Journal of Inorganic Biochemistry 142 (2015) 3946

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Journal of Inorganic Biochemistry

journal homepage: www.elsevier.com/locate/jinorgbio

Semax, an ACTH4-10 peptide analog with high afnity for copper(II) ion
and protective ability against metal induced cell toxicity
Giovanni Tabb a, Antonio Magr a, Alessandro Giuffrida a, Valeria Lanza a, Giuseppe Pappalardo a,
Irina Naletova b, Vincenzo Giuseppe Nicoletti b, Francesco Attanasio a,, Enrico Rizzarelli a,c
a
CNR-Istituto di Biostrutture e Bioimmagini, Via P. Gaifami 18, 95126 Catania, Italy
b
Dipartimento di Scienze Biomediche, Universit Degli Studi di Catania, Viale A. Doria 6, 95125 Catania, Italy
c
Consorzio Interuniversitario C.I.R.C.S.M.B., Via C. Ulpiani 27, 70125 Bari, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Heptapeptide Semax, encompassing the sequence 4-7 of N-terminal domain of the adrenocorticotropic hormone
Received 5 August 2014 (ACTH) and a C-terminal Pro-Gly-Pro tripeptide, belongs to a short regulatory peptides family. This compound
Received in revised form 5 September 2014 has been found to affect learning processes and to exert marked neuroprotective activities on cognitive brain
Accepted 11 September 2014
functions. Dys-homeostasis of metal ions is involved in several neurodegenerative disorders and growing
Available online 28 September 2014
evidences have showed that brain is a specialized organ able to concentrate metal ions. In this work, the metal
Keywords:
binding ability and protective activity of Semax and its metal complexes were studied. The equilibrium study
Copper clearly demonstrated the presence of three complex species. Two minor species [CuL] and [CuLH1] co-exist
Semax together with the [CuLH2]2 in the pH range from 3.6 to 5. From pH 5 the [CuLH2]2 species becomes
Speciation predominant with the donor atoms around copper arranged in a 4 N planar coordination mode. Noteworthy, a
Spectroscopy reduced copper induced cytotoxicity was observed in the presence of Semax by MTT [3-(4,5-dimethylthiazol-
Voltammetry 2-yl)-2,5-diphenyltetrazolium bromide] assay on a SHSY5Y neuroblastoma and RBE4 endothelial cell lines.
Cell viability 2014 Published by Elsevier Inc.

1. Introduction Heptapeptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro) is an analog of

ACTH/MSH-like peptides, usually known as melanocortins, repre-
sent a class of endogenous regulatory peptides [1,2]. Adrenocorticotro-
phic hormone (ACTH), melanocyte-stimulating hormone (MSH), their
fragments and synthetic analogs belong to this class of peptides. These
compounds have shown marked actions on central nervous system
(CNS) function with neurotrophic, nootropic and neuroprotective ef-
fects [35]. These peptides act on learning, motivational processes and
concentration and attention abilities [6,7], with behavioral effects not
associated with their hormonal activity but as the result of their direct
actions on the CNS [810]. Use of short ACTH fragments and their ana-
logs lacking hormone activity allows discriminating between endocrine
properties and neurotrophic effects. Structuralfunctional studies have
shown that as in the case of the behavioral effects, the neurotrophic
activity is due to the N-terminal part of the ACTH molecule [4]. The
most marked effects on behavior were seen with the fragment
ACTH(4-10), the ACTH(4-7) fragment being the shortest sequence
required to affect mammalian behavior with the same capability of
ACTH(4-10) [8]. A number of ACTH analogs have been synthesized.
Some of these compounds are more stable and more effective than the
natural N-terminal ACTH fragments [1113].
Corresponding author. Tel.: +39 095 7338439.
E-mail address: francesco.attanasio@cnr.it (F. Attanasio).
the ACTH (4-10) fragment (Met-Glu-His-Phe-Arg-Trp-Gly), completely
devoid of the hormonal activity associated with the full-length ACTH
molecule; the Pro-Gly-Pro fragment is responsible for its metabolic sta-
bility and its higher resistance against enzymatic cleavage in compari-
son with the analog fragment [1418] and also shows some regulatory
activity [15,19,20]. Analogous to other ACTH fragments, this peptide
stimulates learning and memory formation in rodents and humans
[15,21,22]. In numerous in vitro and in vivo experiments, nootropic, neu-
roprotective and anti-inammatory effects of this peptide have been
observed [19,23,24]. Semax activates the expression of neurotrophic
factors and their receptors in a rat model of cerebral ischemia [25], mod-
ulates the levels of vascular endothelial growth factor (VEGF-A) mRNA
in the frontal cortex and the hippocampus [22], promotes neuronal sur-
vival under hypoxia and improves brain circulation in experimental an-
imals and humans [16,26]. Semax affects cognitive brain functions by
modulating the expression and the activation of the hippocampal
brain derived neurotrophic factor (BDNF)/trk B system [27] and induces
rapid gene- and region-specic changes in neurotrophin gene expres-
sion in normal rat brain [28].
The involvement of bio-metals in neurodegenerative diseases, mem-
ory and cognitive processes has been reported [2931]. It has been
shown the key role of metal ions in the Alzheimer disease (AD)
[3234] and the copper(II) effects on the neurotoxicity [3537].
Furthermore, copper and zinc metallostasis alterations [38] have been

