Coordination abilities of neurokinin A and its derivative and products of metal-catalyzed oxidation

Article abstract of DOI:10.1016/j.jinorgbio.2010.03.016

The classical tachykinins, substance P, neurokinin A and neurokinin B are predominantly found in the nervous system where they act as neurotransmitters and neuromodulators. Significantly reduced levels of these peptides were observed in neurodegenerative diseases and it may be suggested that this reduction may also result from the copper(II)-catalyzed oxidation. The studies of the interaction of copper(II) with neurokinin A and the copper(II)-catalyzed oxidation were performed. Copper(II) complexes of the neurokinin A (His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH₂) and acetyl-neurokinin A (Ac-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH₂) were studied by potentiometric, UV-Vis (UV-visible), CD (circular dichroism) and EPR spectroscopic methods to determine the stoichiometry, stability constants and coordination modes in the complexes formed. The histidine residue in first position of the peptide chain of neurokinin A coordinates strongly to Cu(II) ion with histamine-like {NH₂, N_Im} coordination mode. With increasing of pH, the formation of a dimeric complex Cu₂H₂L₂ was found but this dimeric species does not prevent the deprotonation and coordination of the amide nitrogens. In the Ac-neurokinin A case copper(II) coordination starts from the imidazole nitrogen of the His; afterwards three deprotonated amide nitrogens are progressively involved in copper coordination. To elucidate the products of the copper(II)-catalyzed oxidation of the neurokinin A and Ac-neurokinin A, liquid chromatography-mass spectrometry (LC-MS) method and Cu(II)/hydrogen peroxide as a model oxidizing system were employed.Oxidation target for both studied peptides is the histidine residue coordinated to the metal ions. Both peptides contain Met and His residues and are very susceptible on the copper(II)-catalyzed oxidation.

Full text of DOI:10.1016/j.jinorgbio.2010.03.016

Journal of Inorganic Biochemistry 104 (2010) 831–842
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Coordination abilities of neurokinin A and its derivative and products of
metal-catalyzed oxidation
b
a
b
Teresa Kowalik-Jankowska a, , Elżbieta Jankowska , Zbigniew Szewczuk , Franciszek Kasprzykowski
⁎
a
Faculty of Chemistry, University of Wrocław, Joliot-Curie 14, 50-383 Wrocław, Poland
b
Faculty of Chemistry, University of Gdańsk, Sobieskiego 18, 80-952 Gdańsk, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
The classical tachykinins, substance P, neurokinin A and neurokinin B are predominantly found in the
nervous system where they act as neurotransmitters and neuromodulators. Significantly reduced levels of
these peptides were observed in neurodegenerative diseases and it may be suggested that this reduction
may also result from the copper(II)-catalyzed oxidation. The studies of the interaction of copper(II) with
Received 21 January 2010
Received in revised form 23 March 2010
Accepted 26 March 2010
Available online 11 April 2010
neurokinin
A and the copper(II)-catalyzed oxidation were performed. Copper(II) complexes of the
neurokinin A (His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2) and acetyl-neurokinin A (Ac-His-Lys-Thr-
Asp-Ser-Phe-Val-Gly-Leu-Met-NH2) were studied by potentiometric, UV–Vis (UV–visible), CD (circular
dichroism) and EPR spectroscopic methods to determine the stoichiometry, stability constants and
coordination modes in the complexes formed. The histidine residue in first position of the peptide chain
of neurokinin A coordinates strongly to Cu(II) ion with histamine-like {NH2, NIm} coordination mode. With
increasing of pH, the formation of a dimeric complex Cu2H2L2 was found but this dimeric species does not
prevent the deprotonation and coordination of the amide nitrogens. In the Ac-neurokinin A case copper(II)
coordination starts from the imidazole nitrogen of the His; afterwards three deprotonated amide nitrogens
are progressively involved in copper coordination. To elucidate the products of the copper(II)-catalyzed
oxidation of the neurokinin A and Ac-neurokinin A, liquid chromatography-mass spectrometry (LC–MS)
method and Cu(II)/hydrogen peroxide as a model oxidizing system were employed.
Keywords:
Neurokinin A
Copper(II) complexes
Stability constants
Copper(II)-catalyzed oxidation
Products of oxidation
Oxidation target for both studied peptides is the histidine residue coordinated to the metal ions. Both
peptides contain Met and His residues and are very susceptible on the copper(II)-catalyzed oxidation.
© 2010 Elsevier Inc. All rights reserved.
1. Introduction
NK2 and NK3, which bind preferentially to SP, NKA, and NKB,
respectively, although each of these peptides is able to bind each of
Tachykinins (TKs) are a family of closely related peptides whose best
known members are substance P (SP), neurokinin A (NKA) and
neurokinin B (NKB). For many years, the tachykinins were considered
almost exclusively as peptides of neuronal origin. SP and NKA are found
in the central nervous supplying a number of peripheral tissues [1,2]. SP
and NKA are released from nerve ending at both the spinal cord and the
peripheral level and play a role as excitatory neurotransmitters [2,3].
In mammals, tachykinins act as neurotransmitters, paracrine or
endocrine factors and neuroimmunomodulators [4] and have roles in
the nervous system, gastrointestinal tract [5] and cardiovascular
system [6]. Important actions include vasodilatation, plasma extrav-
asation, smooth muscle, contraction secretion, neuronal excitation
and processing of sensory information; they also have immune and
pro-inflammatory actions [7,8].
the receptors albeit with lower affinity [9].
The tachykinin peptides are characterized by a common C-terminal
sequence, Phe-X-Gly-Leu-Met-NH2, where X represents either
an aromatic (Phe, Tyr) or a branched aliphatic (Val, Ile) amino acid.
The C-terminal region or the message domain is considered to be
responsible for activating the receptor. The divergent N-terminal
region or the address domain varies in amino acid sequence and length
and is postulated to play role in determining the receptor subtype
specificity [10].
Neurokinin A is a decapeptide found in mammalian neuronal
tissue, with the sequence His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-
NH2. It was first isolated from the porcine spinal cord [11]. The
structure of NKA has been strongly conserved during evolution. NKA
from reptiles and chicken are identical to mammalian NKA, and the
frog, teleost, and alasmobranch NKA isolated differ by only two amino
acids [12].
Tachykinins are believed to exert most of their actions via the 3
tachykinin receptors that have been cloned and characterized: NK1,
There are a number of studies that have specifically addressed the
question of tachykinin levels in Parkinson's disease (PD) [13].
