise noted.
3,4-Diaminobenzoic acid was purchased by Fluka and was
purified by chromatography on silica gel with acetone as the eluent.
Bovine serum albumin was purchased by SigmaeAldrich. TLC was
carried out on silica gel plates (Merck 60-F254).
Even though the amount of MnLeBSA adduct formed is low in
the experimental condition of the Fridovich assay, data suggested
that SOD activity was due to the adduct formation. The adduct
could have a higher activity than that observed in the reaction
mixture where the main species is MnL. Using the Fridovich assay
the concentration of the species formed can not be easily forced.
Fridovich assay results obtained for some salen type manganese
(III) complexes in the presence of albumin [22,31,49] have been
reported to verify the stability of the complexes in the presence of
biological ligands. Any modification of the SOD activity of those
complexes in the presence of BSA has never been reported. On the
other hand, the binding of Mn salen complexes with the HSA has
4.1.1. Synthesis of N,N0-bis(salicylidene)-3,4-diaminobenzoic acid (L)
3,4-diaminobenzoic acid (200 mg, 1.31 mmol) was dissolved in
anhydrous methanol (10 ml). Salicylaldehyde (280 ml, 2.63 mmol)
was added and the mixture was stirred for 6 h under nitrogen
streaming. A precipitate was formed after 30 min. After 5 h, the
product was collected by filtration, washed with cool methanol and
dried. Yield: 85%. ESI-MS: m/z ¼ 361.1 [L þ H]þ. UVeVis: (MeOH)
l
/nm (
e
/Mꢀ1 cmꢀ1) 202 nm (34026), 273 (27048), 334 (18480).
1H NMR: (CD3OD, 500 MHz)
d
(ppm) 8.88 (1H, s, iminic proton),
8.86 (1H, s, other iminic proton), 8.05 (1H, d, H-6 of benzoic acid
ring), 8.02 (1H, dd, H-2 of the benzoic acid ring), 7.58 (2H, m, H
ortho to the OH), 7.46 (2H, m, H para to the OH), 7.42 (1H, m, H meta
to the COOH), 6.08 (4H, m, H meta to the OH).
3.0
2.5
2.0
1.5
1.0
0.5
0.0
4.1.2. Synthesis of manganese(III) complex of L (MnL)
L (70 mg, 0.19 mmol) was dissolved in absolute ethanol (10 ml).
The initial yellow-orange solution turned dark brown after Mn
(CH3COO)3 (45 mg, 0.19 mmol) was added and a precipitate was
formed in 20 min. The reaction was stirred for 3 h under reflux.
Then, the precipitate was filtered off, washed with cool methanol
and dried in vacuo. Yield: 92%. ESI-MS: m/z ¼ 413.1 [MnL þ H]þ.
UVeVis: (MeOH) l/nm (e
/Mꢀ1 cmꢀ1): 211 (41280), 249 (41724), 335
(28145), 435 (7916), 549 (495).
4.2. Spectroscopic measurements
0
5.0x10-7
10-6
1.5x10-6 2.0x10-6 2.5x10-6 3.0x10-6
4.2.1. NMR spectroscopy
1H NMR spectra were recorded at 25 ꢃC in D2O or CD3OD with
a Varian UNITY PLUS spectrometer at 499.883 MHz. The 1H NMR
spectra were obtained by using standard pulse programs from
Varian library. In all cases the length of 90ꢃ pulse was 7 ms. The 2D
concentration (M)
Fig. 8. The SOD-like activity of MnL (:) and MnLeBSA adduct (C) in the Fridovich
assay (NBT ¼ 250 M). V0 is the reduction rate of the NBT and Vcat is the rate of
reduction of the NBT in the presence of the complex.
m

V. Oliveri, G. Vecchio / European Journal of Medicinal Chemistry 46 (2011) 961e965
965
[4] R. Ricoux, M. Allard, R. Dubuc, C. Dupont, J.-D. Maréchal, J.-P. Mahy, Org.
Biomol. Chem. 7 (2009) 3208e3211.
[5] A. Mahammed, Z. Gross, J. Am. Chem. Soc. 127 (2005) 2883e2887.
[6] A. Pordea, M. Creus, J. Panek, C. Duboc, D. Mathis, M. Novic, T.R. Ward, J. Am.
Chem. Soc. 130 (2008) 8085e8088.
experiments were acquired using 1K data points, 256 increments
and a relaxation delay of 1.2 s.
4.2.2. Mass spectrometry
The ESI-MS measurements were performed on a Finnigan LCQ-
Duo ion trap electrospray mass spectrometer.
[7] Q. Raffy, R. Ricoux, J.-P. Mahy, Tetrahedron Lett. 49 (2008) 1865e1869.
[8] M.T. Reetz, N. Jiao, Angew. Chem. Int. Ed. 45 (2006) 2416e2419.
[9] S. Abe, T. Ueno, Y. Watanabe, Top. Organomet. Chem. 25 (2009) 25e43.