http://dx.doi.org/10.1016/j.jinorgbio.2014.09.008
0162-0134/ 2014 Published by Elsevier Inc.
40 G. Tabb et al. / Journal of Inorganic Biochemistry 142 (2015) 3946
found to dys-regulate expression levels of learning and memory related
proteins as nerve growth factor (NGF) and BDNF [3944] and to interact
with these neurotrophins and their peptide fragments [4548]. Though
numerous investigations have been performed to understand the
Semax physiological activity and mechanisms of action, the potential
role of bio-metals in tuning or driving the biological activities of this
peptide, underlying its neuroprotective and nootropic action, has not
been reported.
In this work, copper(II) complexes with Semax were characterized by
means of potentiometry, spectroscopic methods (UV-visible (UV-vis),
Circolar Dichroism (CD) and ESR (electron spin resonance)), electrospray
ionization mass spectrometry (ESI-MS), and voltammetry and the antiox-
idant activity of copper(II)Semax system was also measured in vitro.
Noteworthy, Semax showed to affect the copper induced citotoxicity on
SHSY5Y neuroblastoma and RBE4 endothelial cell lines.
2. Experimental
2.1. Materials
All reagents and solvents were purchased commercially and used
as received unless otherwise noted. Semax was purchased from
Sigma-Aldrich.
2.2. Potentiometric titrations
Potentiometric measurements were carried out using a home-
assembled fully automated apparatus sets (Metrohm E654 pH-meter,
combined micro pH glass electrode, Orion 9103SC, Hamilton digital dis-
penser, Model 500) controlled by the appropriate software setup in our
laboratory. The titration cell (2.5 ml) was thermostatted at 25 0.2 C.
Titrated solutions were kept under an argon atmosphere and KOH solu-
tions were used (0.1 M) as titrating solutions and were added through a
Hamilton buret equipped with 1 cm3 syringe. The ionic strength of all
solutions was 0.1 M in KNO3.
KOH 0.1 M was employed to titrate either solutions of the Semax pep-
tide (protonation constants) or Semax with copper(II) (complexation
constants). The peptide concentration ranged from 1 to 2 103 M in
both series of experiments. At least three independent titrations were
performed. Starting pH value was adjusted to 2.4 by adding HNO3
0.2 M. Further details are reported elsewhere [49]. To obtain the proton-
ation and complexation stability constants, the potentiometric data
were rened using the HYPERQUAD program [50]. Errors in stability con-
stant values are reported as three times standard deviations.
The formation reaction equilibria of Semax peptide with protons and
copper(II) are given in Eq. (1):
pCu qL rH Cup Lq Hr 1 with sweep rate of 1020 mV s 1 were run in the same potential
in which L is the peptide under study. The stability constant pqr is
dened in Eq. (2) (charges are omitted):
h i
p q r
pqr Cup Lq Hr = Cu  L  H 2 using methylviologen redox couple, MV2+/MV+,0.446 V vs. NHE [53].
The species distribution as a function of the pH was obtained by
using the computer program Hyss [51].
2.3. Spectroscoscopic studies
2.3.1. UVVis measurements
UVVis spectra were recorded at 25 C, by using an Agilent 8453
spectrophotometer. The concentrations of the Semax peptide and
copper(II) used to record absorption spectra were the same as those
for the potentiometric titrations. Combined spectroscopic and potentio-
metric metal-complex titrations were performed into a quartz cuvette
with a 1 cm path length to get the spectrum in the visible region at
each pH value simultaneously. These experiments were replicated at
least three times for each copper(II)peptide system. Spectroscopic
data were processed by using the HYPERQUAD program [50].
2.3.2. Circular dichroism spectroscopy (CD)
CD spectra were obtained at 25 C under a constant ow of nitrogen
on a Jasco model 810 spectropolarimeter, which was calibrated with an
aqueous solution of (1R)-()-10-camphorsulfonic acid. Measurements
were carried out in water at different pH values using 1 cm pathlength
cuvettes. The CD spectra of the copper(II) complexes in the pH range
of 3.57.1 were obtained in the 190800 nm wavelength regions. All
the solutions were freshly prepared using double distilled water. The
copper(II) ion and peptide concentrations used for the acquisition of
the CD spectra in the visible region were identical to those used in the
potentiometric titrations. Far UV CD spectra were acquired by using
copper(II) ion and peptide concentrations ranging from 5.0 106 to
1.0 105 M. The difference spectra were obtained by the subtraction
of Semax spectra from those obtained after copper(II) addition.
2.3.3. Electron spin resonance spectroscopy (ESR)
A Bruker Elexsys E500 CW-ESR spectrometer driven by a PC running
the XEpr software and equipped with a Super-X microwave bridge op-
erating at 9.39.5 GHz and a super high sensivity SHQE cavity was used
throughout this work. All ESR spectra of frozen solution of copper(II)
complexes were recorded at 150 K by means of a ER4131VT variable
temperature apparatus. In order to increase spectral resolution, a little
amount of methanol (not exceeding 10%) was added to the copper(II)
complex aqueous solutions after adjusting the pH to the desired value,
which were prepared by using 63Cu(NO3)2 0.05 M. ESR magnetic pa-
rameters were calculated from the 2nd and the 3rd line to get rid of
second order effects [52].
2.4. Voltammetric study
Cyclic voltammograms (CV) of the copper(II) complexes in solution
(1 10 3 M, 0.1 M KNO3 as ground electrolyte) were recorded by
means of a BAS CV-50 W voltammetric analyzer equipped with a C3
cell stand driven by a standard PC. The same complex solutions utilized
for potentiometric measurements were used for voltammetric studies.
These solutions were analyzed by using a 2 ml BAS glass cell with a
three electrode assembly: a glassy carbon working electrode (3 mm
diameter), a platinum auxiliary electrode and a standard Ag/AgCl
reference electrode. Complex solutions were degassed by using
ultrapure nitrogen bubbled through a three bubbler set. The square
wave voltammetry (SWV) experiments were recorded in the region
from + 500 to 900 mV, using 25 mV applied pulse value, avoiding
more intense pulses which caused the current peak to broaden. CV
range of SWV experiments and all experiments were carried out at
25 C. All potentials in the paper are referred to Ag/AgCl reference
