Tyrosine nitration in peptides by peroxynitrite generated in situ in a light-controlled platform: Effects of pH and thiols

Article abstract of DOI:10.1016/j.jinorgbio.2014.04.018

Peroxynitrite has been shown to play a critical role in inflammation and affords 3-nitrotyrosine as the hallmark product. The reported methods of generating this reactive nitrogen species in situ often fails to provide a high and steady flux of peroxynitrite resulting in poor yields of 3-nitrotyrosine. Herein we report a two-component peroxynitrite-generating platform in which this anion is produced in a biomimetic fashion and under the control of visible light. Incorporation of the nitric oxide- and superoxide-generating components in polymer matrices allows easy alterations of pH in the reaction wells of this platform. We have demonstrated very efficient nitration of tyrosine by peroxynitrite at different pH values and with varying concentrations of carbonate. In addition to tyrosine, a set of tyrosine-containing peptides was also studied. Presence of glutathione in the reaction wells increases the extent of tyrosine nitration in such peptide substrates presumably by raising the lifetime of nitric oxide in the reaction medium. When a cysteine residue was included in the sequence of the peptide, the extent of nitration of the tyrosine residue was found to depend on the position of the cysteine residue with respect to tyrosine. The extent of tyrosine nitration is strongly attenuated when the cysteine residue is directly adjacent to the tyrosine. This effect has been attributed to an intramolecular radical transfer mechanism. Taken together, results of this study demonstrate the potential of this light-controlled platform as a convenient bioanalytical tool in studying the reactions of peroxynitrite under widely varying conditions.

Full text of DOI:10.1016/j.jinorgbio.2014.04.018

Tyrosine nitration in peptides by peroxynitrite generated in situ in a light-
controlled platform: Effects of pH and thiols
Tara R. deBoer, Rafael I. Palomino, Sharon O. Idiga, Glenn L. Mill-
hauser, Pradip K. Mascharak
PII:
DOI:
Reference:
S0162-0134(14)00123-8
doi: 10.1016/j.jinorgbio.2014.04.018
JIB 9518
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
31 January 2014
25 April 2014
28 April 2014
Please cite this article as: Tara R. deBoer, Rafael I. Palomino, Sharon O. Idiga, Glenn L.
Millhauser, Pradip K. Mascharak, Tyrosine nitration in peptides by peroxynitrite gen-
erated in situ in a light-controlled platform: Effects of pH and thiols, Journal of Inorganic
Biochemistry (2014), doi: 10.1016/j.jinorgbio.2014.04.018
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ACCEPTED MANUSCRIPT
Tyrosine nitration in peptides by peroxynitrite generated
in situ in a light-controlled platform: Effects of pH and thiols
Tara R. deBoer, Rafael I. Palomino, Sharon O. Idiga, Glenn L. Millhauser, and Pradip K.
Mascharak*
Department of Chemistry and Biochemistry
University of California Santa Cruz, Santa Cruz, CA 95064
*
Corresponding author: Tel 1-831-459-4251
FAX: 1-831-459-2935
E-mail address: pradip@ucsc.edu
Keywords: Photoactive metal nitrosyl, Peroxynitrite, Nitric oxide, Superoxide, Tyrosine
nitration
Highlights:





Peroxynitrite was generated in situ within wells of a light-controlled platform
A photoactive metal nitrosyl complex provides the optimal NO flux
Effective tyrosine nitration in model peptides were demonstrated
The platform allowed studies under different pH, CO and thiol concentrations
2
A cysteine neighbor strongly attenuates the extent of tyrosine nitration
1

ACCEPTED MANUSCRIPT
ABSTRACT
Peroxynitrite has been shown to play a critical role in inflammation and affords 3-nitrotyrosine
as the hallmark product. The reported methods of generating this reactive nitrogen species in situ
often fails to provide a high and steady flux of peroxynitrite resulting in poor yields of 3-
nitrotyrosine. Herein we report a two-component peroxynitrite-generating platform in which this
anion is produced in a biomimetic fashion and under the control of visible light. Incorporation of
the nitric oxide- and superoxide-generating components in polymer matrices allows easy
alterations of pH in the reaction wells of this platform. We have demonstrated very efficient
nitration of tyrosine by peroxynitrite at different pH values and with varying concentrations of
carbonate. In addition to tyrosine, a set of tyrosine-containing peptides was also studied.
Presence of glutathione in the reaction wells increases the extent of tyrosine nitration in such
peptide substrates presumably by raising the lifetime of nitric oxide in the reaction medium.
When a cysteine residue was included in the sequence of the peptide, the extent of nitration of
the tyrosine residue was found to depend on the position of the cysteine residue with respect to
tyrosine. The extent of tyrosine nitration is strongly attenuated when the cysteine residue is
directly adjacent to the tyrosine. This effect has been attributed to an intramolecular radical
transfer mechanism. Taken together, results of this study demonstrate the potential of this light-
controlled platform as a convenient bioanalytical tool in studying the reactions of peroxynitrite
under widely varying conditions.
2