Mauborgne et al. first demonstrated that there were reduced levels
(approximately 30%) of substance P in the substantia nigra and pallidum
⁎
Corresponding author. Tel.: +48 71 375 7231; fax: +48 71 328 2348.
E-mail address: terkow@wchuwr.chem.uni.wroc.pl (T. Kowalik-Jankowska).
0162-0134/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2010.03.016

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T. Kowalik-Jankowska et al. / Journal of Inorganic Biochemistry 104 (2010) 831–842
in patients with PD [13]. Substance P, neurokinin A, neuropeptide K
(NPK), and neurokinin B were measured in both control (neurologically
normal) and Huntington's disease patients [14]. The studies suggest that
the neuropeptides might be important in the etiology of neurodegener-
ative disease or might be useful diagnostic markers. The putative role for a
variety of neuropeptides in degenerative neurological disorders is the
subject of extensive and excellent prior reviews [15–19]. Although there
is no direct evidence for a causative role of a particular neuropeptide in a
particular neurodegenerative disease, certain neuropeptide levels are
relatively consistently reported to be altered in certain disorders.
Oxidative stress is considered a major contributor to the patho-
genesis of a number of pathological processes leading to atheroscle-
rosis, inflammatory conditions, multiple system atrophy and several
neurodegenerative diseases. There is increasing evidence that oxida-
tive stress results from either excessive reactive oxygen species (ROS)
production or compromised anti-oxidant defences [20–23]. In general,
the ROS are formed ubiquitously in biological systems by both
enzymatic and metal-catalyzed oxidation (MCO) reactions [24,25].
The MCO reaction involves reduction of Fe(III) or Cu(II) by a suitable
electron donor such as NADH, NADPH, ascorbate, or mercaptane. Fe(II)
and Cu(I) ions bound to specific metal-binding sites on proteins react
with H2O2 to generate •OH [26,27]. A feature of metal-catalyzed
oxidations is the site-specific nature of the reaction, i.e. specific amino
acid residues located at the metal-binding sites are generally altered
[28]. The amino acid residues that are most susceptible to metal-
catalyzed oxidations are His, Arg, Lys, Pro, Met and Cys [28,29]. A factor
influencing the susceptibility of these amino acid residues to metal-
catalyzed oxidation is their ability to form complexes with metals such
as Cu(II) or Fe(III). It is within these complexes that reactive oxygen
species are generated, and oxidation of specific amino acid residues
occurs in what is referred to as a “caged” process [30].
The present paper reports the results of combined spectroscopic
and potentiometric studies on the copper(II) complexes of the
neurokinin A and its N-acetyl derivative. The peptides studied here
are: neurokinin A (NKA), HKTDSFVGLM-NH2 and Ac-neurokinin A
(Ac-NKA), Ac-HKTDSFVGLM-NH2. To determine the involvement of
the NH2-amino group in the coordination of metal ions, the N-
terminal group was blocked by acetylation. Neurokinin A and Ac-
neurokinin A coordination of copper(II) lead to the generation of ROS
involving the reduction of the oxidation state of the coordinated Cu
(II) to Cu(I) and the oxidation of the peptides in the presence of
hydrogen peroxide according to the site-specific mechanism. In this
work, we examine the metal-catalyzed oxidation of the neurokinin A
and its derivative, by the Cu(II)/H2O2 system. We would like to
demonstrate the relationship between the binding sites of copper(II)
ions and the oxidation products of the studied ligands.
tion time-of-flight mass spectrometry (MALDI-TOF MS) and analytical
RP-HPLC using a C8 Kromasil column (4.6×250 mm, 5 μm) or C18
XTerra column (4.6×150 mm, 5 μm). As a mobile phase 30 min linear
gradient of 5–100% B, where A: 0.1% aqueous trifluoroacetic acid
(TFA), B: 80% acetonitrile (ACN)/H2O+0.1%TFA, was used.
Analytical data were as follows: NKA: Rt =15.4 min (Kromasil),
Mw obtained 1133.4, Mw calculated 1133.4, AcNKA: 15.6 min
(Kromasil), Mw obtained 1175.6, Mw calculated 1175.4.
2.2. Potentiometric measurements
Stability constants for proton and Cu(II) complexes were calcu-
lated from pH-metric titrations carried out in an argon atmosphere at
298 K using a total volume of 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 1.5×10−3 M; metal to ligand molar ratio 1:1.1 for
both peptides; ionic strength 0.10 M (KNO3); Cu(NO3)2 was used as
the source of the metal ions; pH-metric titration on a MOLSPIN pH-
meter system using a Russell CMAW 711 semi-micro combined
electrode, calibrated in concentration using HNO3 [32], number of
titrations equal 2; method of calculation SUPERQUAD [33]. The
samples were titrated in the pH region 2.5–10.5. Standard deviations
(values) quoted were computed by SUPERQUAD and refer to random
errors only. They are, however, a good indication of the importance of
the particular species involved in the equilibria.
2.3. Spectroscopic measurements
Solutions were of similar concentrations to those used in
potentiometric studies. For the water solutions containing copper
(II) ions and the studied ligands, the precipitation was observed
(in potentiometric studies the electrolyte KNO3 was used, and the
precipitation was not observed). Addition of ethylene glycol to the
samples (ethylene glycol–water 1:2, v/v) prevented the precipitation
and for neurokinin A the solutions were clear in whole pH range while
for Ac-neurokinin A the solubility was lower. The aggregation of the
peptide may be important in the solubility of the peptide. A solvent
effect on solubility and aggregational properties of polypeptides was
observed [34].
Absorption spectra (UV–visible) 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 spectro-
polarimeter in the 750–250 nm range. The values of Δε (i.e. εl −εr)
and ε were calculated at the maximum concentration of the particular
species obtained from potentiometric data. Electron paramagnetic
resonance (EPR) spectra were performed in ethylene glycol–water
(1:2, v/v) solution at 77 K on a Bruker ESP 300E spectrometer at the
X-band frequency (∼9.45 GHz) and equipped with the Bruker NMR
gaussmeter ER 035M and the Hewlett-Packard microwave frequency
counter HP 5350B. 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. Material and methods
2.1. Synthesis of the peptides
Synthesis of peptide amides: His1-Lys-Thr-Asp-Ser-Phe-Val-Gly-
Leu-Met10-NH2 (NKA), Ac-His1-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-
Met10-NH2 (AcNKA) was performed on a solid-phase using Fmoc
strategy with continuous-flow methodology (9050 Plus Millipore
Peptide Synthesizer) on a polystyrene/polyethylene glycol copolymer
resin (TentaGel R RAM Resin, substitution 0.18 mmol/g) [31]. Acetyla-
tion of the N-terminal amino group was performed on the resin using
1 M acetylimidazole in dimethylformamide (DMF). All peptides were
cleaved from the resin and deprotected by 2 h shaking in a mixture
containing trifluoroacetic acid, phenol, triisopropylsilane and water
(88:5:2:5, v/v).