electrode, + 0.225 V vs. normal hydrogen electrode (NHE), unless
otherwise stated. The Ag/AgCl electrode potential was checked by
In order to have a correct idea of the geometry of these copper(II)
complex species, an optimization procedure of their structural features
was performed with the use of MOMEC97 [54,55] and HYPERCHEM
[56].
2.5. ESI-MS experiments
ESI-MS spectra were recorded on a Finnigan LCQ-Deca ion trap
electrospray mass spectrometer (Bremen, Germany). Solutions of
copper(II) complexes were introduced into the ESI source through a
fused silica capillary (100 m i. d.) from a Hamilton syringe. Water solu-
tions of copper(II) complexes (2.0 106 M, pH = 7.1 and pH = 4.2)
were employed using copper(II) in its natural abundance in order to
 G. Tabb et al. / Journal of Inorganic Biochemistry 142 (2015) 3946
take advantage of the isotopic distribution in the interpretation of mass
spectra. The experimental conditions for all the spectra, acquired in pos-
itive and negative mode, were optimized as follows: needle source volt-
age 3.6 kV, ow rate 3 l min 1, nitrogen sheath gas ow rate 18.0
(arbitrary units), capillary temperature 180 C, and capillary voltage
12 V and 15 V in negative mode.
2.6. Superoxide dismutase (SOD) activity
The SOD activity was determined by using the xanthine (Xa)/xanthine
oxidase (XOD) system as superoxide radical source and Nitro Blue
Tetrazolium chloride (NBT) as detector molecule, according to
Beuchamp and Fridovich method [57]. The reduction of NBT was
followed spectrophotometrically by monitoring the absorbance at
560 nm either in the presence or absence of the investigated complex
for 600 s. The IC50 (the concentration which causes the 50% inhibition
of reduction of NBT) of the copper(II) complex at pH 7.4 was deter-
mined. Briey, the mixture was prepared in phosphate buffer (PB,
10 mM, pH 7.4) by adding Xa (20 M) and NBT (250 M). The superox-
ide production was started by adding the proper amount of XOD able to
determine a A560 ~ 0.024. All measurements were carried out at 25
0.2 C using 1 1 cm thermostatted cuvettes in which solutions were
magnetically stirred. To rule out false positive a cross-check of the po-
tential interference of the test compounds on the superoxide generating
Xa/XOD system was also carried out by following (at 295 nm) the uric
acid production coming from Xa oxidation. The assay was performed
in triplicate for each concentration of the metal complex which was
added in a concentration ranging from 0.05 to 10 M.
2.7. Trolox equivalent antioxidant capacity (TEAC) assay
The TEAC assay evaluates the radical scavenger capacity of a com-
pound by studying the changes of the absorbance of the 2,2-azino-
bis(3-ethylbenzothiazoline-6-sulphonic acid) radical cation (ABTS.+).
The ABTS radical cation decoloration assay was adapted for using
Varioskan Flash microplate reader (Thermo). In order to produce
ABTS.+, ABTS acid (7 mM) was dissolved in water and added with
potassium persulphate solution (2.5 mM). The mixture was allowed to
stand at room temperature in the dark for 1216 h, until it turned
dark blue. The solution was diluted before use to obtain the absorbance
of 0.7 at 734 nm on the plate reader. The 6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid (Trolox) standards (nal
concentration ranging from 0 to 600 M) and test compound in PB
were added to each well and nally added of ABTS.+ solution of the
proper concentration (total volume of 250 l). The ABTS radical reduc-
tion was proportionally dependent on the scavenging ability of the
test compound and caused the colorimetric absorbance variation
which was followed for 6 min. Absorbance was plotted vs. test com-
pound concentration and the resulting slope was normalized with
respect to that obtained for Trolox within the same concentration
range to give the Trolox-equivalence (TEAC) value for each time point
(1, 3, 6 min). All determinations were carried out three times.
2.8. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
assay
The effect of Semax and copper(II)Semax complex on cell viability
was tested on two proliferating cell line cultures: SHSY5Y (neuroblasto-
ma) and RBE4 (endothelial), at 6070% conuence. The SHSY5Y cells
were cultivated in Dulbecco's modied Eagle's medium (DMEM;
Lonza) supplemented with 10% fetal bovine serum (FBS; Lonza) and
100 g ml1 streptomycin, and incubated in a humidied atmosphere
of air/CO2 (95:5) at 37 C in an incubator (Heraeus Hera Cell 150). The
endothelial cell line RBE4 was cultivated in F10 medium (Lonza) sup-
plemented with 20% plasma derived serum (PDS; Lonza), 100 g ml1
streptomycin, heparin 80 g/ml, endothelial cell growth supplement
41
(ECGS) 75 g/ml, and 2 mM L-glutamine. Cells were treated with
Semax, CuSO4, Semax in the presence of CuSO4 or copper(II)Semax
complex for 48 hr. Cell viability was dened as the reduction in MTT.
Living, metabolically active cells reduce the soluble yellow tetrazolium
salt MTT, yielding dark blue water-insoluble formazan crystals. MTT
was dissolved at a concentration of 2 mg/ml in phosphate-buffered sa-
line (PBS) and DMEM supplemented with 100 g ml1 streptomycin.
The cells were incubated for 1.5 h in 300 l of MTT solution per well in
a humidied atmosphere of air/CO2 (95:5) at 37 C in an incubator.
This solution was then replaced with 300 l of DMSO per well and incu-
bated for 15 min at 37 C to dissolve the crystals. Finally, absorbance at
569 nm was measured by a microplate reader spectrophotometer
(Multiskan Ascent).
3. Results and discussion
3.1. Protonation constants and conformational features of Semax
Protonation macroconstants of Semax were determined by potenti-
ometric titration. The peptide shows four proton accepting centers
(LH4) and the related constant values are reported in Table 1. The
resulting species distribution is shown in Fig. S1. The methionine N-
terminal amino group is the most basic center (log K = 7.71), followed
by the N-imidazole of the His residue that shows a log K value of 6.61,
similar to those found for His-containing peptides [58]. Also the proton-
ation value of the -glutamyl carboxylic group (log K = 4.31) nicely
agrees with that found for other peptides containing glutamic residue
[46,59], while the C-terminal carboxylic group shows the lowest
protonation constant (log K = 2.99). Taking in consideration that
macroconstant values are determined for this system, the presence of
