Copper(II) complexes of alloferon 1 with point mutations (H1A) and (H9A) stability structure and biological activity

Article abstract of DOI:10.1016/j.jinorgbio.2014.05.012

Mono- and polynuclear copper(II) complexes of the alloferon 1 with point mutations (H1A) A¹GVSGH⁶GQH⁹GVH¹²G (Allo1A) and (H9A) H¹GVSGH⁶GQA⁹GVH ¹²G (Allo9A) have been studied by potentiometric, UV-visible, CD, EPR spectroscopic and mass spectrometry (MS) methods. To obtain a complete complex speciation different metal-to-ligand molar ratios ranging from 1:1 to 4:1 for Allo1A and to 3:1 for Allo9A were studied. The presence of the His residue in first position of the peptide chain changes the coordination abilities of the Allo9A peptide in comparison to that of the Allo1A. Imidazole-N3 atom of N-terminal His residue of the Allo9A peptide forms stable 6-membered chelate with the terminal amino group. Furthermore, the presence of two additional histidine residues in the Allo9A peptide (H⁶,H¹²) leads to the formation of the CuL complex with 4N {NH₂,N_Im-H ¹,N_Im-H⁶,N_Im-H¹²} binding site in wide pH range (5-8). For the Cu(II)-Allo1A system, the results demonstrated that at physiological pH 7.4 the predominant complex the CuH _{- 1}L consists of the 3N {NH₂,N^-,CO,N _Im} coordination mode. The inductions of phenoloxidase activity and apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH 7.4 were studied. The Allo1A, Allo1K peptides and their copper(II) complexes displayed the lowest hemocytotoxic activity while the most active was the Cu(II)-Allo9A complex formed at pH 7.4. The results may suggest that the N-terminal-His¹ and His⁶ residues may be more important for their proapoptotic properties in insects than those at positions 9 and 12 in the peptide chain.

Full text of DOI:10.1016/j.jinorgbio.2014.05.012

Copper(II) Complexes of Alloferon 1 with Point Mutations (H1A) and (H9A)
Stability Structure and Biological Activity
Agnieszka Matusiak, Mariola Kuczer, Elz˙bieta Czarniewska, Grzegorz
Rosin´ski, Teresa Kowalik-Jankowska
PII:
DOI:
Reference:
S0162-0134(14)00150-0
doi: 10.1016/j.jinorgbio.2014.05.012
JIB 9532
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Journal of Inorganic Biochemistry
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Revised date:
Accepted date:
4 February 2014
22 May 2014
23 May 2014
Please cite this article as: Agnieszka Matusiak, Mariola Kuczer, Elz˙bieta Czarniewska,
Grzegorz Rosin´ski, Teresa Kowalik-Jankowska, Copper(II) Complexes of Alloferon 1 with
Point Mutations (H1A) and (H9A) Stability Structure and Biological Activity, Journal
of Inorganic Biochemistry (2014), doi: 10.1016/j.jinorgbio.2014.05.012
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ACCEPTED MANUSCRIPT
Copper(II) Complexes of Alloferon 1 with Point Mutations (H1A) and (H9A)
Stability Structure and Biological Activity
Agnieszka Matusiaka, Mariola Kuczera, Elżbieta Czarniewskab, Grzegorz Rosińskib and
Teresa Kowalik-Jankowskaa*
a Faculty of Chemistry, University of Wrocław, 14 Joliot-Curie, 50-383 Wroclaw (Poland)
b
Departament of Animal Physiology and Development, Institute of Experimental Biology,
Adam Mickiewicz University, 89 Umultowska, 61-614 Poznań (Poland)
___________________________________________________________________________
* Corresponding author. Tel: +48 71 3757231; fax +48 71 328 23 48.
E-mail address: teresa.kowalik-jankowska@chem.uni.wroc.pl (T. Kowalik-Jankowska)
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ABSTRACT
Mono- and polynuclear copper(II) complexes of the alloferon 1 with point mutations
(H1A) A1GVSGH6GQH9GVH12G (Allo1A) and (H9A) H1GVSGH6GQA9GVH12G (Allo9A)
have been studied by potentiometric, UV-visible, CD, EPR spectroscopic and mass
spectrometry (MS) methods. To obtain a complete complex speciation different metal-to-
ligand molar ratios ranging from 1:1 to 4:1 for Allo1A and to 3:1 for Allo9A were studied .
The presence of the His residue in first position of the peptide chain changes the coordination
abilities of the Allo9A peptide in comparison to that of the Allo1A. Imidazole-N3 atom of N-
terminal His residue of the Allo9A peptide forms stable 6-membered chelate with the terminal
amino group. Furthermore, the presence of two additional histidine residues in the Allo9A
peptide (H6,H12) leads to the formation of the CuL complex with 4N {NH2,NIm-H1,NIm-
H6,NIm-H12} binding site in wide pH range (5-8). For the Cu(II)-Allo1A system, the results
demonstrated that at physiological pH 7.4 the predominant complex the CuH-1L consists of
the 3N {NH2, N-,CO,NIm} coordination mode. The inductions of phenoloxidase activity and
apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at
pH 7.4 were studied. The Allo1A, Allo1K peptides and their copper(II) complexes displayed
the lowest haemocytotoxic activity while the most active was the Cu(II)-Allo9A complex
formed at pH 7.4. The results may suggest that the N-terminal-His1 and His6 residues may be
more important for their proapoptotic properties in insects than those at positions 9 and 12 in
the peptide chain.
Keywords: Alloferon, copper, biological activity, mono- and polynuclear complexes,
apoptosis
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1. Introduction
Alloferon was originally isolated from the experimentally infected insect, a blow fly
Calliphora vicina and consists of 13 amino acids: HGVSGHGQHGVHG. It has been
demonstrated that alloferon stimulates the natural cytotoxicity of human peripheral blood
lymphocytes and enhances antitumor and antiviral activities through the induction of INF
synthesis [1]. Lee et al. have reported that alloferon has dual functions: one is direct inhibition
of the replication of Kaposi’s sarcoma-associated herpesvirus and the other is effective
eradication of virus-infected cells through the activation of natural killer (NK) cells [2].
Alloferon 1 monotheraphy demonstrated moderate tumoristatic and tumoricidal activities
comparable with low dose chemotherapy. When alloferon 1 and the cytotoxic drugs were
combined in a regime of pulse immunochemotherapy the combination antitumor activity
evidently exceeded that of the treatments applied individually [3]. Thus, alloferon 1 is interest
as a potential anticancer drug. It was also reported that alloferon has no toxicity to normal
cells and does not affect the growth of normal cells [4]. It is suggested, that alloferon might be
useful as an immune-modulating reagent to increase NK cell cytotoxicity to NK-resistant
human cancer cells [5]. Chernysh et al. are reported that alloferon 1 and especially its novel
structural analog reffered to as allostatine are worthy of futher consideration as potential
anticancer drugs [6]. Allostatin seems to be particularly perspective for adjuvant cancer
immunotherapy. Since alloferon was originally identified as an anticancer reagent through the
activation of the antitumor immune system, it is expected that alloferon might effectively
prevent the development of skin cancer from UVB irradiation by preventing the malignant
transformation of skin cells and by activating the antitumor immune systems in the skin. The
preventive effect of alloferon on the development of skin cancer by UVB irradiation is under
investigation [4]. In the literature is presented the opinion that the presence of a His residue in
the peptide chain of a suitable structure may endow such a peptide with potent antiviral
capabilities [7]. Therefore, the studies of alloferon 1 analogues modified at position 1 were
performed. In these analogues the His residue at position 1 was replaced by other basic amino
acid (Lys, Arg) and by the hydrophobic amino acid (Ala) [8]. The substitution of His by Ala
at position 1 resulted in a complete loss of activity against studied viruses, while the
replacement of His by Lys at position 1 leads to a compound with strong antiviral activity [8].