2.4. ESI-MS measurement
The mass spectra were obtained on a Bruker MicrOTOF-Q
spectrometer (Bruker Daltonik, Bremen, Germany), equipped with
Apollo II electrospray ionization source with ion funnel. The mass
spectrometer was operated in the positive ion mode. Mass resolution
15,000 FWHM. The instrumental parameters were as follows: scan
range m/z 400–2300, dry gas–nitrogen, temperature 200 °C, reflector
voltage 1300 V, detector voltage 1920 V. The sample was dissolved in
water at pH 10.5 with the concentration 10−4 M (pH value was
adjusted using NaOH) and infused at a flow rate of 3 µL/min. Before
analysis the instrument was calibrated externally with the Tunemix™
The resulting crude peptides were purified by reversed-phase
high-performance liquid chromatography (RP-HPLC) using a C8 semi-
preparative Kromasil column (25×250 mm, 7 μm). The purified
peptides were analyzed by matrix-assisted laser desorption/ioniza-

T. Kowalik-Jankowska et al. / Journal of Inorganic Biochemistry 104 (2010) 831–842
833
Table 1
mixture (Bruker Daltonik, Germany) in quadratic regression mode.
The mass accuracy for the calibration was better than 5 ppm. In MS/MS
(Tandem Mass Spectrometry Analysis) mode, the quadruple was used to
select the precursor ions, which were fragmented in the hexapole
collision cell, generating product ions that were subsequently mass
analyzed by the orthogonal reflectron TOF mass analyzer. For CID MS/MS
(Collision-Induced Dissociation Tandem Mass Spectrometry Analysis)
measurements, the collision energy over the hexapole collision cell was
set to 21 eV, and argon was used as collision gas.
Protonation constants for neurokinin A and Ac-neurokinin A and comparable peptides
at 298 K and I=0.10 M (KNO3).
Peptide/logβ
HL
H2L
H3L
H4L
H5L
Neurokinin A
9.72
0.02
7.43
7.32
7.44
10.11
0.01
19.05
0.01
26.46
0.02
31.89
0.02
35.61
0.02
HSDGI-NHa2
HGGb
13.00
12.77
13.02
19.25
0.01
16.54
15.86
16.30
25.61
0.02
HAAb
Ac-neurokinin A
29.53
0.02
Ac-HGGGWGQ- 6.41
NH2
2.5. Materials used in the oxidation process
Ac-HGGGd
Ac-GGGHd
6.59
7.03
9.96
9.79
Deionized and triply distilled water was used, and the phosphate
buffer was treated with Chelex 100 resin (sodium form, Sigma-
Aldrich) to remove trace metals. Hydrogen peroxide was purchased
from Fluka (Perhydrol, 30%) and EDTA and Cu(NO3)2 were purchased
from POCH. 0.10 M stock solutions of EDTA and hydrogen peroxide in
a phosphate buffer (pH 7.4) were prepared.
log K
NH2-Lys and OH-Ser
NH2-
NIm
COO−
terminal
Neurokinin A
HSDGI-NH2
HGG
9.72
9.33
7.41
7.43
7.32
7.44
5.43
5.57
5.45
5.58
6.36
6.41
3.72
3.54
3.09
3.28
3.92
HAA
2.5.1. Oxidation of the neurokinin A and its acetyl-derivative and LCMS
analysis
Ac-neurokinin A 10.1
Ac-PHGGGWGQ-
NH2
19.14
Copper(II)-catalyzed oxidation of peptide in the presence
of hydrogen peroxide was monitored by analytical RP-HPLC on Varian
ProStar 240 station using XTerra C18 4.6×150 mm column (Waters)
in 30 min linear gradient 5–100% B, where A: 0.1% aqueous
TFA, B: 0.1% TFA in 80% ACN. A reaction mixture (0.5 cm3) containing
5×10−4 M of peptide and metal to ligand molar ratio 1:1.1 in a 0.1 M
phosphate buffer (pH 7.4) was incubated at 37 °C for 24 h in the
presence of hydrogen peroxide at metal to hydrogen peroxide molar
ratio 1:1 and 1:2. The reaction was started by the addition of hydrogen
peroxide, which was freshly prepared. After incubation, the reaction
was stopped by the addition of EDTA to a final complex to EDTA molar
ratio 1:5. The chelating agent EDTA inhibits the oxidation of the
peptide by removing Cu(II) from the peptide. Oxidized and digested
peptides were lyophilized, dissolved in 0.1% trifluoroacetic acid and
desalted on 10 μl ZipTipC18 columns (Omnix, Varian). The columns
were prepared by wetting with 50% acetinitrile and equilibrated with
0.1% trifluoroacetic acid. Each sample was loaded onto ZipTip column,
the column washed with 0.1% TFA to remove salts, and then the
peptides were eluted with 0.1% formic acid in 80% acetonitrile. The
obtained samples were then the subject of LC-ESI-MS analysis.
Acetonitrile, water and formic acid of LC/MS grade were purchased
from Sigma. Positive ion electrospray mass spectrometric analysis was
carried out using a Shimadzu ion trap time-of-flight mass spectrom-
eter (LC–MS IT TOF) at unit resolution. The source temperature was
200 °C, the electrospray voltage was −1700 V. Separation and mass
analysis of oxidized and digested peptides were carried out using a
Phenomenex Jupiter Proteo90A analytical column (2×150 mm, 4 μm)
with a linear gradient 0–30% B in 12.5 min followed by gradient 30–
100% B in 7.5 min (buffer A: 0.2% formic acid/water, buffer B: 0.2%
formic acid/ACN; flow rate 0.2 ml/min). The injection volume was
80 μl and the temperature in which the analysis proceeded was 40 °C.
Data were acquired and analyzed using LC Solution software provided
by Shimadzu.
Ac-HGGG
Ac-GGGH
6.59
7.03
3.37
2.76
a ref. [51], b ref. [50], c ref. [54], d ref. [57].