isomers cannot be ruled out.
Far-UV CD spectra show that the peptide adopts predominantly a
random coil conformation over the pH range investigated (Fig. 1a). A
minimum at 204 nm and a shoulder between 220 and 230 nm are
present and the slight conformational changes occurring on increasing
pH values may be attributed to the deprotonation of glutamic and
histidyl residues. Copper(II) addition induces conformational changes
which can be seen in the CD band proles (see Fig. 1b): in particular,
increasing the pH value over 4.4, a red-shift of the minimum toward
210 nm and an intensity decrease of the shoulder between 220 and
230 nm are observed. The appearance of a negative band at around
275 nm could be assigned to ligand to metal charge transfer (LMCT)
transitions [60,61].
The spectral pattern obtained by subtracting the CD traces recorded
in the absence of metal from those obtained in the presence of
copper(II) (see inset Fig. 1b) gives an indication of the alteration in
the conformation caused by copper(II) complexation over pH 4.0.
Indeed, we observe that the resulting CD curves give a positive ellipticity
around 197 nm and a negative signal centered around 220 nm and
275 nm. Overall, these CD bands indicate the induction of an ordered
structure upon metal coordination resembling that one reported for
peptides adopting turn conformations in solution [6264].
Table 1
Protonation constants (log pqr) and pK values for
peptide (T = 25 C, I = 0.1 M KNO3).a
Species Log
LH 7.71 (1)
LH2 14.32 (1)
LH3 18.63 (1)
LH4 21.62 (1)
pK NH+ 7.71
3
pK ImHis 6.61
pK COOH 4.31
pK COOH 2.99
a
Standard deviations (3 values) are given in
parentheses.
42 G. Tabb et al. / Journal of Inorganic Biochemistry 142 (2015) 3946
Fig. 1. Far-UV CD spectra recorded in aqueous solution at different pH values. From top to
bottom (arrow): (a) Semax, pH 3.5, 4.0, 4.4, 4.9, and 7.0; (b) copper(II)Semax complexes
(M/L = 1, [L] = 1 105 M) pH 4.0, 4.4, 4.9, and 7.0. Inset: difference spectra of
copper(II)Semax complexes after subtraction of Semax spectra.
3.2. ESI-MS characterization
ESI-MS technique allows the transfer of intact metal complexes from
the solution state into gas phase, where the mass assignment gives
direct information on the metal complex stoichiometry [65].
The formation of the copper(II) complexes of the Semax peptide at
1:1 metal to ligand ratio is clear in all the ESI-MS spectra carried out at
different pH values. The spectrum obtained at pH 7.0 in positive mode
is reported in the supporting information (Fig. S2). Two main signals as-
sociated with the un-complexed peptide are shown at m/z = 814.1 and
m/z = 852.2 corresponding to [Semax]H+ and [Semax]K+ systems
respectively. The signals observed at m/z = 875.1, m/z = 897.1 and
m/z = 913.1 are correlated to the copper complex species [CuL] with
H+, Na+, and K+ respectively.
The ESI-MS experiments were also carried out at pH 4.5 and ac-
quired in the negative mode. The related spectrum is reported in
Fig. 2. Two main signals corresponding to [CuLH1] and [CuLH2]2
metal complex species were found at m/z = 873.1 and m/z = 436.0 re-
spectively, in addition to the signal concerning the un-complexed
Semax peptide at m/z = 812.2.
3.3. Speciation, stability constants and coordination mode of Cu(II) complex
with Semax: spectroscopic and voltammetric characterization
The stability constant values of the copper(II) complexes with
Semax are listed in Table 2, whereas the species distribution diagram
is reported in Fig. 3. Semax starts to bind Cu(II) around pH 3.6; three
complex species are present in the system over the whole pH range ex-
plored. The rst species is [CuL], which starts to form at around pH 3.6.
Due to the low formation percentage it is not possible to determine the
Fig. 2. Negative ESI-MS spectrum obtained from 2 105 M of copper(II)Semax com-
plexes in aqueous solution pH = 4.2.
CD and UVVis parameters of [CuL] species. The quite high log value
(log 110 = 8.62) cannot be justied considering only the involvement
of two coordinating nitrogen atoms; thus, the coordination of the oxy-
gen atom belonging to a carboxyl group moiety can be involved. The co-
ordination environment is represented by a nitrogen atom coming from
the methionine amino group, an imidazole nitrogen atom of the histi-
dine residue and by the glutamil -carboxylate. The complex species
[CuLH1] starts to form around pH 3.7 and coexists with [CuL] species
over nearly two pH units. Also for this species it is not possible to asso-
ciate spectroscopic parameters because of the low formation percentage
and the concomitant presence of two other species. The pK value
(pK0/1 = 4.19) calculated for [CuLH1] is the typical value deter-
mined by the deprotonation of a peptide nitrogen atom with high afn-
ity for the metal ion and its involvement in the complexation with
copper(II), suggesting that [CuLH 1] is a 3 N donor atoms species
{NH2, N, Nim} [47,66].
Increasing the pH up to 4.0 the species [CuLH2]2 starts to form,
prevailing up to pH 4.5 and becoming the only species present at the
physiological pH value. Its pK value (pK1/2 = 4.31), similar to that
observed for the previous complex species, indicates that the coordina-
tion of another peptide nitrogen atom with high afnity for copper(II)
occurred, suggesting that [CuLH2]2 species is featured by a 4 N coor-
dination mode {NH2, 2 N, Nim} [47,67] and the formation of the
deprotonated metal complex species is not cooperative.
The two amide nitrogen deprotonations result in characteristic
changes on the UVVis, CD and ESR spectra (Table 3), indicating the in-
crease of the ligand-eld strength around the metal ion. The dd transi-
tions (max = 520 nm) and the appearance of intense, nearly symmetric
couplets both in the dd and UV regions (Fig. 4a) are in favor of the
albumin-like (NH2, N, N, NIm) coordination in [CuLH2]2 [6870].
The involvement of the N-terminal amino group and the amide
Table 2
Stability constants (log pqr) and pK values of copper(II)
complexes with Semax; [L] = 1 103 M; molar ratio
1:1 (T = 25 C, I = 0.1 M KNO3).a
Species (pqr) Log
[CuL] 8.62 (4)
[CuLH1] 4.43 (3)
[CuLH2]2 0.12 (1)
pK (0/1) 4.19
pK (1/2) 4.31
pK(n/m) values reect the pK value of copper(II) complexes.
a
Standard deviations (3 values) are given in
parentheses.
 G. Tabb et al. / Journal of Inorganic Biochemistry 142 (2015) 3946 43