ACCEPTED MANUSCRIPT
1
. Introduction
−
Peroxynitrite (ONOO , PN) is a short-lived reactive nitrogen species (RNS) that is generated

−
from precursor radicals nitric oxide (NO) and superoxide (O ) at a diffusion-controlled rate [1-
2
6
]. It is estimated that under pro-inflammatory conditions, this reactive anion is produced at a
rate of 50-100 μM per min in cellular compartments [4]. Further, steady state concentrations of
PN in the nanomolar range can persist under such conditions for hours. The reactive nature of
this species contributes to a diverse chemistry implicated in a wide range of diseased states from
Alzheimer’s to atherosclerosis [7-13].
The product 3-nitrotyrosine (3-NT) has been identified as a hallmark footprint of PN in vivo,
following the seminal work by Beckman and coworkers [14-16]. In general, nitration of a
tyrosine residue is a selective process that can effectively modify the activity of biomolecules
such as enzymes and proteins to induce inauspicious effects [1,3,17-20]. The mechanism of
tyrosine nitration by PN is a highly dynamic reaction that is strongly affected by numerous
factors including pH and CO concentration [2,21,22]. At physiological pH (pH 7.4), the anionic
2
form of PN predominates (~80%) and reacts rapidly with CO (present in mM concentrations) to
2
−
yield the nitrosoperoxocarbonate intermediate (ONOOCO ). A significant portion of this anion
2

−
(
~33%) undergoes homolysis to yield the strong oxidant carbonate radical (CO ) and nitrating
3

agent nitrogen dioxide ( NO ), both of which participate in the nitration of tyrosine. The extent
2
of nitration by this mechanism is strongly pH-dependent.
In earlier studies, investigation into PN-dependent nitration afforded contradictory results
depending on the mode of PN synthesis [23-25]. Due to the transient nature of this reactive
species, strongly alkaline stock solutions of PN are commonly synthesized to maximize its
lifetime [26,27]. While bolus addition of this stock solution can easily be incorporated into
3

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buffered solutions, PN rapidly isomerizes (to nitrate) and/or decomposes to other species [28].
Such instability prevents studies on the effects of prolonged exposure of a steady flux of PN (and
its precursor radicals) on biological substrates [29]. Comparative studies to elicit nitration effects
of PN generated by more biomimetic co-generative methods (instead of chemically synthesized
sources) have also afforded divergent results due to difficulties in controlling such two-
components systems [23,30].
In the present work, we employed a two-component multi-well PN-generating platform to
demonstrate effective nitration of tyrosine by a sustained level of PN generated in situ under the
control of light [31]. Within this system, tyrosine and tyrosine-containing model peptides were
exposed to PN under different conditions to probe the effects of pH, glutathione (GSH), CO2

−
concentrations, and relative ratios of precursors NO and O2 on this nitration reaction. The
multi-well feature of the applied platform allows for multiple studies to be completed in
synchrony. In addition to systematically modifying the milieu within selected wells to observe
Figure 1 here
modifications to tyrosine nitration as a function of intermolecular effects, we designed three
model peptides to estimate the intramolecular effect of a thiol on tyrosine nitration upon
exposure to PN. Interestingly, the cysteine residue promoted slight enhancement when separated
from the tyrosine residue by several amino acid residues while a strong inhibitory effect was
observed when the two residues were next to each other.
2
. Materials and Methods
2
.1 Materials
4