[10] T. Ueno, T. Koshiyama, M. Ohashi, K. Kondo, M. Kono, A. Suzuki, T. Yamane,
Y. Watanabe, J. Am. Chem. Soc. 207 (2005) 6556e6562.
[11] P. Rousselot-Pailley, C. Bochot, C. Marchi-Delapierre, A. Jorge-Robin, L. Martin,
J.C. Fontecilla-Camps, C. Cavazza, S. Manage, ChemBioChem. 10 (2009) 545e552.
[12] R.M. Wang, T. Komatsu, A. Nakagawa, E. Tsuchida, Bioconjug. Chem. 16 (2005)
23e26.
[13] J.M. McCord, I. Fridovich, Free Radic. Biol. Med. 5 (1988) 363e369.
[14] S. Moncada, R.M.J. Palmer, E.A. Higgs, Pharmacol. Rev. 43 (1991) 109e142.
[15] K. Pong, Exp. Opin. Biol. Ther. 3 (2003) 127e139.
[16] B. Halliwell, J.M.C. Gutteridge (Eds.), Free Radicals in Biology and Medicine,
Oxford University Press, NJ, USA, 1999, pp. 617e859.
[17] H. Maeda (Ed.), Controlling Oxidative Stress: Therapeutic and Delivery
Strategies in Advanced Drug Reviews, vol 61, 2009, pp. 285e366.
[18] D. Salvemini, D.P. Riley, S. Cuzzocrea, Nat. Rev. Drug Discov. 1 (2002) 367e374.
[19] I. Batinic-Haberle, J.S. Reboucas, I. Spasojevic, Antioxid. Redox Signal. 13
(2010) 877e918.
[20] S. Melov, S.R. Doctrow, J.A. Schneider, J. Haberson, M. Patel, M. Patel,
P.E. Coskun, K. Huffman, D.C. Wallace, B. Malfroy, J. Neurosci. 21 (2001)
8348e8353.
[21] M.A. Sharpe, R. Olloson, V.C. Stewart, J.B. Clark, Biochem. J. 366 (2002) 97e107.
[22] S.R. Doctrow, K. Huffman, C.B. Marcus, W. Musleh, A. Bruce, M. Baudry,
B. Malfroy, in: H. Sies (Ed.). Adv. Pharmacol.38, 1996, pp. 247e269.
[23] R. Liu, I.Y. Liu, X. Bi, R.F. Thompson, S.R. Doctrow, B. Malfroy, M. Baudry, Proc.
Natl. Acad. Sci. U.S.A. 100 (2003) 8526e8531.
4.2.3. UV/Visible, fluorescence and CD spectroscopy
UV/Visible spectra were recorded with Agilent 8452A diode
array spectrophotometer.
CD measurements were performed under a constant flow of
nitrogen on a JASCO model J-810 spectropolarimeter. MnL stock
solutions were prepared in MeOH/water (4:6). The final concen-
tration of MeOH did not exceeded 5% and did not affect the
measurements. The spectra were recorded at 25 ꢃC, on freshly
prepared solutions. 0.1 cm or 1.0 cm cuvettes were used.
All fluorescence spectra were recorded on LS-55 spectrofluo-
rimeter (PerkineElmer) equipped with 1.0 cm quartz cells and
a thermostat bath. All studies were performed at 25 ꢂ 1 ꢃC using
5 nm excitation and emission slit widths. The fluorescence spectra
were recorded in the range of 390e550 nm with lex ¼ 335 nm.
SigmaPlot (SPSS Science Chicago) was used to fit the equation
(1), using the Levenberg-Marquadt algorithm.
4.3. Superoxide dismutase assay
[24] I. Anderson, C. Adinolfi, S. Doctrow, K. Huffman, K.A. Joy, B. Malfroy, P. Soden,
H.T. Rupniak, J.C. Barnes, Biochem. Soc. Symp. 67 (2002) 141e149.
[25] A.J. Bruce, B. Malfroy, M. Baudry, Proc. Natl. Acad. Sci. USA 93 (1996)
2312e2316.
[26] K. Pong, S.R. Doctrow, M. Baudy, Brain Res. 881 (2000) 182e189.
[27] M.W. Brazier, S.R. Doctrow, C.L. Masters, S.J. Collins, Free Radic. Biol. Med. 45
(2008) 184e192.
[28] Y. Rong, S.R. Doctrow, G. Tocco, M. Baudry, Proc. Natl. Acad. Sci. 96 (1999)
9897e9902.
[29] K.I. Ansari, J.D. Grant, S. Kasiri, G. Woldemariam, B. Shrestha, S.S. Mandal,
J. Inorg. Biochem. 103 (2009) 818e826.
[30] A. Puglisi, G. Tabbì, G. Vecchio, J. Inorg. Biochem. 98 (2004) 969e976.
[31] V. Lanza, G. Vecchio, J. Inorg. Biochem. 103 (2009) 381e388.
[32] M.J. Hawkins, P. Soon-Shiong, N. Desaia, Adv. Drug Deliv. Rev. 60 (2008)
876e885.