Fig. 3. Species distribution diagram for copper(II) complexes in aqueous solution with
Semax in the pH range 39. (CL = CM = 1 103 M).

nitrogens is evident for the presence of the diagnostic charge trans-

fer transition bands in CD spectra centered at around 270 nm
(NH2 copper(II)) and 310 nm (N copper(II)) (Fig. 4b) [71].
However, the Hamiltonian parameters drawn out from the frozen
solution ESR spectrum at pH 7 are g|| = 2.176 and A|| = 0.0210 cm1
(Fig. 5). These parameters are characteristic for the albumin-like bind-
ing mode and indicate that Semax formed a very strong planar four ni-
trogen coordination environment around copper(II) ion, in line with the
low max wavelength obtained from visible spectra [7275].
Due to the concomitant low formation percentages of the copper
complex species existing below pH 5 it was not possible to assess the
ESR parameters at a unique species. In fact, according to the solution
Fig. 4. Near-UV CD spectra recorded in aqueous solution of copper(II)Semax system at
speciation study, a clear overlap between the signals of hexaquo copper
pH values of 4.3, 5.5, and 7.1: (a) dd transitions (arrow from top to bottom); (b) CT tran-
ion and the rst two copper(II) complexes with Semax can be seen in sitions (arrow from bottom to top). (M/L = 1, [L] = 1 105 M).
the acquired spectra pH 3.54 (Fig. 5). Recently, voltammetric studies
have been reported, which have shown to be very useful in the assign-
ment of the coordination geometries to single species that form in the
same pH range [75]. protonated ligand present in a slight excess. The donor set would in-
The square wave voltammogram run at pH 3.6 (see Fig. 6ab) volve the amino terminal nitrogen of the methionine, the histidine im-
showed a peak at +0.024 V due the reduction of Cu(II) hexaaquo ion idazole nitrogen and an oxygen coming from the glutamic carboxylic
[76] followed by a small peak at about 0.240 V which is ascribable moiety, the equatorial coordination being completed by a water oxygen
to [CuL] species. The moderately negative formal potential suggests atom. A further water oxygen atom is expected to coordinate apically to
that the bound metal ion experiences a distorted square based pyrami- form a distorted square pyramid coordination polyhedron.
dal coordination geometry. In fact, the formal potentials of copper(II) As the pH is slightly raised to 4.2, besides the Cu(II) hexaaquo ion, two
complexes having tetragonally elongated pseudo-octahedral geome- peaks centered at 0.240 V and 0.468 V were recorded (Fig. 6cd).
tries fall in a more negative redox potential region, but, if they present These peaks were attributed to the [CuL] and the [CuLH1] species,
slightly compressed pseudo-octahedral stereochemistries, the poten- respectively. With respect to the [CuL] species, a deprotonation of an
tials become a little more positive [75]. Copper(II) complexes which amide nitrogen occurs and leads to the formation of a chelate ring. The
have tetrahedral environments or square planar complexes severely formation of a chelate is reected on the redox potential which becomes
perturbed by a tetrahedral eld showed positive formal potentials. In more negative.
between there is a region (in the eld of negative redox potentials, The strong eld experienced by copper(II) in the [CuLH2]2 hin-
but less negative of those found for tetragonally elongated pseudo- ders its ease of reduction as witnessed by the very negative formal po-
octahedral complexes) which contains square-based pyramidal struc- tential obtained for this species. In fact, the SWV performed on a
tures [75]. A broad peak is also present at about 0.7 V the intensity solution containing an equimolar amount of copper(II) and Semax
of which rapidly decreases as the pH is raised. This peak practically dis- at pH 7 gave a formal potential of 0.585 V (Fig. 7a) which falls
appeared at about pH 4.5 and may be attributed to the reduction of the in the negative region where potentials belonging to planar and

Table 3
Spectroscopic parameters of copper(II) complexes with Semax. [L] = 1 103 M; molar ratio 1:1.

pH Species UVvis CD ESR

(nm) (, M1 cm1) (nm) (, M1 cm1) g|| A|| (cm1)

4.3 Cu, [CuL], [CuLH1] 272 (2.87); 310 (1.19); 482 (0.63); 554 (0.66)
5.57.1 [CuLH2]2 520 (102) 274 (3.46); 312 (1.5); 482 (0.77); 556 (0.77) 2.176 210 104
44 G. Tabb et al. / Journal of Inorganic Biochemistry 142 (2015) 3946

Fig. 5. Frozen spectra ESR spectra of copper(II) complex with Semax in aqueous solution at
1:1 Cu:L ratio and at different pH values.

pseudo-octahedral complexes gather [75]. As can be seen from cyclic
voltammogram of this species the reduction wave is completely lacking
of the corresponding re-oxidation step (Fig. 7b). This irreversible behav-
ior is in perfect agreement with the strong equatorial eld imposed by
ligand donor atoms which hinder the reduction and the accommoda-
tion of Cu+ which prefers trigonal, tetrahedral or linear environment
[77]. The SWV peak potentials found for [CuLH1] and [CuLH2]2
species are well separated by about 0.12 V. If the formation of
[CuLH 2 ] 2 species would has been occurred in cooperative fashion
only one peak potential (or two very close ones, such as differing by
0.030.05 V) should be recorded. Therefore a cooperative process
Fig. 7. Square wave (a) and cyclic (b) voltammograms of 1 mM aqueous solution of
copper(II)Semax complex species at 1:1 Cu:L ratio and at pH 7. SW voltammogram
was run at 15 Hz (step 2 mV) whereas CV was run at 0.02 V s1. 100 mM KNO3 was
used as supporting electrolyte.
for the formation of the deprotonated metal complex species can be
ruled out.
3.4. Antioxidant properties
The antioxidant feature of Semax and the superoxide dismutase mi-
metic activity of the copper(II) complexes with Semax were determined
(Table 4). The ligand Semax did not show any antioxidant activity.
Conversely, a negligible stoichiometric scavenging activity is shown by
its complex (IC50 equal to 6.7 M). For comparison, the values of IC50
of SOD native enzyme and CuHPO4 under our experimental conditions
were 0.014 M and 1.0 M, respectively [78].
Further, the protective effect of Semax against ABTS radical cation
was also evaluated by the TEAC assay. The results shown in Table 4 sug-
gest that Semax peptide has a radical scavenger capacity quite limited
and probably due to a poor stoichiometric interaction with the ABTS
radical. This limited radical scavenger ability is abolished after complex-
ation with copper(II). Due to the strength of the coordination equatorial
plane that features the copper(II) complex with Semax, its reduction
redox potential is quite negative, meaning that the copper(II) reduction
is unfavored. In addition, no interaction between [CuLH2]2 and
Trolox radical occurred.

Table 4
Kinetic rate constants (kcat, M1 s1), IC50 (M) and TEAC values (at 1, 3, and 6 min) for
Semax and copper(II)Semax complex.