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All chemicals used were analytical or ACS grade and received from chemical suppliers, unless
otherwise stated. Platform components tetramethyl orthosilicate (TMOS), hypoxanthine (HX),
and xanthine oxidase (XO) were purchased from Sigma-Aldrich. All peptides synthesized were
assembled on a Rink-amide ChemMatrix® resin purchased from Sigma-Aldrich, and required
amino acids were purchased from AAPPTec.
2
.2 Preparation of the PN-generating platform
Prefabricated 24-well plates were synthesized as previously described [31]. Five-fold increase
of all components was required to compensate for the increased well size in the present work. In
short, to a stock solution of hydrolyzed tetramethyl orthosilicate solution [Mn(Papy )(NO)]ClO
3
4
(
PaPy = N,N´-bis(2-pyridylmethylamine-N-ethyl-2-pyridine-2-carboxamide, (Mn(NO)) [32] was
3
added and allowed to stir until the nitrosyl was completely dissolved. A batch of 600l of this
stock Mn(NO) solution was then added to individual wells and allowed to dry and age for 1
week prior to use. The final Mn(NO)-encapsulated sol-gel discs (Mn(NO)SG) were stored at 4
o
C in the dark prior to use.
In a second 24-well plate, a batch of 800l of a xanthine oxidase/catalase containing sol-gel
(XO/CATSG) mixture were plated into individual wells and allowed to dry and age for 1.5
weeks. Prior to their use, these enzyme encapsulated sol-gel discs were washed three times and
their activity was evaluated by electronic absorption spectroscopy [31]. Construction of the PN-
generating platform was completed by adding a single Mn(NO) sol-gel disc to each well of a 24-
well plate with XO/CATSG layered at the bottom (Fig. 1). Generation of PN within the wells
was initiated with the addition of hypoxanthine (HX) and concomitant illumination of the plate
with broadband light (10 mW, Electrofiberoptics Corporation 410 illumination). Because the
photoactive metal nitrosyl exhibits photobands in both visible and UV regions, a broadband light
5

ACCEPTED MANUSCRIPT
source was employed to trigger the release of NO. Optimization of NO release from the Mn(NO)
sol-gel under exposure to this low power light source was completed using a NO electrode

−
(
[
Innovative Instruments) to assure 1:1 flux ratio of NO and O2 within the wells of the platform
31]. The kinetics of PN formation within the individual wells was followed (quantitated to be
4
.42 ± 0.22 M per min) using the PN probe coumarin-7-boronic acid [33].
2
.3 Peptide Synthesis and Purification
All peptides were synthesized using Fmoc synthesis on a Liberty 1 Peptide Synthesizer with
Discover® microwave platform (CEM, Matthews, NC). The syntheses were performed on a 0.1
mmol scale, and monitored with PepDriver software. The deprotection of the Fmoc group was
achieved using a solution of 20% piperidine in dimethylformamide (DMF). Solutions of 0.625 M
diisopropylcarbodiimide and 1.25 M hydroxybenzotriazole in DMF were used to activate the
carboxy-termini. All coupling reactions were performed at 4 equivalents of Fmoc-amino acid,
and each amino acid was double coupled and capped with a 10% acetic anhydride solution. Fully
synthesized peptides were washed with dichloromethane and split into two reaction vessels for
cleavage.
A cleavage cocktail consisting of 15 mL of trifluoroacetic acid, 0.5 mL each of 1,2-
ethanedithiol and liquefied phenol, and 1 mL of triisopropylsilane, was added to each vessel of
dried resin and reacted for 2 h followed by filtration. The filtrate was added to 90 mL of cold,
dry diethyl ether for precipitation. The precipitate was collected by centrifugation, and the ether
was discarded. The resulting pellet was dissolved in 20 mL of 1:1 H O/acetonitrile (1% formic
2
acid) and lyophilized.
Peptides were purified by reversed phase (RP)-HPLC on a Vydac (Hesperia, CA) preparative
C18 column. Fractions were collected and analyzed by electron spray ionization mass
6