In the second group of analogues, modified at position 6 (His residue) of the alloferon peptide
chain, it was found that all analogues showed the proapoptotic effect of 50 – 220 % relative to
alloferon. The most active was alloferon with point mutation (H6A), Allo6A [9].
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Many essential metal ions act as the important factor influencing the structure of
natural and synthetic oligopeptides, and as a consequence, they may have a critical impact on
their biological activity [10]. Moreover, the copper-peptide complex may be more resistant to
enzymatic degradation in comparison to that of free ligand [11]. The pharmacological tests
reveal that the coordination effect may improve the biological activity of the ligand [12].
Complex species are built around 4d or 5d metals: e.g. palladium and platinum [13,14],
cadmium [15], gold [16], nickel(II) and copper(II) [12,17]. The activity may be also linked to
the lipophilicity of the complexes: the higher the lipophilicity, the easier the penetration in the
cell through the cellular membrane [18].
It was found that insect peptides as Neb-colloostatin [19], alloferon, and its selected
analogues cause apoptosis in hemocytes of T. molitor [9]. Furthermore, the studies
demonstrates that many Cu(II) complexes exhibit cytotoxic activity through cell apoptosis
[20-23]. Also, heavy metals are known to be typical stimuli to trigger apoptosis in vertebrate
and invertebrate cells [24-26]. The induction of apoptosis in vivo in insect cells of Tenebrio
molitor by the alloferon mutants (H6A) and (H12A) and their copper(II) complexes at pH 7.4
was determined [27]. The biological studies show that Cu(II) ions in vivo did not cause any
apparent apoptotic features in T. molitor cells but Cu(II) complexes of alloferon induce
apoptosis [27]. It was also reported that Cu(II) strongly induces apoptosis in vitro in insect
cells of Aedes albopictus [25].
Now, in this paper we report the synthesis of two, terminally free peptides containing
three separate histidyl residues and the studies on their copper(II) complexes. The peptides
involved in the study are mutants (H1A) and (H9A) of alloferon 1, H-His1-Gly-Val-Ser-Gly-
His6-Gly-Gln-His9-Gly-Val-His12-Gly-OH (Allo1). The peptides which are studied contain
three histidine residues (H6,H9,H12) Allo1A (H1A) and (H1,H6,H12) Allo9A (H9A). The
copper(II) complexes were studied by the combined application of potentiometric equilibrium,
spectroscopic UV-visible (UV-vis), CD and EPR and mass spectrometry methods in solution.
The induction of apoptosis in vivo in insect cells of Tenebrio molitor by the ligands
studied and their copper(II) complexes at pH 7.4 was determined. Moreover, the effects of the
alloferon 1, alloferon 1 mutants and their Cu(II) complexes on the phenoloxidase enzyme
activity in T. molitor and on myocardial activity in vitro on the heart of T. molitor were
studied. The coordination abilities and biological activities of the alloferon mutants (H1A)
and (H9A) are compared to those of the (H6A), (H12A) [27] and (H1K) [28] analogues. The
importance of the position of the His residue in the peptide chain of alloferon 1 on its
biological activity is suggested.
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2. Experimental
2.1. Materials
The peptides were synthesized according to a procedure reported below. As a metal
ion source Cu(NO3)2 * 3 H2O was used. KNO3, KOH and HNO3 were Merck products.
2.2. Peptide syntheses, purification and characterization
Peptides were synthesized by the classical solid phase method according to the Fmoc-
procedure as described previously [27,28]. Amino acids were assembled on a Fmoc-Gly-
Wang resin. As a coupling reagent 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU) in the presence of 1-hydroxybenzotriazole (HOBt) was used.
The N-Fmoc (Fmoc, 9-fluorenylmethoxycarbonyl) group was removed with 20% piperidine in
N, N-dimethylformamide (DMF). The peptide-resin was cleaved with trifluoroacetic acid
(TFA) in the presence of ethanedithiol (EDT). All peptides were purified by preparative
HPLC on a Varian ProStar, column -Tosoh Biosciences ODS-120T C18 (ODS 300 x 21.5
mm). Water–acetonitrile gradients containing 0.1% TFA at a flow rate of 7 ml/min were used
for purification with UV detection at 210/254 nm. Analytical HPLC was performed using a
Thermo Separation Products with a Vydac ProteinRP C18 column (4.6 mm × 250 mm)
(Grace, Deerfield, IL, USA) with a linear gradient from 0 to 100% B in 60 min, flow rate
1 ml/min, solvent A = 0.1% TFA in water, solvent B = 0.1% TFA in 80% acetonitrile/water,
UV detection at 210 nm). The molecular weights of the synthesized compounds were
confirmed by ESI-MS using Apex-Qe Ultra 7T FT-ICR instrument (Bruker Daltonic, Bremen,
Germany). Analytical data were as follows:
Allo1A (H1A): Rt = 11.7 min, [M+H]+ MS = 1199.573 Da, Mcalc = 1199.567 Da; [M+2H]2+,
MS = 600.295 Da, Mcalc = 600.287 Da; [M+3H]3+ MS = 400.533 Da, Mcalc = 400.527 Da;
Synthesis of Allo1A was carried out on a 0.25 mmol scale, yielding 280 mg (94%) dry crude
peptide (74% purity). From a preparative HPLC run of 50 mg of crude Allo1A, 15 mg (30%)
of pure peptide was obtained (> 98% purity).
Allo9A (H9A): Rt = 13.0 min, [M+H]+ MS = 1199.6205 Da, Mcalc = 1199.567 Da; [M+2H]2+,
MS = 600.307 Da, Mcalc = 600.287 Da; [M+3H]3+ MS = 400.531 Da, Mcalc = 400.527 Da;
Synthesis of Allo9A was carried out on a 0.25 mmol scale, yielding 275 mg (91%) dry crude
peptide (81% purity). From a preparative HPLC run of 50 mg of crude Allo1A, 18 mg (36%)
of pure peptide was obtained (> 98% purity).
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Monoisotopic mass of the indicated ion formed by the peptide calculated using the mMass
program.
2.3. Potentiometric measurements
Stability constants for proton and Cu(II) complexes were calculated from pH-metric
titrations carried out in the argon atmosphere at 298 K using a total volume of 1.3 – 2 ml.
Alkali was added from a 0.250 ml micrometer syringe which was calibrated by both weight
titration and the titration of standard materials. Experimental details: ligand concentration
0.001 M for Allo1A and Allo9A, metal-to-ligand molar ratio 1:1, 2:1, 3:1 for both systems
and 4:1 for Cu(II)-Allo1A (concentration of stock solution of copper(II) ions 0.05 M); the
ionic strength 0.10 M (KNO3); pH-metric titration on a MOLSPIN pH-meter system using a
Russell CMAW 711 semimicro combined electrode, calibrated in concentration using HNO3
[29]; number of titrations = 2; method of calculation SUPERQUAD [30] and HYPERQUAD
[31]. The samples were titrated in the pH region 2.5 – 10.5. Standard deviations (values)
quoted were computed by SUPERQUAD and HYPERQUAD and refer to random errors only.
They are, however, a good indication of the importance of the particular species involved in
the equilibria. The purities and exact concentration of the solutions of the ligand were
determined by the method of Gran [32].
The formation reaction equilibrium of ligands with protons and copper(II) ions are given in
Equation (1):
p Cu + q H + r L = CupHqLr
(1)
in which L are the peptides under study. The stability constant βpqr is defined in Equation (2):
βpqr = [CupHqLr] / [Cu]p . [H]q . [L]r
(2)
2.4. Spectroscopic measurements
Solutions were of similar concentrations to those used in potentiometric studies.