SOD activity TEAC values

IC50 (M) Kcat 1 min 3 min 6 min

Fig. 6. Square wave and cyclic voltammograms of 1 mM aqueous solution of copper(II)
SOD 0.014 (0.003) 1.5 (0.3) 109
Semax complex species at 1:1 Cu:L ratio and at pH 3.6 (ab) and 4.2 (cd). Asterisks indi-
Semax n.a. n.a. 0.14(2) 0.15(3) 0.15(2)
cate a residual reduction of the ligand. SW voltammograms were run at 15 Hz (step 2 mV)
Cu(II)Semax (1:1) 6.68 (0.05) 2.2 (0.3) 106 n.a. n.a. n.a
and CV were run at 0.02 V s1. 100 mM KNO3 was used as supporting electrolyte.
 G. Tabb et al. / Journal of Inorganic Biochemistry 142 (2015) 3946
Fig. 8. Dose response experiment measuring the protective effect of Semax against Cu2+
toxicity at the ratio Semax/Cu2+ 1:1. SHSY5Y (neuroblastoma) cells were incubated
with increasing concentrations of CuSO4 or CuSO4 in the presence of Semax for 48 h
after which cell viability was measured using MTT assay and performed in triplicate.
Results are presented as mean SEM. Asterisk (*) represents the correlation signicant
at the p 0.05 level.
3.5. Biological assays
Neuroblastoma (SHSY5Y) cell cultures treated with the peptide and
its copper(II) complex at different concentrations (range 1 200 M)
don't undergo toxic effect at each concentration used (Fig. S3). Semax,
furthermore, showed a signicant and dose-dependent protective effect
against copper toxicity (Fig. 8). The signicant reduction of viability ob-
served after cell culture treatment for 48 h with 100 and 200 M CuSO4
( 30% and 50% respectively) was abolished by the presence of the
peptide at the same concentration. We observed the same effects on en-
dothelial RBE4 cells (Fig. S4). The observed copper(II) mediated toxicity
is related to the high chelating ability of Semax.
4. Conclusions
Semax is a synthetic analog of the ACTH(4-10) with neuroprotective
and nootropic properties. Its potential chelating ability has not been
reported.
In this work, thermodynamic and spectroscopic parameters indicate
that Semax peptide is able to complex copper(II) in an albumin-like
mode starting from pH 4.5, showing a very strong afnity. The ligand
Semax structure, rened by MOMEC97, shows a sort of pocket ready
to host the metal (Fig. S5a). This structure is stabilized by electrostatic
interactions and by a hydrogen bond between the peptidic hydrogen
of the Phe and the amino nitrogen of Met. An accurate inspection of
the rened structure of the [CuLH2]2 species reveals a further stabi-
lization by a couple of hydrogen bonds: the rst between the peptidic
hydrogen of the Phe and the adjacent carbonyl oxygen of Pro, while
the second interaction is between the peptide hydrogen of the
Gly(6) and the carbonyl oxygen of Met (Fig. S5b). This additional non-
covalent interaction accounts for the high stability of the copper (II)
complex species.
[CuH2L]2 is the only, very stable complex species detectable at
physiological pH with a very strong ligand eld around the metal ion.
In this respect, from the stability constants data reported in Table 2,
we calculated a value of 1.3 1015 M for the conditional dissociation
constant (cKd) of this system at pH 7.4 by using a method reported else-
where [79]. This value is comparable to that observed for other peptide
having similar amino acid sequences as reported in literature [80,67].
Finally, the data obtained on the copper chelating activity in the cell
culture medium reported here suggest that Semax might have a role in
the copper homeostasis. Furthermore, considering the irreversible be-
havior of copper(II)Semax complex in the redox cycling and the
45
growing evidence on the role of redox active metal ions in several neu-
rodegenerative disorders, memory and cognitive processes, the poten-
tial role of Semax in tuning or minimizing metal induced oxidative
stress will be object of further investigations.
Acknowledgments
This work was supported by MIUR FIRB_MERIT RBNE08HWLZ for
nancial support. Authors wish to thank Ms Daniela Gemma Cartia for
text correction and manuscript preparation.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jinorgbio.2014.09.008.
References