ACCEPTED MANUSCRIPT
spectrometry (ESI-MS) on a Micromass (Wythenshawe, UK) ZMD mass spectrometer to
confirm the correct molecular weight. Fractions containing the correct molecular weight were
combined and lyophilized. Further purification was performed on a semi-preparative scale as
needed (HPLC chromatogram of all synthesized peptide shown in Supplementary Information,
Fig. S1 and Fig. S2). Peptides were quantified by their UV absorbance at 280 nm.
2
.4 Circular Dichroism
Far-UV CD measurements were performed with an AVIV 60DS spectrophotometer using a
.1 cm path length cuvette. Peptides were prepared at 150 μM concentration in 50 mM
0
phosphate-buffered saline (PBS) at pH 7.4. Spectra were recorded from 260 to 200 nm with a
step size of 1 nm, a bandwidth of 1.5 nm, and an averaging time of 8 s. An average of 16 scans
was obtained for each spectrum.
3
. Experiments
3
.1 Nitration of free tyrosine by PN under varied pH conditions
In a prefabricated 24-well PN-generating plate, three wells were prepared under three pH
conditions (0.01M phosphate buffer, pH 7.4, 7.0, and 6.5). To each well, stock solutions of
tyrosine, NaHCO and hypoxanthine were added to achieve a final concentration of 1 mM, 1.25
3
mM and 0.4 mM, respectively. Immediately following the addition of these stock solutions the
multi-well plate was illuminated with a low power broadband light source and allowed to mix on
a plate rocker for 30 min. Termination of the reaction was completed by simply removing the
reaction mixture from the plate and then transferring the analyte into a 1 mL centrifuge tube.
This solution was centrifuged down to ensure absence of free-floating particulates. Samples were
7

ACCEPTED MANUSCRIPT
then analyzed by electronic absorption spectroscopy (Cary 5000 Spectrophotometer) and ESI-
MS to confirm and quantitate 3-NT formation.
3
.2 Nitration of tyrosine by PN within a model peptide
In a prefabricated 24-well PN-generating platform, investigation into the nitration of a model
tyrosine-containing peptide (Y1, sequence AAAAAKKAAAYAAAKK) by PN was performed
at pH 7.4) by preparing three wells with 0.25 mM of the peptide, 0.4 mM HX, and 4 mM
(
NaHCO . The plate was allowed to undulate on a plate rocker for 30 min while being illuminated
3
by a low power broadband light source. The well content was then transferred to a 0.5 mL
Eppendorf tube, centrifuged, and then analyzed by electronic absorption spectroscopy and mass
spectrometry. Additionally, the effect of CO on this PN-dependent reaction was evaluated by
2
modulating the concentration of NaHCO (0 mM, 1.25 mM, and 4 mM) in wells prepared as
3
described above.
3
.3 Nitration of tyrosine by PN within Peptide Y1 in the presence of GSH
Similar to the experimental system described above, three wells were prepared with 0.25 mM
of the peptide, 0.4 mM HX, and 4 mM NaHCO and supplemented with glutathione (1 mM).
3
After 30 min of undulation and illumination, the reactions was terminated and evaluated as
discussed above.
3
.4 Modification of cysteine within a model peptide
In a prefabricated 24-well PN-generating platform, investigation on the modification of a
model cysteine-containing peptide (C1, sequence AAAAAKKAAACAAAKK) by PN was
performed by preparing three wells with 0.25 mM of the peptide, 0.4 mM HX, and 4 mM
NaHCO . Solutions were treated and evaluated as discussed above. Additionally, control wells
3
8

ACCEPTED MANUSCRIPT
were prepared where C1 was exposed to NO only (these wells excluded HX). Cysteine
nitrosylation was then quantified by electronic absorption spectroscopy (max= 336 nm, = 920
-1
-1
M cm ) and employed as a correction factor.
3
.5 Nitration of tyrosine by PN within a model peptide containing cysteine
Three peptides were designed with varied relative distance between a cysteine and tyrosine
residues:
CY1,
sequence
AAACAAAKKAAAYAAAKK;
CY2,
sequence
AAAKAAACKAAAYAAAKK; and CY3, sequence AAAKKAAACYAAAKK. In the
prefabricated 24-well PN-generating platform, the intramolecular effect of thiols on the nitration
of tyrosine in these peptides by PN was monitored. Three wells were prepared with 0.25 mM of
the peptide, 0.4 mM HX, and 4 mM NaHCO . Solutions were treated and evaluated as discussed
3
above. Additional control studies were completed with peptide CY1, where the peptide was

−
exposed to NO or O . All other previously described parameters and protocols were maintained
2

−
in these studies unless otherwise noted. Control studies on the effects of NO or O2 were
performed by removing the Mn(NO)SG or XO/CATSG disc from the wells.
The standard deviation values reported for the nitration studies described below (in well
volume of 500 l, pH 7.4) were calculated using results obtained from a minimum of three
independent measurements. The nitration of both free tyrosine and the tyrosine-containing
peptides where calculated based on the concentration of PN generated within the wells of the
platform.
4
. Results
4
.1 Effects of pH on tyrosine nitration
In order to establish the utility of our two-component PN-generating platform as an alternative
tool to study biochemical modifications induced by this RNS, we first attempted to monitor the
9