Absorption spectra (UV-vis) were recorded on a Cary 50 “Varian” spectrophotometer in the
850 – 300 nm range. Circular dichroism (CD) spectra were recorded on a Jasco J-715
spectropolarimeter in the 750 – 250 nm range. The spectra were scanned at the pH and
stoichiometric M:L values adequate to obtain the maximum formation of the particular
species but also in this condition other species coexist in a smaller concentration. The values
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of Δε (i.e. εL-εR) and ε were calculated at the maximum concentration of the particular species
obtained from potentiometric data. EPR spectra were performed in the ethylene glycol-water
(1:2, v/v) solution at 77 K on a Bruker ELEXYS E 500 spectrometer equipped with NMR
teslameter ER 036TM and frequency counter E 41 FC at the X-band frequency (~9.45 GHz).
The spectra were analyzed by using Bruker’s WIN-EPR SimFonia software, version 1.25.
Copper(II) stock solution was prepared from Cu(NO3)2 * 3 H2O.
2.5. ESI-MS measurements
The mass spectra were obtained on a Bruker MicrOTOF-Q spectrometer (Bruker
Daltonik, Bremen, Germany), equipped with Apollo II electrospray ionization source. The
mass spectrometer was operated in the positive ion mode. The instrumental parameters were
as follows: scan range m/z 400–2300, dry gas - nitrogen, temperature 200 ºC, reflector voltage
1300 V, detector voltage 1920 V. The samples 1:1, 2:1, 3:1 metal-to-ligand molar ratios for
both systems and also 4:1 for Cu(II)-Allo1A at pH about 7 were prepared in water and
infused at a flow rate of 3 µl/min. The instrument was calibrated externally with the
Tunemix™ mixture (Bruker Daltonik, Germany) in quadratic regression mode.
2.6. Biological studies
Insects: A stock culture of T. molitor was maintained at the Department of Animal Physiology
and Development as described previously [33]. Studies were carried out on 4-days old adult
male beetles. As the mealworm parents’ age is important for the developmental features of
their offspring [34-36] all insects in our experiments derived from less then 1 month old
parents. Alloferon 1, respective analogues and their copper(II) complexes were injected in
MOPS buffer at pH 7.4 (Sigma-Aldrich, MOPS – 3-(N-morpholino)propanesulfonic acid)
[37].
Phenoloxidase activity studies
The beetles were anaesthetised with CO2, washed in distilled water and disinfected
with 70% ethanol. Alloferon or its analogue were injected (2 µl, in dose of 10 nmole per
insect) through the ventral membrane between the second and the third abdominal segments
towards the head with a Hamilton syringe (Hamilton Co.). The control insects were injected
with the same volume of physiological saline. All peptides solutions were sterilised through
the 0.22 μm pore filter membrane (Millipore) and all injections were performed in sterile
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conditions. Haemolymph of control and injected insects were taken 1 h after injection for the
phenoloxidase activity studies. Before haemolymph collection, the beetles were anaesthetised
again with CO2, washed in distilled water and disinfected with 70% ethanol. Upon cutting off
a tarsus from a foreleg, haemolymph samples (1 µl), were collected with “end to end”
microcapillaries (Drummond). Phenoloxidase activity was measured by a slight modification
of the procedure described by Sorrentino et al. [38]. Briefly, 1 µl of fresh whole haemolymph
samples were placed on a white filter paper (Whatman No 52) soaked in 10 mM sodium
phosphate buffer (pH = 6,6) containing DL-DOPA (2 mg/ml; Sigma Aldrich). Samples were
incubated for 30 min at room temperature and next, they were air-dried and scanned by
CANON Lide110 (600 dpi, 8 bits, gray scale). Images were transformed, so that the darker
samples correspond to the highest PO activity. Images of samples were analyzed with
computer program Image J (ver. 2). The distribution of the parameters PO activity was normal,
so Student’s t-test for paired samples was used.
In vivo haemocyte bioassay
The beetles were anaesthetised with CO2, washed in distilled water and disinfected
with 70% ethanol. Alloferon or respective analogue and Cu complexes of these peptides were
injected (2 µl, in dose of 10 nmole of peptide per insect) as describes above. The control
insects were injected with the same volume of physiological saline. Before haemolymph
collection, the beetles were anaesthetised again with CO2, washed in distilled water and
disinfected with 70% ethanol. Both control and injected insects were taken 1 h after injection,
and the haemocytes were prepared according to the method of Czarniewska et.al.[19]. The
prepared haemocytes were used for the appropriate microscopic analysis (active caspase, F-
actin microfilament staining and nuclei detection). The presence of active caspases was
searched for by using a sulphorhodamine derivative of valylalanylaspartic acid fluoromethyl
ketone, a potent inhibitor of caspase activity (SR-VAD-FMK) (in accordance to the
manufacturer’s instructions of the sulphorhodamine multi-caspase activity kit, AK-115,
BIOMOL, PA). Haemocytes from the control and alloferon, its analogues and Cu(II)
complexes of these peptides-injected insects were allowed to adhere for 15 min. Next, they
were rinsed with physiological saline, incubated in reaction medium (1/3x SR-VAD-FMK)
for 1 h at room temperature in the dark, rinsed again three times with wash buffer for 5 min at
room temperature and finally fixed in 3.7% paraformaldehyde for 10 min. For visualizing
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F-actin microfilaments, haemocytes that first had been stained for caspase activity were
permeabilized in 3.7% paraformaldehyde in physiological saline containing 0.1% Triton
X-100 for 5 min at room temperature. Next, the haemocytes were washed in physiological
saline and stained with Oregon Green® 488 phalloidin (Invitrogen) for 20 min at room
temperature in the dark, in accordance to the manufacturer’s instructions. After washing again
in physiological saline, the haemocytes were stained with freshly prepared solutions of DAPI
(Sigma) in physiological saline. Incubation in the dark lasted for 5 min. Thereafter, the
haemocytes were washed once with distilled water, they were mounted using a mounting
medium, and examined with a Nikon Eclipse TE 2000-U fluorescence microscope. The
intensity of the apoptosing cells was measured by using NIS-Element AR 3.1 programme.
The changes in fluorescence intensity were used to quantify the activity of caspases. Data are
shown as mean±SD. The percentage of apoptotic was calculated as apoptotic haemocytes to
total haemocytes in each ([number of fluorescent apoptotic cells/number all cells] × 100%).
In vitro semi-isolated heart bioassay
In order to determination influence on myocardial activity of alloferon, its analogues
and their copper(II) complexes, the microdensitometric method described by Rosiński and
Gade was used [39]. All experiments were carried out on semi-isolated heart of one-month
old adults of the beetle T. molitor. After anaesthesia insects were decapitated and the legs,
elytra and wings were removed. For heart preparation only abdomen was used. With surgical
tools the sclerotized ventral part of cuticle was removed by cutting it along the abdomen about
1 mm from lateral part of it. Only the last segment of the sclerotized cuticle was left. Finally
fat body, gut and reproductive organs were removed. During the preparation and after it, the
isolated heart was washed by saline solution. Semi-isolated heart was placed in incubation
chamber and continuously perfused by saline solution for 15 min to return it to the basic heart
beating rhythm. For myocardial activity recording, the computer program LARWA (designed
in our Lab) was used. The tested compounds were applied in 10 μl volume at concentration
0.1 μM and 10 μM to application port of microperfusion system. Changes in the frequency
and amplitude contractions of heart were recorded with computer programme ANALIZA
(designed in our lab.). Changes in the heart contractions are presented. Changes were as ratio
of average frequency of control (30 s before application of tested compounds) to average
frequency after application of tested compounds (during 30 s after application). For statistical
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analysis one-way ANOVA (analysis of variance) conjugated with Tukey’s range test was
used.