(log K=5.43) are comparable to those of the peptides containing the
N-terminal His residue (HSDGI-NH2, HGG, HAA, Table 1). For N-acetyl
derivative of neurokinin A the protonation constant of the imidazole
nitrogen of the His residue is about one order of magnitude higher in
comparison to those of the peptides with free-amine group, but these
values are comparable to those of the peptides containing N-blocked
amine group (Table 1). The protonation constants the β-carboxylate
of the Asp residue in both peptide amides differ only slightly, and they
are close to those expected for comparable peptides [35,36].
Potentiometry detects a range of Cu(II) complexes with the
formation constants reported in Table 2. The values of log K* are the
protonation corrected stability constants which are useful to compare
the ability of ligands to bind a metal ion [37], and they are given in
Table 3. The spectroscopic properties of the major complexes are
given in Table 4.
3.2. Determination of the additional deprotonation site in neurokinin A
and its derivative
Nonenzymatic intramolecular reactions can result in the deamida-
tion, isomerization, and racemization of protein and peptide aspar-
aginyl and aspartyl residues via succinimide intermediates [38]. This
five-membered succinimide ring is unstable under physiological
conditions and is hydrolyzed to give a mixture of aspartyl and
isoaspartyl peptides. This post-translation modification is suspected
to contribute to the aging of proteins and to protein folding disorders
such as Alzheimer's disease. This modification occurs spontaneously
under physiological conditions whose rate is affected by both its
amino acid sequence and three-dimensional structure [39]. It should
be mentioned that this additional deprotonation is also observed for
the fragments of neuropeptide gamma (tachykinin family group)
where the HKTDSFVGLM sequence exists [40]. The presence of the
TDS-sequence in these peptides may also suggest the formation of the
lactones and their alkaline hydrolysis [41]. The MS spectra of
the neurokinin A and its acetyl derivative do not show molecular
ions [M+2H–H2O] with changing of pH suggesting lack of the
formation of the lactone or succinimide rings in the peptides studied.
The obtained ESI-MS spectrum for the Ac-neurokinin A (Fig. 1) shows
3. Results and discussion
3.1. Protonation constants
Calculated protonation constants for the studied and comparable
ligands are given in Table 1. Neurokinin A and its N-acetyl derivative
contain five and four, respectively, dissociable protons. Deprotonation
of the lysyl group takes place at around pH 9–10, but for both peptides
additional deprotonation with pK value 9–10 was observed (Table 1).
For neurokinin A the protonation constants of the: N-terminal
amino group (log K=7.41), the imidazole nitrogen of the His residue

834
T. Kowalik-Jankowska et al. / Journal of Inorganic Biochemistry 104 (2010) 831–842
Table 2
Stability constants of copper(II) complexes of neurokinin A, Ac-neurokinin A and comparable peptides at 298 K and I=0.10 M (KNO3).
Peptide/logβ
CuH3L
CuH2L
CuHL
CuL
CuH−1
L
CuH−2
L
CuH−3
L
CuH−4
L
Cu2H2L2 or Cu2H−2L2
Neurokinin A
HSDGI-NHa2
HGGb
31.88 0.02 27.90 0.01
12.86 0.02 4.01 0.03 −5.06 0.02 −14.82 0.02 −25.53 0.03 44.86 0.03
12.47
8.69
8.46
8.57
−4.01
−6.85
−6.37
8.63
7.24
7.09
HAAb
Ac-neurokinin A
Ac-PHGGGWGQ-NHc2
Ac-HGGGd
23.83 0.02 17.84 0.02 10.82 0.02 2.27 0.03 −7.20 0.05 −16.79 0.05
3.69
4.20
4.56
−2.80
−2.40
−2.35
−8.99
−9.36
−9.03
−17.97
−18.28
−17.45
−28.92
Ac-GGGHd
a,b,c,d,
as in Table 1.
a dominant signal for the monosodiated doubly charged form
(599.285 m/z), whereas the monosodiated singly charged
species is almost absent. Peaks corresponding to diprotonated
(588.295 m/z) and oligosodiated forms are also present on the
spectrum. The most abundant peaks at 588 m/z and 599 m/z,
corresponding to the [M+2H]2+ ion and its monosodiated counter-
part [M+H+Na]2+ was chosen as a precursor for the fragmentation
by CID. The fragmentation pattern of the doubly protonated peptide
(Fig. 2a) is dominated by doubly protonated a and b fragments
(according to nomenclature developed by Roepstorff and Fohlman
[42]). No y series, containing C-terminal tail were observed. This is due
to the presence of basic amino acid residues (that are accessible for
protonation) on N-terminal side of the peptide only. In contrast to the
CID spectrum obtained for fragmentation of the double protonated
peptide, [M+2H]2+, sodiated ions become the major type of the
fragment ions resulted from the CID of the doubly charged mono-
sodiated species [M+H+Na]2+. This suggest that the sodium salts are
relatively stable in the performed CID conditions. It is known that
structure of the sodiated peptide ions may affect the CID MS/MS
fragmentation pattern. Alkali metal ions interact selectively with polar
functional groups to form a chelate coordination structure of the
peptide–metal ion complex and can form fixed charges of the peptide
backbone. MS/MS analysis of metaliated peptides is known method of
localization of the peptide groups involved in interaction with the metal
[43]. Recently, Bensadek and co-workers found that the fragmentation
of the sodiated peptides gives rise to sodium cationized y ions, since the
sodium cation is chelated at the C-terminus of tested peptides [44]. In
this paper we performed the CID analysis of the doubly charged
monosodiated species [M+H+Na]2+ to estimate possible binding site
of sodium ion to the peptide. We noticed a striking difference in the
fragmentation patterns between sodiated and non-sodiated peptide
ions (Fig. 2a and b) suggesting that the presence of the sodium cation
selectively interacts with the peptide. The CID of the doubly charged
monosodiated species [M+H+Na]2+ resulted in the formation of
sodiated y ions in the m/z range above the precursor and a
characteristic an/bn (n=6–10) pair in the low m/z range. Only double
charged sodiated a6–10 and b6–10 double charged ions in the in the range
of m/z 376–600 were detected during CID of the doubly charged
monosodiated species [M+H+Na]2+, what suggests, that the metal
ion is localized in a polar group of one of six amino acid residues
situated on N-terminal peptide part. On the other side, formation of
singly charged sodiated y7–y9 ions suggests that the sodium adduct is
located rather on the C-terminal heptapeptide. The presence of
the single charged non-sodiated b2 and b3 ions at 308.17 m/z and
409.22 m/z, respectively further confirms that the sodium ion is not
localized on the first three N-terminal residues. The identified
fragments presented above indicate that the sodium adduct may be
attached to Ser5 or Asp4 side chains. These suggestions are in agreement
with previous investigations performed by Feng and co-workers [45].