[1] I. Gantz, T.M. Fong, Am. J. Physiol. Endocrinol. Metab. 284 (2003) E468E474.
[2] M.E. Hadley, R.T. Dorr, Peptides 27 (2006) 921930.
[3] C. Caruso, L. Carniglia, D. Durand, T.N. Scimonelli, M. Lasaga, Melanocortins: anti-
inammatory and neuroprotective peptides, in: L.M. Martins, S.H.Y. Loh (Eds.),
Neurodegeneration, InTech, 2012, pp. 93120.
[4] E.M. Hol, W.H. Gispen, P.R. Bar, Peptides 16 (1995) 979993.
[5] F.L. Strand, Ann. N. Y. Acad. Sci. 897 (1999) 116.
[6] P.E. Gold, R.L. Delanoy, ACTH modulation of memory storage processing, in: J.L.
Martinez Jr., R.A. Jensen, R.B. Messing, H. Rigter, J.L. McGaugh (Eds.), Endogenous
Peptides and Learning and Memory Processes, Academic Press, New York, 1981,
pp. 7997.
[7] H.M. Greven, D. de Wied, Front. Horm. Res. 4 (1977) 140152.
[8] D. de Wied, Behav. Brain Res. 83 (1997) 8390.
[9] D. de Wied, A. Witter, H.M. Greven, Biochem. Pharmacol. 24 (1975) 14631468.
[10] D. de Wied, W.H. Gispen, Behavioral effects of peptides, in: H. Gainer, J.L. Barker
(Eds.), Peptides in Neurobiology, Plenum Press, New York, 1977, pp. 397448.
[11] A. Witter, H.M. Greven, D. de Wied, Exp. Ther. 193 (1975) 853860.
[12] F.L. Strand, Eur. J. Pharmacol. 405 (2000) 312.
[13] F.L. Strand, Prog. Drug Res. 61 (2003) 137.
[14] V.N. Potaman, L.Y. Alfeeva, A.A. Kamensky, N.G. Levitskaya, V.N. Nezavibatko,
Biochem. Biophys. Res. Commun. 176 (1991) 741746.
[15] I.P. Ashmarin, V.N. Nezavibat'ko, N.G. Levitskaya, V.B. Koshelev, A.A. Kamensky,
Neurosci. Res. Commun. 16 (1995) 105112.
[16] I.P. Asmarin, V.N. Nezavibat'ko, N.F. Miasoedov, A.A. Kamenskii, I.A. Grivennikov,
M.A. Ponomareva-Step-naia, L.A. Andreeva, A.Y. Kaplan, V.B. Koshelev, T.V. Riasina,
Zh. Vyssh. Nerv. Deiat. Im I P Pavlova 47 (1997) 420430.
[17] V.N. Potaman, L.Y. Alfeeva, A.A. Kamensky, V.N. Nezavibat'ko, Peptides 14 (1993)
491495.
[18] Y.A. Zolotarev, O.V. Dolotov, L.S. Inozemtseva, A.K. Dadayan, E.M. Dorokhova, L.A.
Andreeva, L.Y. Alfeeva, I.A. Grivennikov, N.F. Myasoedov, Amino Acids 30 (2006)
403408.
[19] T.P. Storozhevykh, G.R. Tukhbatova, Y.E. Senilova, V.G. Pinelis, L.A. Andreeva, N.F.
Myasoyedov, Bull. Exp. Biol. Med. 143 (2007) 601604.
[20] K.V. Martynova, L.A. Andreeva, P.A. Klimova, Y.G. Kirillova, V.P. Shevchenko, I.Y.
Nagaev, S.I. Shram, V.I. Shvets, N.F. Myasoedov, Russ. J. Bioorg. Chem. 35 (2009)
150156.
[21] A.Y. Kaplan, A.G. Kochetova, V.N. Nezavibat'ko, T.V. Rjasina, I.P. Ashmarin, Neurosci.
Res. Commun. 19 (1996) 115123.
[22] V.V. Stavchanskiia, T.V. Tvorogova, A.Yu. Botsinab, S.A. Limborskaa, V.I. Skvortsovab,
N.F. Myasoedova, L.V. Dergunovaa, Mol. Biol. 47 (2013) 406410.
[23] V.G. Bashkatova, V.B. Koshelev, O.E. Fadyukova, A.A. Alexeev, A.F. Vanin, K.S.
Rayevsky, I.P. Ashmarin, D.M. Armstrong, Brain Res. 894 (2001) 145149.
[24] V.N. Potaman, L.V. Antonova, V.A. Dubynin, D.A. Zaitzev, A.A. Kamensky, N.F.
Myasoedov, V.N. Nezavibatko, Neurosci. Lett. 127 (1991) 133136.
[25] V.G. Dmitrieva, L.V. Dergunova, O.V. Povarova, V.I. Skvortsova, S.A. Limborska, N.F.
Myasoedov, Dokl. Biochem. Biophys. 422 (2008) 261266.
[26] E.V. Yakovleva, V.S. Kuzenkov, V.N. Fedorov, V.I. Skvortsova, V.B. Koshelev, E.I.
Gusev, I.P. Ashmarin, Bull. Exp. Biol. Med. 128 (1999) 806807.
[27] O.V. Dolotov, E.A. Karpenko, L.S. Inozemtseva, T.S. Seredenina, N.G. Levitskaya, J.
Rozyczka, E.V. Dubynina, E.V. Novosadova, L.A. Andreeva, L.Y. Alfeeva, A.A.
Kamensky, I.A. Grivennikov, N.F. Myasoedov, J. Engele, Brain Res. 1117 (2006)
5460.
[28] T.Y. Agapova, Y.V. Agniullin, M.I. Shadrina, S.I. Shram, P.A. Slominsky, S.A.
Lymborska, N.F. Myasoedov, Neurosci. Lett. 417 (2007) 201205.
[29] G. Marx, C. Gilon, ACS Chem. Neurosci. 3 (2012) 633642.
[30] J. Ceccom, F. Cosledan, H. Halley, B. France, J.M. Lassalle, B. Meunier, PLoS ONE 7
(e43105) (2012) 17.
[31] P.A. Adlard, A.I. Bush, J. Alzheimers Dis. 10 (2006) 145163.
[32] G. Eskici, P.H. Axelsen, Biochemistry 51 (2012) 62896311.
[33] A.I. Bush, Trends Neurosci. 26 (2003) 207214.
[34] E. Gaggelli, H. Kozlowski, D. Valensin, G. Valensin, Chem. Rev. 106 (2006)
19952044.
46 G. Tabb et al. / Journal of Inorganic Biochemistry 142 (2015) 3946