3. Results and Discussion
3.1. Protonation constants of the Allo1A (H1A) and Allo9A (H9A)
Protonation constants of the alloferon 1 mutants have been determined by
potentiometric titrations, and the data are included in Table 1. The ligands show five proton-
accepting centers, as expected. The amino group is the most basic center of the peptides
studied. The logK value of the N-terminal amino-group depends on the nature of the N-
terminal amino acid. The imidazole side chain on the His residue makes the amino group of
the peptide more acidic in comparison to those of Gly-Gly-His [40]. The amino group logK
values of the peptides studied are similar to those of the peptides containing His [41-42] or
Ala [43-45] residues at the N-terminal position. The molecules of the peptides contain three
histidyl residues, namely His1, His6 and His12 for Allo9A (H9A) and His6, His9 and His12 for
Allo1A (H1A). Deprotonations of the protonated imidazolium side chains take place in the
pH range from 5 to 7, with the average values changing from (pK values) 6.14 to 6.43 (Table
1). The small differences in the pK values of the two ligands suggest that the deprotonation of
the imidazolium groups completely overlaps. As a consequence, the stepwise constants cannot
be assigned to specific histidyl residues and data can only be considered to be simple
macroscopic thermodynamic parameters. The other protonation constants concern the C-
terminal carboxyl group with the stepwise value (logK) below 3.5 (Table 1) [45]. It should be
mentioned, that the -Gly10-Val-His12-Gly-OH fragment of the peptides studied here (logK
3.16 and 3.14) is the same as for the alloferon 1 (3.19) [46], and logK values of the C-terminal
carboxyl groups are similar to each other.
3.2. Equilibria analysis of the Cu(II) complexes
The stability constants of the copper(II) complexes of the Allo1A and Allo9A have
been determined by potentiometric titrations by using three (Allo9A) and four (Allo1A)
different metal-to-ligand molar ratios (M/L 4:1 for Allo1A, and 3:1, 2:1 and 1:1 for both
peptides). For the systems studied, the precipitation of copper(II) hydroxide was never
observed at any M/L ratios or pH values; this suggests the formation of mono-, bi-, tri-, and
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tetranuclear complexes. In the case of the Allo9A the histidyl residues in positions H1, H6, H12
may form trinuclear Cu(II) complexes while the Allo1A contain three histidyl residues H6, H9,
H12 and N-terminal amino group, therefore, the formation of tetranuclear complexes is
possible. The stability constants of different species can be calculated in the Cu(II)-Allo9A or
Cu(II)-Allo1A systems (Table 1), but it requires the use of the HYPERQUAD computer
program [31]. To check and/or modify the computational models all possible spectroscopic
methods for paramagnetic Cu(II) complexes were applied. UV-vis and EPR spectroscopies
were used to get complementary information about the number of coordinated nitrogen atoms
as well as the geometry of the copper(II) complexes. CD spectra helped us to differentiate
between the different types of nitrogen atoms that participate in metal coordination.
Electrospray ionization mass spectrometry (ESI-MS) measurements aided the interpretation of
the equilibria data by confirming the stoichiometry and binding sites of the metal complex
formed. All spectroscopic data suggest that the amine N-terminal group and the imidazole
residues of the peptides behave as independent metal-binding sites. The best fitting of the
potentiometric data was obtained by invoking the species listed in Table 1.
3.3.Mononuclear Cu(II) complexes of the Allo1A and Allo9A peptides
For the Cu(II)-Allo1A system computer analysis of the potentiometric data has
revealed the presence eight mononuclear complexes: CuH3L, CuH2L, CuHL, CuL, CuH-1L,
CuH-2L, CuH-3L and CuH-4L (charges omitted for simplicity, Figure 1a, Table 1). Figure 1a
shows that the existence of the CuHL, CuL, CuH-1L, CuH-2L and CuH-4L species is
characteristic in the samples containing a 1:1 metal-to-ligand molar ratio. The spectroscopic
and magnetic parameters calculated from the EPR spectra of frozen solutions are listed in
Table 2. For the Cu(II)-Allo1A 1:1 molar ratio system species distribution of the complexes
formed as a function of pH is similar to that of the Cu(II)-AlloK 1:1 system [28]. Moreover,
the spectroscopic data for both systems are also similar to each other. The logK* value
(logK*=logβ(CuHjL) – logβ(HnL) where the index j corresponds to the number of the protons
in the coordinated ligand to metal ion and n corresponds to the number of protons coordinated
to ligand) of the CuHL complex (-10.50) of the Allo1A with the 3N {NH2,CO,2NIm}
coordination mode is similar to that of the CuH(LH) species of the Allo1K (-10.62) [28] with
similar binding sites. This similarity of the logK* for the CuHL (Allo1A) and CuH(LH)
(Allo1K) complexes may suggest the coordination of the histidine residues with the formation
of the macrochelate with similar size ring: (H6,H9), or (H6,H12), or (H9,H12). The EPR
parameters for the CuHL complex g=2.275 and AII=158 G, and d-d transition energy at 628
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nm (Figure S1), compared to that provided by the equation of Prenesti [47] (λmax=626 nm)
correspond to the 3N {NH2,CO,2NIm} binding site (Table 2). The low value of AII (158 G,
Table 2) may reflect deformation of the complex plane expected when a macrochelate ring is
formed. The low values of AII were observed for complexes containing the macrochelate
formed by the histidine residues [48]. The first amide nitrogen deprotonation is slightly
suppressed (6.69, Table 1) in comparison to that of Gly5His (5.48) [48], but is compared to
that of the Allo1K (6.93) [28]. At physiological pH 7.4 the CuH-1L complex dominates with
3N {NH2,N-,CO,NIm} or 4N {NH2,N-,2NIm} coordination modes. The energy of d-d transition
at 607 nm (Table 2, Figure S1) compared to that provided by the equation of Prenesti [47] for
the 3N {NH2,N-,CO,NIm} λmax 599 nm suggests rather the 3N binding site than 4N (556 nm
from equation of Prenesti) for the CuH-1L complex. The amine and amide nitrogen atoms
from N-terminal part of the Allo1A peptide beside the imidazole nitrogen atom of histidine
residue H6, H9 or H12 are coordinated to copper(II) ions. Therefore, the coordination isomers
are likely for the CuH-1L complex. With increasing pH above 7.5 the shift of the d-d transition
energy to higher values (λmax=559 nm, Table 2) indicates deprotonation and coordination of
subsequent amide nitrogens. At pH above 8 the CuH-3L and CuH-4L complexes are present in
solution with the same 4N {NH2,3N-} or 4N {NIm,3N-} coordination modes. The
spectroscopic parameters obtained at pH 9.5 – 10.5 are similar to each other (Table 2). It is
reported that the terminal amino group is a more effective anchor for amide coordination than
the side chain imidazole [49]. Therefore, it is likely that for the CuH-3L and CuH-4L
complexes the 4N {NH2,3N-} binding site is present (Figure S2) . For the CuH-3L complex of
Allo1A peptide logK* value (-20.51, Table 1) is similar to that of the Allo1K (-20.91) [28]
suggesting no effect of the first amino acid residue (A,K) on the stability constant value.
The ESI-MS spectra obtained for the Cu(II)-Allo1A 1:1 metal-to-ligand molar ratio
system recorded in the positive mode show dominant signals for the mononuclear complexes:
[CuHL]3+ (420.8 Da), [CuL]2+ (631.2 Da, Figure S3) and [CuH-1L]+ (1260.5 Da) supporting
the formation of the mononuclear complexes in the MS experimental conditions. The ESI-MS
method has been used in a wide variety of fields to study the formation, stoichiometry, and
speciation of metal complexes of organic ligands [50-52].