The authors studied lithium and sodium ion binding energies of N-
acetyl and N-glycyl amino acids and concluded that the presence of a
coordinating group (e.g., –OH, –CO2H) in the amino acid side chain can
significantly increase the lithium and sodium binding energies.
However, a sodiated singly charged peak at 674.32 m/z corresponds
to [y6Na]+ fragment, that does not contain the aspartic acid residue.
This may indicate that Ser5 present in [y6Na]+ fragment may be
particularly responsible for interaction with the sodium ion. A transfer
of the sodium ion from Asp4 carboxyl group to y6 fragment containing
Ser5 during CID is rather doubtable, and therefore the sodium ion
should be attached to the serine residue before the fragmentation. As
can be expected, no peak corresponding to the [y5Na]+ fragment is
present in the spectrum, confirming that sodium ion is attached to the
serine residue only in [y6Na]+ fragment. These data may suggest a
deprotonation of the hydroxyl-OH group of the Ser residue in the
Table 3
Calculated log K* and deprotonation constants for amide protons (pK) values for Cu(II) complexes with studied and comparable ligands.
Peptide/log K*
1 N {NIm or NH2}
2 N{NIm, NH2}
3 N dimeric{NH2, N−,CO, NIm
}
3 N{NH2, 2N−,CO or COO−
}
4 N{NH2, 3N−
}
}
Neurokinin A
HSDGI-NH2
HGG
−0.01
−0.53
−3.99
−4.31
−4.31
−4.45
−18.92
−17.37
−18.30
−18.95
−13.60
−22.45
−14.17
−13.81
−16.44
HAA
AAAAA-NHa2
−24.41
log K*b
1 N{NIm
}
2 N{NIm, N−
}
3 N{NIm, 2N−
}
4 N{NIm, 3N−
Ac-neurokinin A
Ac-HGGGWGQ-NH2
Ac-HGGG
−1.78
−2.72
−2.39
−2.47
−7.77
−9.21
−8.99
−9.38
−14.79
−15.40
−15.95
−16.06
−23.34
−24.38
−24.87
−24.48
Ac-GGGH
pK1
pK2
pK3
Ac-neurokinin A
Ac-HGGGWGQ-NH2
Ac-HGGG
5.99
6.49
6.60
6.91
7.02
6.19
6.96
6.68
8.55
8.98
8.92
8.42
Ac-GGGH
a ref. [53], b K*=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.

T. Kowalik-Jankowska et al. / Journal of Inorganic Biochemistry 104 (2010) 831–842
835
Table 4
Spectroscopic data for the Cu(II) complexes of neurokinin A and Ac-neurokinin A.
Species
Absorption
pH
CD
EPR
λ (nm)
ε (M−1 cm−1
)
λ (nm)
Δε (M−1 cm−1
)
A||
g||
H2N-HKTDSFVGLM-NH2
CuH3L
3.5
5.5
7.5
163
2.299
2.238
{NH2 or NIm
CuH2L
}
654a
598a
55
98
694a
323d
690a
564a
345d
282bc
+0.397
−0.229
+0.335
−0.060
−0.133
−0.077
189
{NH2, NIm
Cu2H2L2
}
Dimer
{NH2, N−, CO, NIm
}
CuL
8.5
584a
516a
106
58
{NH2, 2N−, CO}
CuH−1L, CuH−2L,
Above
9.0
530a
311e
273b
−0.989
+0.563
−2.300
202
2.179
CuH−3
L
{NH2, 3N−
}
Ac-HKTDSFVGLM-NH2
CuH2L
{NIm,}
CuHL
5.0
6.5
732a
660a
16
313d
+0.049
164
171
2.297
2.242
f
597a
309e
255c
584a
348d
304e
256c
547a
311e
255c
−0.107
+0.043
+0.246
+0.219
+0.036
+0.062
−0.235
−0.372
+0.148
+1.022
{NIm, N−
}
f
f
CuL
8.0
600a
540a
186
205
2.229
2.182
{NIm, 2N−
}
CuH−1L, CuH−2L,
Above
9.0
CuH−3
L
{NIm, 3N−
}
a
d–d transition.
NH2 →Cu(II) charge transfer transition.
NIm (π2)→Cu(II) charge transfer transition.
b
c
d
e
f
N
Im →Cu(II) charge transfer transition.
N-(amide)→Cu(II) charge transfer transition.
ε cannot be estimated because of higher base line, the solutions were not too clear.
ligands studied. It seems that the reason of the low protonation
constant of the hydroxyl OH group of the serine may be presence of the
His, Asp and Ser residues (like in serine proteases) and the secondary
structure of the peptide. Moreover, it should be also mentioned that
this additional deprotonation has not any influence on the coordination
abilities of the neurokinin A and its derivative.
agreement with the previous findings of Sigel and Martin, which state
that ability of metal ions to sense asymmetry on a nearby side chain is
transmitted in the decreasing order: N−(amide)NNHCONCOO−NNH2
[46]. The next species, CuH2L, is detected in the wide 3.5–7.5 pH range as a
major complex (Fig. 3). The EPR parameters gII =2.238 and AII =189 G
(Fig. 4b), the d–d transition energy at 654 nm and the presence in CD
spectra of the NIm →Cu(II) charge transfer transition at 323 nm are con-
sistent with the histamine-like coordination {NH2, NIm} of the neurokinin
A to copper(II) ions (Scheme 1) [47–51]. Coordination of histidine
normally gives charge transfer transitions at c.a. 330 nm [π1(NIm–Cu(II))].
The magnitude and precise energy of the charge transfer transitions for
an imidazole nitrogen to Cu(II) are very sensitive to the position of the
ring plane relative to the complex plane, and thus to the possibility of its
rotation [52]. The value of log K* for 2 N {NH2, NIm} complex of neurokinin
A is comparable to those of the peptides containing the histidine residue
in the first position of the peptide chain (H1) (Table 3).
3.3. Copper(II) complexes
According to potentiometric and spectroscopic results, the neurokinin
A forms with Cu(II) ions the CuH3L, CuH2L, CuL, CuH−1L, CuH−2L,
CuH−3L, CuH−4L and Cu2H2L2 complexes (charges omitted for simplicity,
Table 2). Two protonation constants of the Lys residue and, most likely,
hydroxy group of Ser residue were detected, therefore, the 1 N {NH2, CO
or NIm} complex will be the CuH3L species (Scheme 1). This complex at
pH 3–5 is formed (Fig. 3) with EPR parameters gII =2.299 and AII =163 G
(Fig. 4a, Table 4). In CD spectra the d–d absorption band is not observed
for this complex (concentration of copper(II) about 0.001 M) and it is in
With increasing pH above 6.5 in the CD spectra the NIm →Cu(II)
charge transfer transition at 345 nm is present but the EPR signal
Fig. 1. Mass spectrum of Ac-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2 at pH 10.5.