ACCEPTED MANUSCRIPT
effect of pH on the nitration of free tyrosine by PN. The formation and quantification of 3-NT
formed was first evaluated by electronic absorption spectroscopy (max = 370 nm). Standard
solutions of 3-NT were prepared to optimize the extinction coefficient of this species under the
-1
-1
defined experimental conditions and calculated to be 4015 M cm . As predicted, it was
observed that increased acidity of the well content from pH 7.4 to 7.0 to 6.5 resulted in a general
diminution of tyrosine nitration by PN (Table 1). Additionally, the collected analytes were
studied by mass spectrometry (ESI, positive ion mode), which corroborate 3-NT formation with
+
the presence of the corresponding peak at 227 m/z [M+H] . Much like our previously reported
studies, a peak corresponding to 3,3'-dityrosine (361 m/z) was also observed. However, the
relative abundance of 3-NT was greater than that of the dimer under the defined conditions.

−
Control studies were completed at pH 7.4 where the flux ratio of NO:O2 was changed from 1:1
to 1:2. This flux alterations was implemented by simply incorporating a Mn(NO) sol-gel piece
with one-half the nitrosyl compound loaded into the sol-gel matrix (7.25 mg) [31]. Such changes
resulted in a near elimination in tyrosine nitration (Figure S4).
Table 1 here
4
.2 Nitration of Peptide Y1 and effects of CO and GSH
2
To extend the biochemical complexity of the PN substrate within the prefabricated platform,
we designed and synthesized a model peptide that contained a single tyrosine residue (Y1)
amenable to nitration. Nitration of the single tyrosine residue within the random coiled
(confirmed by CD) peptide was first verified by electronic absorption spectroscopy, with a peak
centered at 370 nm indicating the formation of 3-NT (Fig. 2). Mass spectrometry confirmed the
1
0

ACCEPTED MANUSCRIPT
presence of this modification with a mass increase of 46 Da (−NO ) indicative of nitration of the
2
3+
model peptide ([M+3H] = 521 m/z).
Figure 2 here
Because PN-mediated nitration of tyrosine is dependent on CO , we modulated the NaHCO
2
3
concentration (source of CO ) within three PN- generating wells and observed alterations in the
2
extent of nitration of Y1. In line with other PN studies with peptides and proteins, 250 M of Y1
was employed in these studies. As anticipated, enhancement in nitration of the tyrosine residue
within Y1 was observed under increasing concentrations of CO , with a ~4.5-fold increase in
2
nitration being observed when the concentration of NaHCO was increased from 1.25 mM to 4.0
3
mM (Table 2).
Table 2 here
Next, to investigate the effects of antioxidants on the nitration of tyrosine within the designed
peptide we added the biologically relevant antioxidant thiol GSH into the PN-generating wells.
Incorporation of GSH (1 mM) did not inhibit tyrosine nitration within Y1 and instead resulted in
a slight (~1.1-fold, 39.2 ± 1.2%) enhancement. Analysis of the well content collected from this
experiment by mass spectrometry revealed the selective nitration of Y1, indicated by the
presence of a peak at 521 m/z. Although control studies performed in the absence of Y1 showed
a small peak at 353 m/z correlating to the nitration of GSH in addition to a moderately strong
+
peak at 360 m/z [M+Na] due to S-nitrosylated GSH, the presence of Y1 in the well resulted in
+
the peak at 360 m/z [M+Na] only (Fig. 3).
Figure 3 here
1
1

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4
.3 Thiol-containing peptides and the effect of thiol on tyrosine nitration
The results of the Y1 nitration study in the presence of GSH prompted us to design a series of
cysteine-containing peptides (C1, CY1, CY2, and CY3) to investigate the effect(s) of a thiol
residue on the nitration of a neighboring tyrosine within a peptide chain (Supplementary
Information, Fig. S3). Following exposure of the control peptide C1 to PN within the platform,
the well content was collected and first analyzed by electronic spectroscopy. A peak at 336 nm
was observed, corresponding to S-nitrosylation and/or S-nitration of the thiol residue. The
sample was then processed by mass spectrometry and a prominent peak corresponding to a mass
increase of 30 Da (−NO) confirmed the formation of the S-nitrosylated product.
Investigation into the intramolecular effects of a thiol on neighboring tyrosine residue nitration
was completed using the model peptides CY1, CY2, and CY3, where the cysteine and tyrosine
residues were separated by varied number of amino acids within the primary sequence (Table 3).
When the cysteine and tyrosine residues were separated by eight (CY1) or four (CY2) non-
reactive amino acids, a slight enhancement in tyrosine nitration was observed. However,
placement of these residues directly adjacent to each other resulted in a dramatic attenuation of
tyrosine nitration based on the absorbance at 370 nm corresponding to 3-NT formation within the
respective peptides (Table 3).
Table 3 here
The mass spectrum of the analyte collected from these wells showed formation of distinct
products. In case of CY1 and CY2 peptides, predominant peaks corresponding to mass additions
of 30 Da and 46 Da were present, corresponding to respective S-nitrosylation and tyrosine-
nitration of the peptides. A strong electronic absorption peak at 370 nm confirmed that the extent
of tyrosine nitration was much higher than that of S-nitrosylation of the cysteine residue.
1
2