The stability of the complexes formed in the Cu(II)-Allo9A system was determined by
analysis of the potentiometric studies carried out at 1:1, 2:1 and 3:1 metal-to-ligand molar
ratios. The best model with the stoichiometries (charges have been omitted for clarity)
reported in Table 1 was chosen. From potentiometric data calculations eight mononuclear
complexes are found for the Cu(II)-Allo9A system (Table 1, Figure 1b). In solution
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containing a 1:1 metal-to-ligand molar ratio, the following species are present: CuH3L,
CuH2L, CuHL, CuL, CuH-1L, CuH-3L, CuH-4L and CuL2. Omission of the CuL2 species from
the Cu(II)-Allo9A equilibria caused a deterioration in the goodness of fit between
experimental and measured pH values in the pH range 7 – 9.5. The stability constant of the
CuL2 complex (18.12,
le 1) is compared to that obtained for the CuL2 species of the Cu(II)-His system (17.99) [53]
suggesting the same 4N 2×2N{NH2,NIm-His1} binding sites of Allo9A to copper(II) ions. At
low pH, three protonated complexes CuH3L, CuH2L and CuHL are formed in a strongly
overlapping manner. The EPR data AII=158 G, gII=2.285 (Table 3) at pH=4 and low intensity
CD spectrum reflect mostly to the binding mode in the dominating the CuH2L species, and
suggest histamine-like {NH2,NIm-H1} coordination mode. The next deprotonations result in
characteristic changes on the UV-vis and EPR spectra, indicating the increase of the ligand-
field strength around the metal ion. The subsequent deprotonations CuH2L → CuHL → CuL
with pK values 4.46 and 5.10 (Table 1) correspond very well to the deprotonation and
coordination of the imidazole nitrogen atoms to the copper(II) ions. Between pH 4.5 and 8.5
the CuL complex is the dominant species in the solution. Lack of the d-d transitions in CD
spectra for the complexes formed in the 3.5 – 8.5 pH range may support coordination of the
imidazole nitrogen of the histidine residues rather than amide nitrogens (Table 3) [54,55].
This is indirect proof for coordination of the metal ion by the side chain histidine residues,
which are rather far from the chirality centers of the molecule. In CuL species low value of
AII = 180 G, gII = 2.250 (Figure S4), and the d-d transition energy at 598 nm compared to that
provided by the equation of Prenesti [47], (λmax=577 nm) may be explained by the distortion
of the coordination geometry of copper(II) caused by the formation of a macrochelates. The
thermodynamic stability of this species for Allo9A logβ(CuL) = 12.57 (Table 1) is
comparable to those of the Allo6A (12.33) [28] and alloferon 1 (12.60) [46]. It means that it is
likely that alloferon 1 may coordinate copper(II) ions in the CuL complex by amine and
imidazole H1, H6, H12 and/or H1, H9, H12 nitrogen atoms. The coordination isomers cannot be
excluded (Figure 2). The formation of the Cu2L2 complex is also likely (Figure 2c). Although,
the EPR spectra indicate rather the formation of mononuclear complex than dimer. Moreover,
if two copper(II) ions in dimeric species are located far from each other, the monomeric
spectrum will be also seen. It should be mentioned that His-rich N-terminal metal binding
domain from Haemophilus ducreyi (H1GDH4MH6NH8DTK) forms also 4N {NH2,3NIm}
complex with lower stability constant (11.73) by about 0.8 log units compared to that of
Allo9A [56]. At physiological pH (7.4) the CuL complex with 4N {NH2,NIm-H1,2NIm}
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coordination mode dominates. The 4N coordination mode in CuL species is supported by the
presence of nine superhyperfine lines in EPR spectra for the Cu(II)-Allo9A 1:1 metal-to-
ligand molar ratio at pH 6.5 (Figure S4). The deprotonation and coordination of first amide
nitrogen occurs with pK value 7.77 in comparison to that of the HSDGI-NH2 equals to 6.35
[57]. It is the second proof for the 4N binding sites in the CuL species. It is known that
imidazole-N3 atom of N-terminal His residue (Allo6A, Allo9A and Allo12A) can form stable
6-membered chelate with the terminal amino group. This interaction significantly enhances
the thermodynamic stability of simple mono- and bis(ligand) complexes and suppresses amide
deprotonation [49]. For the Cu(II)-Allo6A, Cu(II)-Allo9A and Cu(II)-Allo12A systems the
formation of the CuL complex with 4N {NH2,NIm-H1,2NIm} coordination mode suppresses the
amide deprotonation to pH above 7.5. The Allo6A, Allo9A and Allo12A form the CuL
complex with similar 4N {NH2,NIm-H1,2NIm} binding modes but different coordination
isomers. The stabilities of the macrochelates follow the order Allo9A (H1,H6,H12)(12.57) ~
Allo6A (H1,H9,H12)(12.33) > Allo12A (H1,H6,H9) (10.90). With increasing pH above 7.5 the
d-d transition energy is shifted to higher values indicating a stronger ligand field around
copper(II) ion resulting from the coordination of amide nitrogens to the metal ion in the
equatorial plane. For the CuH-3L and
CuH-4L complexes the magnetic
parameters gII = 2.218 and AII = 192 G are characteristic of a 4N complex species with a
{NH2,3N-} or {NIm,3N-} binding mode (Figure S4). The absorption maximum at λmax = 550
nm (Table 3) suggests a weak axial interaction (most likely) of the imidazole side chain of the
histidine residue. For the CuH-3L complexes of Allo6A the d-d transition energy was
observed at 525 nm [27], while for the Allo12A similar to that of Allo9A (550 nm) at 547 nm.
If in pH above 9 the 4N {NH2,3N-} complex is formed, the similarities of the d-d transition
energy of the Cu(II)-Allo9A and Cu(II)-Allo12A systems may suggest the axial coordination
of the His6 residue to the copper(II) ions. Above pH 10 the CuH-4L complex is present in
solution. The pK value (10.12, Table 1) for the reaction CuH-3L → CuH-4L + H+ suggests: 1)
the deprotonation and coordination of the amide nitrogen atom, 2) water hydrolysis, or 3)
deprotonation of the pyrrole-type N(1)H group of the coordinated imidazole ring of the
histidine residue, which can lead to the formation of imidazolato-bridged polynuclear species
[58]. However, the parameters of UV-vis, CD and EPR spectra are not altered in solution
above pH 9.5 suggesting the same binding mode in both CuH-3L and CuH-4L complexes.
Therefore, the formation of the CuH-4L species may be assigned to the deprotonation of the
N(1)-pyrrolic nitrogen in the imidazole ring without metal coordination [59].
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The ESI-MS spectra obtained for the Cu(II)-Allo9A 1:1 metal-to-ligand molar ratio
system recorded in the positive mode show dominant molecular ions: [CuHL]3+ (420.8 Da),
[CuL]2+ (630.8 Da) and [CuH-1L]+ (1260.6 Da) supporting the formation of the mononuclear
complexes in the MS experimental conditions.
It should be mentioned that in equimolar solutions the dinuclear complexes are formed
in the range 5 – 20% (Figures 1a and 1b). For the Cu(II)-Allo1A 1:1 system the dimeric
Cu2H-1L species is present in 5% at pH about 5.5, while for the Cu(II)-Allo9A (like to Allo6A
and Allo12A) the dimeric species are present at pH above 7.5.
3.4. Dinuclear complexes of Allo1A and Allo9A
For the Allo1A, monodentate binding of independent amine and histidines through the
NIm-donor atoms should have the Cu2H2L stoichiometry, but stability constants can be
calculated only for Cu2H-1L and the less protonated counterparts. It can be explained by the
preference of the NIm atoms for macrochelation. The major species among the dinuclear
complexes of the Allo1A are Cu2H-5L and Cu2H-7L (Figure 3a). The Cu2H-5L species
predominates around pH 8.5 – 9.0, and the stoichiometry and spectral parameters suggest the
4N {NH2,3N-} 4N {NIm,2N-,NIm} coordination mode. In UV-vis, the d-d transition energy at
582 nm, in CD spectra of the NIm → Cu(II) at 342 nm [60] and N-(amide) → Cu(II) at 307 nm
charge transfer transitions (Table 2), and the broad EPR spectra with parameters AII=200 G,
gII=2.195 are consistent with suggested binding sites for the Cu2H-5L species. The equatorial
arrangement of the donors in the species Cu2H-5L includes likely 4N {NH2,3N-} (517 nm
obtained from equation of Prenesti [47]) and 4N {NIm,2N-,NIm} (542 nm) binding sites. A red
shift of the d-d energy band (582 nm) may result from the axial interaction of the imidazole
nitrogen atom of His residue [61]. Further, two deprotonation reactions take place around pH
9 (pK 9.67 and 9.88) and these are accompanied with the blue shift of the d-d of the energy
band to 556 nm (Table 2). The stoichiometry and all spectral parameters are characteristic of
those reported for the 4N complex of the Cu(II)-Allo1A system. The parameters of UV-vis,
CD and EPR spectra are not altered in basic solution (pH 10 – 11 range) suggesting the same
binding sites in the Cu2H-6L and Cu2H-7L complexes (Figure 3a). Therefore, the formation of
the Cu2H-7L species can be assigned to the further deprotonation of the N(1)-pyrrolic
nitrogen in the imidazole ring without metal coordination [59]. The difference in the
distribution diagrams of the complexes for the Cu(II)-Allo1A and Cu(II)-Allo1K 2:1 metal-to-
ligand molar ratio [28] is in pH range above 7.5. For the Allo1K the Cu2H-3L (Cu2H-4(LH)
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species) complex is dominant while for the Cu(II)-Allo1A the Cu2H-5L. It may result from
the preference of these peptides to different coordination isomers.