836
T. Kowalik-Jankowska et al. / Journal of Inorganic Biochemistry 104 (2010) 831–842
Fig. 2. MS/MS spectrum of doubly charged peptide Ac-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2 (parent ion m/z 589.29) (a). MS/MS spectrum of the doubly charged
monosodiated peptide Ac-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2 ion at m/z 599.29. [ynNa]+ denotes yn ions in which a proton has been exchanged for a sodium cation.
intensity is reduced indicating a possible antiferromagnetic interac-
tion among copper(II) ions in the complex formed (Table 4). In pH
range 6.5–8.5 (Fig. 3), the dimeric Cu2H2L2 species is formed through
Gly-Gly-like coordination, and the imidazole nitrogen acts as bridging
ligand (Scheme 1). The structure of the imidazole-bridging dimer has
been proposed for HG [47], HM [48], HH [49], HGG and HAA [50] and
HSDGI-NH2 [51]. Above pH 8, the band diagnostic of imidazole
nitrogen binding is no longer observed and the CuL complex with
{NH2, 2N−, CO} coordination mode is formed (Scheme 1). The
coordination of the ligand in the Cu(II)–neurokinin A system at pH
above 8.5 follows by sequential deprotonation and coordination of
nitrogen of His1 dramatically changes the coordination, which begins at a
higher pH than that discussed above (Fig. 5). The coordination of the
histidine side chain instead of the amine N would yield 7,5,5-membered
fused chelate rings, resembling that of the prion protein octarepeat metal-
binding site [54,55]. This type of structure has later been confirmed by the
X-ray diffraction analysis of the copper(II) complex of Ac-His-Gly-Gly-
Gly-Trp-NH2, a segment of the octarepeat region [56]. Since then a
number of N-protected peptides with histidine at the N-terminus have
been shown to have similar type of coordination [50,57,58]. The
anchoring site at the imidazole nitrogen is metal-bound in the CuH2L
species (Scheme 2). This complex undergoes stepwise deprotonation
processes giving CuHL, CuL, CuH−1L, CuH−2L, CuH−3L complexes. The
major species in the “physiological” pH, 6–9 is the 3 N {NIm, 2N−} (CuL)
complex. The d–d transition energy at 600 nm, the EPR parameters
gII =2.229, AII =186 G and the presence in the CD spectrum of charge
transfer transitions NIm →Cu(II) at 348 nm and N−(amide)→Cu(II) at
304 nm (Table 4) indicate a 3 N complex with {NIm, 2N−} coordination
mode. With increasing of pH, the shift of the absorption maximum to
540 nm and the EPR parameters gII =2.182, AII =205 G suggest coordi-
nation of the additional nitrogen donor atom with the formation of the
4 N CuH−1L, {NIm, 3N−} complex (Scheme 2). The values log K* for 1 N,
2 N, 3 N and 4 N copper(II) complexes of Ac-neurokinin A are higher by
about 1, 1.5, 0.7 and 1 orders of magnitude, respectively, in comparison to
those of Ac-PHGGGWGQ-NH2 (Table 3). It should be mentioned that the
coordination of the histidine side chain in the CuH−3L complex of Ac-
HGGG peptide yields 7,5,5-membered fused chelate rings, while in the
CuH−3L complex of Ac-GGGH 5,5,6-membered chelate rings. However,
the log K* values for the 1 N-to-4 N complexes of Cu(II)–Ac-HGGG and Cu
(II)–Ac-GGGH systems and the deprotonation constants for amide
protons (pK values) of their Cu(II) complexes are similar to each other
(Table 3).
neighboring peptide nitrogen with the formation of 4 N {NH2, 3N−
}
complex (Scheme 1). The d–d energy at 516 nm, the parameters of CD
spectra and gII =2.179, AII =202 G correspond very well to the 4 N
{NH2, 3N−} coordination mode (Fig. 4c, Table 4). These parameters of
UV–Vis, CD and EPR are not altered in basic solution suggesting the
same coordination binding in the CuH−1L, CuH−2L and CuH−3
L
complexes. Value of log K* for the dimeric complex of neurokininA is
comparable to those of the HGG and HAA and about 1.5 log units lower
in comparison to that of HSDGI-NH2 (Table 3). The value of log K* for
3 N {NH2, 2N−,CO or COO−} species of neurokinin A is comparable to
those of the HGG and HAA, and about three orders of magnitude higher
compared to that of pentaalanine amide (Table 3) [53]. This
stabilization of the CuL complex of neurokinin A may proceed from
the additional coordination of the histidine side-chain residue in axial
position as it was suggested for HGG and HAA [50]. The stabilization of
the 4 N {NH2, 3N−} complex by about two orders of magnitude is also
observed in comparison to that of pentaalanine amide (Table 3). This
stabilization may also result from the axial interaction of the histidine
residue in the CuH−1L complex (Scheme 1).
Six metal complex species can be fitted to the experimental titration
curves obtained for the Cu(II)–Ac-neurokinin A system: CuH2L, CuHL, CuL,
CuH−1L, CuH−2L, CuH−3L (Table 2). The protection of the N-terminal
The CD spectra in the range 190–300 nm of metal-free NKA and
Ac-NKA and complexed ligands at pH 7 indicate an unordered

T. Kowalik-Jankowska et al. / Journal of Inorganic Biochemistry 104 (2010) 831–842
837
Scheme 1. Schematic structures of the Cu(II)–neurokinin A complexes formed with increasing of pH.
structure of peptides with time (aging process) at this pH (data not
shown).
phate buffer at pH 7.4 were made (data not shown). The spectroscopic
data for the Cu(II)–neurokinin A and Cu(II)–Ac-neurokinin A in
phosphate buffer suggest that the coordination modes of these
peptides to copper(II) ions are similar to those obtained in aqueous
solution at pH 7.4. For the Cu(II)–neurokinin A system, the Cu2H2L2
complex dominates at pH 7.4 (Fig. 3) with the coordination of copper
(II) ions by the nitrogen atoms of amine group and imidazole of
histidine (H1) and atoms of amide nitrogen and oxygen of carbonyl
group of the lysine residue (K2). For the Cu(II)–Ac-neurokinin A
3.4. Oxidation of neurokinin A and its derivative
The oxidation of protein causes the modification of amino acids,
protein aggregation, and protein fragmentation [27,28]. The mecha-
nism of generation for the active species from Cu(II)/peptide/H2O2 has
been considered (reactions (1)–(3)) [59,60].