[35] X. Huang, M.P. Cuajungco, C.S. Atwood, M.A. Hartshorn, J.D. Tyndall, G.R. Hanson, K. [56] HYPERCHEM, release 5.1 Hypercube, 1996.
C. Stokes, M. Leopold, G. Multhaup, L.E. Goldstein, R.C. Scarpa, A.J. Saunders, J. Lim, R. [57] C. Beauchamp, I. Fridovich, Anal. Biochem. 44 (1971) 276287.
D. Moir, C. Glabe, E.F. Bowden, C.L. Masters, D.P. Fairlie, R.E. Tanzi, A.I. Bush, J. Biol. [58] S. Timri, C. Kllay, K. Osz, I. Sovago, K. Vrnagy, Dalton Trans. (2009) 19621971.
Chem. 274 (1999) 3711137116. [59] G. Grasso, A. Magr, F. Bellia, A. Pietropaolo, D. La Mendola, E. Rizzarelli, J. Inorg.
[36] G. Meloni, V. Sonois, T. Delaine, L. Guilloreau, A. Gillet, J. Teissie, P. Faller, M. Vasa'k, Biochem. 130 (2014) 92102.
Nat. Chem. Biol. 4 (2008) 366372. [60] J.M. Tsangaris, J.W. Chang, R.B. Martin, J. Am. Chem. Soc. 91 (1969) 726731.
[37] C. Opazo, X. Huang, R.A. Cherny, R.D. Moir, A.E. Roher, A.R. White, R. Cappai, C.L. [61] T.G. Fawcett, E.E. Bernarducci, K. Krough-Jespersen, H.J. Schugar, J. Am. Chem. Soc.
Masters, R.E. Tanzi, N.C. Inestrosa, A.I. Bush, J. Biol. Chem. 277 (2002) 4030240308. 102 (1980) 25982604.
[38] D. Milardi, E. Rizzarelli, Neurodegeneration: Metallostasis and Proteostasis, RSC, [62] A. Perczel, M. Hollosi, in: G.D. Fasman (Ed.), Circular Dichroism and the Conforma-
Cambridge, 2011. tional Analysis of Biomolecules, Plenum Press, New York, 1996, pp. 285380.
[39] W. Wang, J.L. Post, K.E. Dow, S.H. Shin, R.J. Riopelle, G.M. Ross, Neurosci. Lett. 259 [63] R.W. Woody, in: E.R. Blout, F.A. Bovey, M. Goodman, L. Lotan (Eds.), Peptides, Poly-
(1999) 115118. peptides and Proteins, Wiley, New York, 1974, p. 338.
[40] B. Byrkava, J.M. Aletta, J. Neurobiol. 63 (2005) 4961. [64] M.C. Manning, M. Illangasekare, R.W. Woody, Biophys. Chem. 31 (1988) 7786.
[41] S. Kheirvari, K. Uezu, T. Sakai, M. Nakamori, M. Alizadeh, N. Sarukura, S. Yamamoto, J. [65] G. Maccarrone, R. Caruso, A. Contino, A. Giuffrida, M. Messina, V. Cucinotta, Eur. J.
Nutr. Sci. Vitaminol. 52 (2006) 421427. Inorg. Chem. (2009) 26122620.
[42] F.J. Sanchez-Martin, E. Valera, I. Casimiro, J.M. Merino, Brain Res. Bull. 81 (2010) [66] T. Kowalik-Jankowska, E. Jankowska, F. Kasprzykowski, Inorg. Chem. 49 (2010)
458466. 21822192.
[43] C. Corona, F. Masciopinto, E. Silvestri, A. Del Viscovo, R. Lattanzio, R. La Sorda, D. [67] D. Witkowska, S. Bielinska, W. Kamysz, H. Kozlowski, J. Inorg. Biochem. 105 (2011)
Ciavardelli, F. Goglia, M. Piantelli, L.M.T. Canzoniero, S.L. Sensi, Cell Death Dis. 28 208214.
(2010) e91. [68] C. Harford, B. Sarkar, Acc. Chem. Rev. 30 (1997) 123130.
[44] Y. Yang, X.-P. Jing, S.-P. Zhang, R.-X. Gu, F.-X. Tang, X.-L. Wang, Y. Xiong, M. Qiu, X.-Y. [69] T. Gaida, B. Henry, A. Anbry, J.J. Delpuech, Inorg. Chem. 35 (1996) 586593.
Sun, D. Ke, J.-Z. Wang, R. Liu, PLoS ONE 8 (2013) e55384. [70] C. Conato, H. Kozlowski, P. Mlynarz, F. Pulidori, M. Remelli, Polyhedron 21 (2002)
[45] G.M. Ross, I.L. Shamovsky, S.B. Woo, J.I. Post, P.N. Vrkljan, G. Lawrance, M. Solc, S.M. 14691474.
Dostaler, K.E. Neet, R.J. Riopelle, J. Neurochem. 78 (2001) 515523. [71] T. Kowalik-Jankowska, M. Ruta-Dolejsza, K. Wisniewska, L. Lankiewicz, J. Inorg.
[46] A. Travaglia, G. Arena, R. Fattorusso, C. Isernia, D. La Mendola, G. Malgieri, V.G. Biochem. 86 (2001) 535545.
Nicoletti, E. Rizzarelli, Chem. Eur. J. 17 (2011) 37263738. [72] R.P. Bonomo, E. Cucinotta, F. D'Alessandro, G. Impellizzeri, G. Maccarrone, G. Vecchio,
[47] A. Travaglia, A. Pietropaolo, D. La Mendola, V.G. Nicoletti, E. Rizzarelli, J. Inorg. E. Rizzarelli, Inorg. Chem. 30 (1991) 2702713.
Biochem. 111 (2012) 130137. [73] R.P. Bonomo, E. Conte, R. Marchelli, A.M. Santoro, G. Tabb, J. Inorg. Biochem. 53
[48] A. Travaglia, D. La Mendola, A. Magr, V.G. Nicoletti, A. Pietropaolo, E. Rizzarelli, (1994) 127138.
Chem. Eur. J. 18 (2012) 1561815631. [74] R.P. Bonomo, A. De Flora, E. Rizzarelli, A.M. Santoro, G. Tabb, M. Tonetti, J. Inorg.
[49] R.P. Bonomo, V. Cucinotta, A. Giuffrida, G. Impellizzeri, A. Magr, G. Pappalardo, Biochem. 59 (1995) 773784.
E. Rizzarelli, A.M. Santoro, G. Tabb, L.I. Vagliasindi, Dalton Trans. (2005) 150158. [75] G. Tabb, A. Giuffrida, R.P. Bonomo, J. Inorg. Biochem. 128 (2013) 137145.
[50] P. Gans, A. Sabatini, A. Vacca, Talanta 43 (1996) 17391753. [76] R.P. Bonomo, R. Marchelli, G. Tabb, J. Inorg. Biochem. 60 (1995) 205218.
[51] L. Alderighi, P. Gans, A. Ienco, D. Peters, A. Sabatini, A. Vacca, Coord. Chem. Rev. 184 [77] N. Greenwood, A. Earnshaw, Chemistry of the Elements, Pergamon Press, Oxford,
(1999) 311318. 1984.
[52] A. Lund, T.J. Vanngard, Chem. Phys. 50 (1969) 29792980. [78] R.P. Bonomo, V. Bruno, E. Conte, G. De Guidi, D. La Mendola, G. Maccarrone,
[53] A.E. Kaifer, J. Bard, J. Phys. Chem. 89 (1985) 48764880. F. Nicoletti, E. Rizzarelli, S. Sortino, G. Vecchio, Dalton Trans. (2003) 44064415.
[54] P. Comba, T.V. Hambley, N. Okon, MOMEC, A Molecular Modelling Package for Inor- [79] P. Faller, C. Hureau, Dalton Trans. (2009) 10801094.
ganic Compounds, Lauer & Okon, Heidelberg, Germany, 1996. [80] G. Arena, E. Rizzarelli, B. Sarkar, Inorg. Chim. Acta 37 (1979) L555L557.
[55] P. Comba, P.V. Bernardt, Inorg. Chem. 31 (1992) 26382644.