Two dinuclear complexes are the major species in the Cu(II)-Allo9A 2:1 metal-to-ligand
molar ratio system (Figure 3b). The Cu2H-5L complex dominates in the pH range 7.5 – 10.0,
whereas at higher pH values Cu2H-7L species is formed. It should be mentioned that for 2:1
molar ratio system of the Cu(II)-Allo6A the Cu2H-3L and Cu2H-7L species dominate while for
the Cu(II)-Allo12A the Cu2H-2L, Cu2H-4L and Cu2H-6L complexes [27]. The deprotonation
constant values and coordination of the amide nitrogen atoms to copper(II) ions for the
Allo9A are lower in comparison to those of Allo6A and Allo12A (Table 1) [27]. These
differences result from different amino acid residues in the peptide chain near the His residues
involved in the binding of the copper(II) ions (coordination isomers). The stoichiometry of the
Cu2H-5L complex and the spectroscopic parameters of UV-vis, CD and EPR spectra may
suggest the 4N {NH2,3N-} 4N {NIm,2N-,NIm} binding modes around two metal ions (Table 3,
Figure S5). With increasing pH the Cu2H-5L complex lost protons and the Cu2H-6L and the
Cu2H-7L species are formed (Figure 3b). The logK* values of the Cu(II)-Allo9A (-40.86,
Table 1), Cu(II)-Allo6A (-43.86) and Cu(II)-Allo12A (-44.66) systems suggest higher
stability of the Cu2H-6L complex of the Allo9A peptide in comparison to those of the Allo6A
and Allo12A [27]. For the Cu2H-6L and Cu2H-7L complexes, the spectroscopic parameters
suggest the same 4N {NH2,3N-} 4N {NIm,3N-} coordination modes (Table 3). In these
complexes only one imidazole nitrogen atom of histidine residue is coordinated from three
present in the molecule, therefore, the coordination isomers cannot be excluded [62]. The
presence of the coordination isomers and the difference in the ratio of these isomers are well
seen in the difference of the speciation of these Cu(II)-Allo9A, Cu(II)-Allo6A and Cu(II)-
Allo12A 2:1 metal-to-ligand molar ratio systems [27].
The ESI-MS spectra obtained for the Cu(II)-Allo1A 2:1 metal-to-ligand molar ratio
system recorded in the positive mode show dominant molecular ions: [Cu2H-1L]3+ (441.8 Da);
[Cu2H-2L]2+ (661.2 Da, Figure 4); [Cu2H-3L]+ (1321.4 Da), while in MS spectra for the Cu(II)-
Allo9A system only one molecular ion [Cu2H-2L]+2 (661.2 Da) was observed.
3.5. Trinuclear complexes of Allo1A and Allo9A
As it is seen for the Cu(II)-Allo1A 3:1 metal-to-ligand molar ratio system (Figure 5a)
the Cu3H-7L complex is dominant in pH range 6.5 – 9.5. The stoichiometry may suggest likely
coordination modes the 4N {NH2,3N-} (predicted value from equation of Prenesti 517 nm,
[47]) 4N {NIm,2N-,NIm} (542 nm) 3N {NIm,2N-} (584 nm) although the λmax value is equal
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613 nm. This red shifted of the d-d transition in comparison to those predicted indicates the
distortion of square planar coordination geometry around the metal ions [61]. Distorted D4h
geometry around the copper(II) ions is also seen in CD spectra at pH 7 – 9. For the Cu(II)-
Allo1A 3:1 metal-to-ligand molar ratio system three d-d transition bands are observed (Table
2, Figure S6). The logK* values of the Cu(II)-Allo1A (-46.34) and Cu(II)-Allo1K (-49.72)
[28] systems suggest higher stability of the Cu3H-7L complex of the Allo1A peptide in
comparison to that of the Cu3H-7(LH) complex (Lys residue is protonated) of the Allo1K. For
the Allo1K peptide, the Cu3H-5L complex dominates in the 6.5 – 9.5 pH range [28]. The
pK(amide) values in the copper(II) complexes for both peptides are different (Table 1) [28].
These values are lower for the Allo1A complexes. Further deprotonation reactions take place
(Figure 5a) and these are accompanied with the spectral changes (Table 2). At pH above 10.5
the Cu3H-9L and Cu3H-10L complexes are present in solution with 4N {NH2,3N-} 4N
{NIm,3N-} 4N {NIm,3N-} coordination modes.
For the tri- (Allo1A and Allo9A peptides) and tetranuclear complexes of the Allo1A
peptide, significant EPR line broadening is observed. Significant EPR line broadening
suggests some spin-spin interactions between the coordinated Cu(II) ions, indicating that
paramagnetic copper(II) ions are close to each other. The broadening of the EPR line may be
due to dipolar as well as exchange interaction between copper(II) ions [63]. The exchange
interaction produces the collapse of the four hyperfine lines, according to the Anderson
exchange model [64]. Because the observed line width does not change by increasing or
decreasing the concentration (data not shown), this indicates an association of the complexes
into larger aggregates, leading to the concentration-independent line broadening by dipolar
interactions [65].
For the 3:1 metal-to-ligand molar ratio of the Cu(II)-Allo9A system, the Cu3H-6L and
Cu3H-10L complexes dominate (Figure 5b) in the 6.5 – 8 and above 9.5 pH range, respectively.
The stoichiometry of the Cu3H-6L complex and the spectroscopic parameters of UV-vis, CD
and EPR spectra (Table 3) may suggest 3N {NH2,2N-,CO} 3N {NIm,2N-} 3N {NIm,2N-}
binding modes around three metal ions. The deprotonation and coordination of the amide
nitrogen atoms to copper(II) ions for the Allo9A peptide occur at lower pH (Table 1) in
comparison to those of the Allo6A and Allo12A peptides [27]. Therefore, it is clear that the
Cu3H-6L complex of the Allo9A dominates at pH 7.2 – 7.3, while this complex of the Allo6A
and Allo12A dominate at pH 8.0 – 8.5 [27]. The logK* values of the Cu(II)-Allo9A (-40.41),
Cu(II)-Allo12A (-42.03) and Cu(II)-Allo6A (-43.25) systems suggest higher stability of the
Cu3H-6L complex of the Allo9A (H1,H6,H12 are coordinated) in comparison to those of
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Allo12A (H1,H6,H9) and Allo6A (H1,H9,H12). At pH above 9.5 the Cu3H-9L and Cu3H-10L
complexes dominate (Figure 5b). The stoichiometry of the Cu3H-9L complex and the
transition energy of the d-d at 536 nm suggest the 4N {NH2,3N-} 4N {NIm,3N-} 4N {NIm,3N-}
coordination modes (Table 3, Figure S7a). The logK* values of the Cu(II)-Allo9A (-66.23)
and Cu(II)-Allo12A (-70.01) systems suggest higher stability of the Cu3H-9L complex of the
Allo9A in comparison to that of the Allo12A. The Allo9A peptide forms the Cu3H-9L
complex with the formation, likely, the(6,5,5)(7,5,5)(6,5,5) chelate rings, while the Allo12A
forms the this species with the chelate rings (6,5,5)(7,5,5)(7,5,5). Coordination of copper(II)
ions to the imidazole nitrogen atoms promotes the deprotonation and metal binding of amide
groups towards the C-termini in the form of (7,5,5)-membered chelates [66,67]. The
thermodynamic stability of the peptide complexes having this coordination mode is smaller
than those formed in the form of (6,5,5)-membered chelates [67-71].