system at pH 7.4 the CuL complex exists (Fig. 5) with the 3 N {NIm
,
2N−} coordination binding. Therefore, the modification of the
peptides at N-terminal amine group should be expected.
peptide ꢀ CuðIIÞ þ H2O2→peptide ꢀ CuðIÞ þ O−2 • þ 2Hþ
peptide ꢀ CuðIIÞ þ O−2 •→peptide ꢀ CuðIÞ þ O2
ð1Þ
ð2Þ
ð3Þ
The chromatograms of the neurokinin A and Ac-neurokinin A after
24 h incubation at 37 °C for the peptide alone, with Cu(II) only, hydrogen
peroxide and with Cu(II)–H2O2 indicate that for the solution containing
peptide alone and the copper(II) ions with 1:1 peptide to copper(II) molar
ratio were not changed in comparison to the peptide alone before
incubation (Fig. 6). For the solution containing the peptide–hydrogen
peroxide for both systems at 1:1 or 1:2 molar ratio a new peak on the
chromatogram was detected. Liquid chromatography/mass spectrometry
•
peptide ꢀ CuðIÞ þ H2O2→peptide ꢀ CuðIIÞ þ OH þ OH−
In accord with a site-specific mechanism [28,61], suggesting that
the active oxygen species are formed on or near the metal-binding
sites of the protein and react predominantly with amino acid residues
of close proximity the spectroscopic measurements in a 0.1 M phos-

838
T. Kowalik-Jankowska et al. / Journal of Inorganic Biochemistry 104 (2010) 831–842
Fig. 3. Species distribution curves for Cu(II) complexes of neurokinin A. Cu(II) to peptide molar ratio 1:1, [Cu(II)]=0.001 M.
(LC–MS) was the primary analytical tool utilized in the product analysis of
the oxidation reactions. Liquid chromatography (LC) was employed to
monitor and quantitate the reaction products, while mass spectrometry
enabled the structural identification. The desalted reaction mixture of
peptide–hydrogen peroxide systems contains two major components,
one corresponding to the Met sulfoxide (tR=10.5 min for neurokinin A,
tR=11.6 for Ac-neurokinin A, Tables 5 and 6, respectively) and one
corresponding to unoxidized peptide (tR=12.3 min and 13.4 min for
neurokinin A and Ac-neurokinin A, Tables 5 and 6, respectively). The
doubly charged molecular ions with m/z 575.2 Da and 596.8 Da for
neurokinin A and its acetyl-derivative, respectively, were present in mass
spectra (Tables 5 and 6). The oxidation of the methionine residue to the
methionine sulfoxide by hydrogen peroxide alone [62,63] and by metal-
catalyzed oxidation was observed [64–66].
When the copper(II) ions are alone with peptides the modifica-
tions of these peptides don't occur (Fig. 6), but when the copper(II)
ions with hydrogen peroxide are together then further oxidation of
the peptides is observed (Tables 5 and 6). The exposure of the
peptides to Cu(II) and H2O2, resulted in an efficient conversion of the
peptide His residues into asparagines, aspartic acid, aspartylurea,
formylasparagine [67], and 2-oxo-His [68–70]. It is suggested that the
sequence and conformation are important parameters controlling the
metal-catalyzed oxidation of His [71]. For the Cu(II)–neurokinin A
system, mass spectrometry of the LC fraction eluted at 13.3 min
yielded a doubly charged molecular ion with mass of 583.4 Da
supporting the oxidation of the His residue to 2-oxo-His or further
oxidation of Met sulfoxide to sulfone (Table 5). The loss of sulfinic acid
(CH3SOH, 64 Da) from the oxidized methionine residue is diag-
nostic for the oxidized methionine [65]. The masses of [M+H]+ and
[M+2H]2+ molecular ions at 1085.5 and 542.7 Da, respectively,
observed for the LC fraction eluting at 13.3 min may be assigned to
the peptide with loss of CH3SOH from the peptide with oxidized
methionine to sulfoxide (Table 5). The involvement of the histidine in
binding of copper(II) ions is supported by the cleavage of the H1–K2
peptide bond. Mass spectrometry for the LC fraction eluted at
13.0 min yielded masses of [M+H]+ and [M+2H]2+ molecular ions
at 997.5 and 498.7 Da, respectively (Table 5, Fig. 7). These masses may
be assigned to K2–M10 fragment. Under experimental conditions the
other molecular ions of copper(II) catalyzed oxidation products for
neurokinin A were also detected and the modifications of this
neuropeptide are proposed in Table 5.
When the sample of Cu(II)–Ac-neurokinin A–H2O2 1:1:1 and 1:1:2
molar ratio after incubation was loaded onto ZipTip column to remove
salts and was eluted with 0.1% formic acid in 80% acetonitrile the
precipitation of the oxidized Ac-neurokinin A was observed. However,
the obtained sample was the subject of LC-ESI-MS analysis (Table 6).
Mass spectrometry of the LC fraction eluted at 14.6 min yielded a mass
of 1207.5 Da supporting the oxidation of the His residue to 2-oxo-His
or further oxidation of Met sulfoxide to Met sulfone (Table 6). A peak
of the doubly charged [M+2H]2+ molecular ion occurring at m/z
577.4 Da in the LC fraction eluting at 14.9 min (Table 6) may
correspond to the peptide containing the oxidized histidine residue
to aspartic acid. The fragmentation products obtained by cleavage of
the peptide bonds near the His residue were also observed supporting
the involvement of this residue in binding of copper(II) ions (Table 6).
It is well known that physicochemical properties of a protein can be
significantly altered upon covalent modifications. The data for
proteins suggest that modification of amino acid residues (especially
His) leads to alternations in secondary and tertiary structures, and
may increase the exposure of the hydrophobic surface area which
results in consequent noncovalent aggregation and precipitation
[65,72,73].
4. Conclusions
Neurokinin A contains the histidine residue in the first position of
the peptide sequence (His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-
Fig. 4. X-band EPR spectra of 1:1.1 Cu(II)–neurokinin A frozen solution at 77 K at pH 3.5
(a), 5.5 (b) and 10.0 (c).