[33] Y.-J. Hu, Y. Liu, X.-H. Xiao, Biomacromolecules 10 (2009) 517e521.
[34] Y.-J. Hu, Y. Ou-Yang, C.-M. Dai, Y. Liu, X.-H. Xiao, Mol. Biol. Rep. 37 (2010)
3827e3832.
[35] K.P. Bijan, S. Anuva, G. Nikhil, J. Phys. Chem. B 114 (2010) 6183e6196.
[36] H.H. Cai, X. Zhing, P.H. Yang, W. Wei, J. Chen, J. Cai, Colloids Surf. A 372 (2010)
35e40.
[37] N.J. Greenfield, V.M. Fowler, Biophys. J. 82 (2002) 2580e2591.
[38] N.J. Greenfield, Nat. Protoc. 1 (2006) 2733e2741.
SOD-like activity was determined by the indirect Fridovich assay
[51]. Superoxide radical anions were enzymatically generated by
the xanthineexanthine oxidase (XO/XOD) system and spectro-
photometrically detected by monitoring the formation of reduced
NBT at 550 nm. The reaction mixture was composed by NBT
250
10 mM, at pH 7.4. An appropriate amount of xanthine oxidase was
added to 2.0 ml reaction mixture to produce a
A550 nm minꢀ1 of
0.024. This corresponded to a Oꢀ2 production rate of 1.1 mol minꢀ1
mM, xanthine 50 mM, catalase 30 mg/ml in phosphate buffer
D
m
.
The NBT reduction rate was measured in the presence and in the
absence of the investigated complex for 500 s. All measurements
were carried out at 25.0 ꢂ 0.2 ꢃC using 1.0 ꢁ 1.0 cm thermostated
cuvettes in which solutions were magnetically stirred. In separate
experiments urate production by xanthine oxidase was spectro-
photometrically monitored at 295 nm, ruling out any inhibition of
xanthine oxidase activity.
The MnLeBSA adduct was synthesized by incubating equimolar
amounts of BSA and MnL for 10 min in the buffer solution at room
temperature with no further purification.
[39] C.E. Petersen, C.E. Ha, K. Harohalli, D. Park, N.V. Bhagavan, Biochemistry 36
(1997) 7012e7017.
[40] Q. J-Liu, J. Tian, X. Tian, X. Chen, Bioorg. Med. Chem. 12 (2004) 469e474.
[41] M. Takahashi, Y. Matsushita, I. Moriguchi, Chem. Pharm. Bull. 42 (1996)
937e940.
[42] H. Kohita, Y. Matsushita, I. Moriguchi, Chem. Pharm. Bull. 42 (1994) 937e940.
[43] Y. Ni, S. Su, S. Kokot, Anal. Chim. Acta 580 (2006) 206e215.
[44] Y. Peng, F. Huang, J. Wen, B. Huang, X. Ma, Q. Wang, J. Coord. Chem. 61 (2008)
1503e1512.
The I50 (the concentration which causes the 50% inhibition of
NBT reduction) of manganese(III) complexes at pH 7.4 was deter-
mined. Three determinations were carried out.
Acknowledgments
[45] Z. Liu, G.B. Robinson, E.M. Gregory, Arch. Biochem. Biophys. 315 (1994) 74e81.
ꢀ
[46] I. Spasojevic, I.-B. Haberle, R.D. Stevens, P. Hambright, A.N. Thorpe,
The authors wish to thank Tiziana Campagna for her technical
assistance. This research is funded by MIUR (2008R23Z7K).
J.G. Pedatsur Neta, I. Fridovich, Inorg. Chem. 40 (2001) 726e739.
[47] S.R. Doctrow, K. Huffman, C.B. Marcus, G. Tocco, E. Malfroy, C.A. Adinolfi,
H. Kruk, K. Baker, N. Lazarowych, J. Mascarenhas, B. Malfroy, J. Med. Chem. 45
(2002) 4549e4558.
[48] I. Batinic-Haberle, J.S. Reboucas, I. Spascjevic, Antioxid. Redox Signal. 13
(2010) 877e917.
[49] Z.X. Liu, G.B. Robinson, E.M. Gregory, Arch. Biochem. Biophys. 15 (1994) 74e81.
[50] F. Bellia, D. La Mendola, C. Pedone, E. Rizzarelli, M. Saviano, G. Vecchio, Chem.
Soc. Rev. 38 (2009) 2756e2781.
References
[1] P. J-Taquet, C. Frochot, V. Manneville, M. Barberi-Heyob, Curr. Med. Chem. 14
(2007) 1673e1687.
[2] Y. Lu, N. Yeung, N. Sieracki, N.M. Marshall, Nature 460 (2009) 855e862.
[3] Q. Raffy, R. Ricoux, E. Sansiaume, S. Pethe, J.-P. Mahy, J. Mol. Catal. A 317
(2010) 19e26.
[51] C. Beauchamp, I. Fridovich, Anal. Biochem. 44 (1971) 276e287.