The ESI-MS spectra obtained for the Cu(II)-Allo1A and Cu(II)-Allo9A 3:1 metal-to-
ligand molar ratio system recorded in the positive mode show dominant molecular ion
[Cu3H-4L]2+ (692.7 Da). For the Cu(II)-Allo1A 4:1 metal-to-ligand molar ratio tetranuclear
complexes in MS experiment were not observed.
3.6. Tetranuclear complexes of the Allo1A peptide
Table 1 and the corresponding speciation curve (Figure 6) reveal that tetranuclear
species are also formed in the copper(II)-Allo1A system, similar to that of the Cu(II)-Allo1K
system [28]. As it is seen on Figure 6, tetranuclear species are formed at pH above 6, similar
to that of Allo1K system [28]. However, for the Cu(II)-Allo1A system the Cu4H-9L species
exists in solution in wide 6 – 10 pH range, while for the Cu(II)-Allo1K the Cu4H-8(LH) (Lys
residue is protonated). For the Cu4H-9L species of the Cu(II)-Allo1A system the d-d transition
energy at 611 nm and CD spectra (Table 2, Figure S8) suggest the 4N {NH2,3N-}, 3×3N
{NIm,2N-} binding modes around four metal ions. The logK* values of the Cu(II)-Allo1A
(-61.98) and Cu(II)-Allo1K (-64.89) systems suggest higher stability of the Cu4H-9L complex
of the Allo1A peptide in comparison to that of the Allo1K [28]. With increasing pH (8.5 – 11)
three deprotonation reactions take place and these are accompanied with the spectral changes
characteristic for the 4N {NH2,3N-}, 2×4N {NIm,3N-}, 3N {NIm,2N-,OH-} binding sites for the
Cu4H-12L species (Figures S7b, S8 and S9).
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4. Biological Activity
4.1. Phenoloxidase activity assay
The innate immune system in insects is composed of a large variety of specific and
nonspecific responses that are activated in response to the presence of foreign agents. One
important element in such responses is the enzyme phenoloxidase (PO) [72]. The research has
documented PO as an important tool used against several pathogens (bacterial gram + and -,
fungal and viral) [73]. Phenoloxidase produces indole groups, which are subsequently
polymerized to melanin. Melanin fills many roles in invertebrates besides pigmentation.
Several studies have shown that mechanical injury or the presence of macropathogens results
in melanin deposition around the damaged tissue or foreign object. This process is commonly
known as melanization [73,74]. Phenoloxidase is a member of the tyrosinase group whose
main function is to oxidize phenols. All tyrosinases have in common a binuclear type 3
copper center within their active site. This copper center is surrounded by three histidine
residues [75]. Phenoloxidases are expressed as inactive zymogens (propos) in all insects and
are converted to active PO when required [72]. Soderhall et al. have reported that the proPO
system of invertebrate hemocyte lysates are activated specifically by either β-1,3-glucan from
fungal cell wall or lipopolysaccharides and peptidoglycans from bacterial cells [76]. It has
found that such factors as temperature and calcium concentration also effect proPO system
activation [77,78]. Now we would like to known if the peptides studied (alloferon and its
analogues) and their copper(II) complexes may activate phenoloxidase in the Tenebrio
molitor.
Figure 7a shows the effect of tested compounds on humoral response in T.molitor.
Injections of alloferon did not have a statistically significant effect on the phenoloxidase
activity when compared to control beetles. However, phenoloxidase activity was significantly
lower in beetles receiving analogue Allo1A or Allo9A. On the other hand, the analogue
Allo1K injection slightly increased level of phenyloxidase activity in T. molitor. Generally,
copper(II) complexes of all studied analogues significantly increased phenoloxidase activity
in T. molitor, but the highest increase in enzyme activity was observed after injection of
Cu(II)–Allo9A. As it is seen on Figure 1b, at pH 7.4 the CuL complex dominates in solution
with the 4N {NH2,NIm-H1, NIm-H6, NIm-H12} coordination mode. In this complex three His
residues of the Allo9A are coordinated to copper(II) ions. It is the first report that analogues
of alloferon have immunomodulation activity in insects.
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4.2. In vivo haemocyte bioassay
The representative images in Figures 7b and 8 show the effect of the studied peptides
and their copper(II) complexes on the T. molitor haemocytes. The in vivo bioassay on the T.
molitor haemocytes showed that Allo9A and its Cu(II) complex were potent because one hour
after injection of the 10 nM solution of Allo9A or its copper(II) complex haemocytes’
viability and morphology was drastically changed. The most active was Cu(II)-Allo9A
(Figure 7b), it retained more than 100% of activity of alloferon, whereas analogue Allo9A
showed 12% of pro-apoptotic activity of alloferon (Table 4). Control haemocytes formed
extensive filopodia, in which the F-actin microfilaments were detected supcortically after the
cells had been allowed to adhere to the cover slips. In all studied individuals, injection of
native peptide, Allo9A or Cu(II)-Allo9A caused failure of F-actin organization in haemocytes.
They lost their regular F-actin staining pattern as a result of depolymerization of F-actin
microfilaments. Moreover, 1-hour exposure of the Tenebrio molitor haemocytes to the
studied peptides induced in these cells malformations that are typical for an apoptotic
morphology, such as cell shrinkage, rounding up and fragmentation of nuclei (Figure 8).
Analogue Allo1A did not change haemocytes’ viability and morphology compared with
control haemocytes. However, its copper(II) complex strongly stimulated the filopodia
formation as shown in Figures 8D and 8E. Analogue Allo1K and its Cu(II) complex show
weak pro-apoptotic effect (21% and 18% of alloferon activity, respectively) on haemocytes of
T. molitor. However, these compounds caused the F-actin cytoskeleton changes, but the effect
of Cu(II)-Allo1K was stronger than Allo1K. In the T. molitor haemocytes, F-actin was found
aggregating into distinctive patches after injection of Allo1K or its copper(II) complex
(Figures 8J and 8K). The F-actin cytoskeleton changes may be responsible for the observed
inhibition of adhesion of haemocytes and for the inhibition of filopodia formation. Moreover,
the percentage of apoptotic cells increased after injection of Cu(II)-Allo9A analogue (55% of
haemocytes displayed caspase activity) when compared to Allo9A peptide (20% of activated
haemocytes). It is interesting that similar data we observed for Cu(II)-Allo12A [27].
Additionally, as shown in Figure 7b injection of Cu(II)-Allo1A and Cu(II)-Allo1K resulted in
a decreased the rate of apoptotic haemocytes. These peptides displayed the lowest
hemocytotoxic activity among tested copper(II) complex of alloferon analogues. These results
indicated that the N-terminal and H6 histidine residues are more important for the pro-
apoptotic properties in insects than histidine at positions 9 and 12 of the peptide chain of
alloferon 1. It should be mentioned that three histidine residues of the Allo9A {NH2,NIm-H1,
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NIm-H6, NIm-H12} or Allo12A {NH2,NIm-H1, NIm-H6, NIm-H9} are coordinated to the copper(II)
ions.