T. Kowalik-Jankowska et al. / Journal of Inorganic Biochemistry 104 (2010) 831–842
839
Fig. 5. Species distribution curves for Cu(II) complexes of Ac-neurokinin A. Cu(II) to peptide molar ratio 1:1, [Cu(II)]=0.001 M.
NH2) and forms monomeric and dimeric complexes of copper(II) ions.
The CuH2L complex with the histamine {NH2, NIm} type coordination
mode predominates at low and wide 3.5–7.5 pH range. In the pH 6.5–
8.5 range the formation of a dimeric complex Cu2H2L2 was found in
which the coordination of copper(II) ions is glycylglycine-like, while
the fourth coordination site is occupied by the imidazole nitrogen
atom, forming a bridge between two copper(II) ions. The formation of
this dimeric species does not prevent the deprotonation and
coordination of the sequential amide nitrogens and at pH above
8.0 the 4 N {NH2, 3N−} complex is formed. The coordination of
the histidine side chain instead of the amine NH2 nitrogen yields
7,5,5-membered fused chelate rings for the CuH−1L complex of Cu
(II)–Ac-neurokinin A system. The copper(II) ion is coordinated by the
imidazole nitrogen atom of histidine and deprotonated amide
nitrogens from the next Lys, Thr and Asp residues.
For the neurokinin A and its acetyl derivative the additional
deprotonation at pH 9–10 was detected. The MS/MS experiments
suggest a deprotonation of hydroxy group of Ser residue.
For the neurokinin A and Ac-neurokinin A-hydrogen peroxide
systems at 1:1 and 1:2 molar ratio the oxidation of the methionine
residue (M10) to methionine sulfoxide was observed. For the both
studied peptides the coordination of the copper(II) ions by the
Scheme 2. Binding modes of the species formed in the copper(II)–Ac-neurokinin A system.

840
T. Kowalik-Jankowska et al. / Journal of Inorganic Biochemistry 104 (2010) 831–842
Fig. 6. Chromatograms of the neurokinin A after 24 h incubation at 37 °C for the peptide alone (first from bottom), peptide with copper(II) (1:1 molar ratio) (second from bottom),
peptide with hydrogen peroxide (1:2 molar ratio) (second from top) and peptide with copper(II) and hydrogen peroxide (1:1:2 molar ratio) (first from top). Solution contained
0.1 M phosphate buffer (pH 7.4); concentrations of peptide and Cu(II) 0.50 mM. Incubation at 37 °C.
histidine occurs, therefore, in the Cu(II)–peptide–hydrogen peroxide
1:1:1 and 1:1:2 molar ratio systems, oxidation of histidine to 2-oxo-
histidine may be suggested. Fragmentations by the cleavage of the
peptide bonds near the His residue were also detected.
Hydrogen peroxide is produced in vivo [74] and if both H2O2 and
copper(II) ions are available in vivo, then radicals will form. The
dominant risk factor associated with the neurodegenerative diseases is
increasing age. Several studies in animals and humans have reported a
Table 5
Products of oxidized neurokinin A (HKTDSFVGLM-NH2) in Cu(II)–neurokinin A-H2O2 system analyzed by LCMS spectra.
Determination of peptide modification
Charge
MWcalcd (Da)
14.6
tR(min)
MWobsd (Da)
10.3
10.5
11.9
12.3
13.0
13.3
13.9
Neurokinin A
1
2
1133.6
567.8
1133.6
567.8
Neurokinin A:H2O2 (1:1)
Oxidation of Met→sulfoxide
2
3
575.8
384.2
575.2
383.8
Cu(II):neurokinin A:H2O2 (1:1:1)
Oxidation of Met→sulfoxide
1
2
2
1149.6
575.8
1149.6
575.3
Oxidation of Met→sulfoxide and
583.8
583.4
His→2-oxo-His or Met→sulfone
Oxidation of Met→sulfone, cleavage of the H–K peptide bond (K2–M10
)
2
1
2
1
2
1
2
1
2
2
515.2
1012.5
516.8
Oxidation of Met→sulfoxide, cleavage of the H–K peptide bond (K2–M10
)
1011.4
506.3
993.4
498.7
1085.5
542.7
507.2
996.5
Cleavage of H1–K2peptide bond (K2–M10
)
997.5
498.7
499.2
1085.6
543.8
1040.6
521.3
Loss of CH3SOH fragment from peptide with oxidized Met→sulfoxide
Oxidation of Met→sulfoxide, loss of
1038.5
519.7
CH3SOH fragment, decarboxylation of D4 and deamination of H1
Oxidation of Met→sulfoxide and
561.3
560.7
559.8
652.4
His→2-oxo-His or Met→sulfone, decarboxylation of D4 and deamination of H1
Cleavage of D4–S5peptide bond,S5–M10 fragment
1
652.1

T. Kowalik-Jankowska et al. / Journal of Inorganic Biochemistry 104 (2010) 831–842
841
Table 6
Products of oxidized Ac-neurokinin A (Ac-HKTDSFVGLM-NH2) in Cu(II)–Ac-neurokinin A–H2O2 system analyzed by LCMS spectra.
Determination of peptide modification
Charge
MWcalcd (Da)
tR (min)
MWobsd (Da)
14.6
11.6
13.4
14.9
15.1
Ac-Nurokinin A
1
2
1175.4
588.7
1175.6
588.8
Ac-neurokinin A:H2O2 (1:1 and 1:2)
Oxidation of Met→sulfoxide
1
2
1191.4
596.7
1192.6
596.8
Cu(II):Ac-neurokinin A:H2O2 (1:1:1)
Oxidation of Met→sulfoxide and
His→2-oxo-His or Met→sulfone
Oxidation of His→Asp
1
2
2
1
1207.4
604.7
577.7
768.0
1207.5
604.8
603.4
577.4
767.4
Cleavage of T3–D4peptide bond,(D4–M10
Cu(II):Ac-neurokinin A:H2O2 (1:1:2)
Oxidation of Met→sulfoxide and
His→2-oxo-His or Met→sulfone
)
1
1
1
1207.4
604.7
856.1
1207.6
604.4
Cleavage of K2–T3peptide bond,(T3–M10
)
855.3
Oxidation of Met→sulfone, decarboxylation of D4
Fig. 7. Mass spectrum of the LC fraction eluted at 13.0 min for the Cu(II)–NKA–H2O2 1:1:1 molar ratio system after 24 h incubation. Solution contained 0.1 M phosphate buffer
(pH 7.4); concentration of the neurokinin A, Cu(II) and hydrogen peroxide 0.50 mM. Incubation at 37 °C.
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Acknowledgment
Financial support from the Polish State Committee for Scientific
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