4.3. In vitro semi-isolated heart bioassay
The knowledge about copper metabolism in the cardiovascular system is very limited,
but many clinical and experimental studies have produced data regarding the change in copper
concentrations in the blood under cardiovascular disease conditions, including coronary artery
disease [79], myocardial infarction [80], diabetic cardiomyopathy [81], cardiac hypertrophy
[82], atherosclerosis [83], and other cardiovascular diseases [84]. However, it is difficult to
extrapolate the relationship between the increase or decrease in serum copper concentrations
and the progression of cardiovascular diseases [85]. The effects of the peptides on the heart
contractile activity of Tenebrio molitor were established [86,87]. The biological investigation
of alloferon and its selected analogues on the myotropic activity in insects were performed [9].
Alloferon induced the cardiostimulatory effect on the heart of Zophobas atratus, however, the
heart of Tenebrio molitor was insensitive to alloferon. Now, we tested the analogues of
alloferon 1 and their copper(II) complexes on the heart of Tenebrio molitor.
The bioassay in vitro on the heart of T. molitor showed that application of alloferon
analogues or their copper(II) complexes didn’t change heartbeat frequency and power of
contractions. All tested were inactive on the myocardium of this beetle at the 10-7 and 10-5 M
concentration (Figure 9). However, copper(II) complexes of Allo1A, Allo9A and Allo1K are
slightly more active than the free ligands itself at the 10-5 M concentration.
5. Conclusion
Here we report the characterization of the copper(II) complexes with the alloferon 1
mutants Allo1A and Allo9A by means of potentiometry, CD, EPR and UV-vis spectroscopic
techniques, and ESI-MS spectrometry. The Allo1A peptide forms the mononuclear copper(II)
complexes similar to those of the Allo1K [28]. The stability constants of mononuclear
copper(II) complexes for both ligands are similar to each other. The CuHL and CuL
complexes (CuH2L and CuHL of Allo1K) dominate in wide 4 – 8 pH range with 3N
{NH2,CO,2NIm} binding sites. At physiological pH 7.4 the CuH-1L complex is present in
solution with 3N {NH2,N-,CO,NIm} coordination mode. The stability of polynuclear
complexes of the Allo1A peptide is higher in comparison to those of the Allo1K. Different
distribution diagrams obtained for the 2:1, 3:1 and 4:1 metal-to-ligand molar ratios for both
peptides (Allo1A and Allo1K) result likely from the presence of the coordination isomers and
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the difference in their ratio. It should be mentioned that the mononuclear complexes of the
Allo1A and Allo1K are similar to those of alloferon 2 [46]. The presence of the His residue in
first position of the Allo9A, Allo6A, Allo12A and alloferon 1 changes their coordination
modes to copper(II) ions in comparison to those containing Lys or Ala amino acid residues.
The Allo9A, Allo6A and Allo12A peptides form the CuL complex in wide 4.5 – 8.5 pH range
with the 4N {NH2,NIm-H1,2NIm} binding mode. The stability of this complex follows the
order Allo9A (H1,H6,H12; 12.57) ~ Allo6A (H1,H9,H12; 12.33) > Allo12A (H1,H6,H9; 10.90).
It should be mentioned that the stabilization of the copper(II) complexes may result not only
from the chelate size but also from structural organization of the peptide in the complex [88].
For the metal-to-ligand 2:1and 3:1 molar ratios the di- and trinuclear complexes dominate at
pH above 5.5 and 6.5, respectively. For these peptides different dominant complexes in
solution are observed because of the presence of different coordination isomers.
The peptides tested did not cause changes in the frequency (chronotropic response) or
amplitude (inotropic response) of heart contractions although they cause the hemocytotoxic
effect. Lack of heart response to alloferon and its analogues suggests that it does not affect the
mechanical and bioelectrical function of myocardium and does no cytotoxic influence to cells
in this tissue.
The Allo1A and Allo1K and their copper(II) complexes displayed the lowest
haemocytotoxic activity among tested copper(II) complex of alloferon analogues. The most
active was the Cu(II)-Allo9A complex formed at pH 7.4 with 4N {NH2,NIm-H1,NIm-H6,NIm-
H12} binding site. Similar data were observed for the Cu(II)-Allo12A system at pH 7.4 with
similar binding site but different coordination isomer. It may suggest that the copper(II)
complex with the coordination of histidine residues H1, H6 and H9 or H12 of the peptide chain
has higher proapoptotic properties in insects than those no containing His residue at first
position. The coordination to copper(II) ions of the N-terminal amine group and imidazole
nitrogen atoms of three His residues creates the macrochelates, the structure of which may be
responsible for the biological activity.
Results from all three bioassays indicate that the action of alloferon analogues is
tissue-specific and relies on modulation of immune system function in T. molitor.
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6. Abbreviations
HBTU – 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
HOBt – 1-hydroxybenzotriazole
Fmoc – 9-fluorenylmethoxycarbonyl
DMF – N,N-dimethylformamide
TFA – trifluoroacetic acid
EDT – 1,2-ethanedithiol
ESI-MS – Electrospray Ionization Mass Spectrometry
FT-ICR – Fourier Transform Ion Cyclotron Resonance
CD – Circular Dichroism
MS – Mass Spectrometry
MOPS – 3-(N-morpholino)propanesulfonic acid
NK- Natural Killer
UVB – Ultraviolet B (280-315 nm)
UV-vis – UV-visible
Mcalc – molecular mass calculated
DL- DOPA – DL-3,4-dihydroxyphenylalanine
PO – phenolooxidase
SR-VAD-FMK – sulforhodamine-Val-Ala-Asp-fluoromethylketone
DAPI - 4',6-diamidino-2-phenyloindole
MFI - Mean Fluorescence Intensity
SD – Standard Deviation
Acknowledgments: Financial support from the Polish Ministry of Science and Higher
Education Grant NN 204085638 is gratefully acknowledged. The authors thank to Arkadiusz
Urbański and Szymon Chowański for their technical assistance in the biological experiments.
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List of Figures
Figure 1. Species distribution diagram for Cu(II) complexes with a) Allo1A, b) Allo9A
Charges are omitted for clarity. [L] = 0.001 M, M/L molar ratio = 1:1.
Figure 2. Schematic of the CuL complex species: a) Allo9A; b) Allo6A; c) Cu2L2 complex of
the Allo9A.
Figure 3. Species distribution diagram for Cu(II) complexes with a) Allo1A, b) Allo9A.
Charges are omitted for clarity. [L] = 0.001 M, M/L molar ratio = 2:1.
Figure 4. ESI mass spectrum for the Cu(II)-Allo1A and Cu(II)-Allo9A systems at 2:1 molar
ratio in water solution at pH about 7. Experimental and simulated spectra for the Cu2H-2L
molecular ion with m/z 661.2 Da.
Figure 5. Species distribution diagram for Cu(II) complexes with a) Allo1A, b) Allo9A
Charges are omitted for clarity. [L] = 0.001 M, M/L molar ratio = 3:1.
Figure 6. Species distribution diagram for Cu(II) complexes with Allo1A. Charges are omitted
for clarity. [L] = 0.001 M, M/L molar ratio = 4:1.
Figure 7. (a) Humoral response expressed as phenoloxidase (PO) activity in T.molitor. Values
are given as mean ±SE. *Significant differences in PO activity between treated groups were
observed (F=7.095, p<0.0001); (b) The rate of apoptotic haemocytes to total haemocytes
after injection of T. molitor. * Data not include cells which have been fragmented into
apoptotic bodies. **Data from literature [27].
Figure 8. Representative fluorescence microscopic images showing in vivo induced apoptotic
effects in T. molitor haemocytes. A, PS; B, MOPS; C, MOPS + Cu(II); D, Allo1; E, Cu(II)-
Allo1; F, Allo1A; G, Cu(II)-Allo1A; H, Allo9A; I, Cu(II)-Allo9A; J, Allo1K; K; Cu(II)-
Allo1K). All haemocytes were stained with SR-VAD-FMK reagent for caspase activity
detection (red color), DAPI for cell nuclei detection (blue color) and Oregon Green–phalloidin
for staining of the F-actin cytoskeleton (green color). Arrows show aggregation of F-actin (G,
H, J) and apoptotic bodies (I). Scale bars indicate 10 µm in all panels.
29