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Interestingly, the mass spectrum of the content of the well where CY3 was exposed to PN
showed mass additions of 16 Da, 30 Da, and 46 Da corresponding to oxidation, S-nitrosylation
and S-nitration of the peptide substrate (Fig. 4). The nitrosylation/nitration of the cysteine
residue was indicated by a strong absorption band at 330 nm. In addition, the absorption at 370
nm arising from nitration of tyrosine was diminished significantly compared to those observed
with CY1 and CY2.
Figure 4 here

−
Finally, in order to verify that both NO and O2 were required to promote tyrosine nitration,
the peptide CY1 was allowed to react with each species individually. When CY1 was exposed to
NO, a peak at 330 nm in the absorbance spectrum corresponding to S-nitrosylation was
observed. In contrast, no absorption in the 300-600 nm region was noted when this peptide was

−
exposed to O2 (Fig. 5). No absorption corresponding to 3-NT (370 nm) was observed in both
cases.
Figure 5 here
5
. Discussion
In this work we have employed a light-activated biomimetic PN-generating platform [31] to
study the chemistry of tyrosine nitration mediated by PN. This platform releases sustained fluxes

−
of NO, O , and PN upon exposure to light and addition hypoxanthine. It also allows studies
2
with PN under varied pH conditions. With alternative sources such as authentic PN solutions
and SIN-1, alkaline conditions are often required to study the reactions of PN. When the strongly
alkaline authentic PN solutions are added to buffered media, rapid decomposition of PN results

−
in high concentrations of secondary radical species such as NO and CO . However, such rapid
2
3
1
3

ACCEPTED MANUSCRIPT
burst of radicals fails to take into consideration the formation of pathological concentration of

−
PN from rising levels of NO and O2 in biological milieu [1,34]. Because changes in fluxes of
these precursors can lead to very different product formations [31], we have utilized our two-
component platform that generates a sustained flux of PN in a biomimetic fashion to explore the
reactions of PN with tyrosine and tyrosine-containing peptides under different pH conditions,
bicarbonate concentrations, and in the presence of thiols.
The biomimetic generation of PN from the two precursors in the wells of our multi-well
platform is quite effective. Exposure of 1 mM of tyrosine to the ~4 M per min flux of PN,
generated for 30 min, afforded 46.5 M of 3-NT (38.5 ± 0.4% nitration based on PN
concentration). This yield is particularly notable when compared to ~1% tyrosine nitration
obtained with 1 mM of SIN-1 (and comparable CO concentration) after 12 h as reported by
2
Mayer and coworkers [23]. Further, when a comparative study was performed where 1 mM
tyrosine was exposed to 500 M SIN-1 for 30 min in the presence of 1.25 mM NaHCO3
(comparable to the free tyrosine study completed within the wells of the platform), the resulting
absorbance spectrum lacked an absorption peak corresponding to 3-NT formation. It is important
to note that the presence of urate in our wells does not appear to significantly attenuate the extent
of tyrosine nitration. Presumably, both the steady and high concentration of PN and the slow
reaction rate of PN with urate [2] give rise to this advantage in the present platform. Also,

−
sustained release of NO, O , and PN within the wells avoids the use of rapid stirring to assure
2
adequate exposure of a substrate to the RNS, as is required in studies where authentic PN is
employed. The present PN-generating plate requires only undulation to promote uniformity
throughout the wells. Further, because the well volume and respective sol-gel units can be easily

−
adjusted to release desired concentrations of NO and O , our platform can release much higher
2
1
4

ACCEPTED MANUSCRIPT
fluxes of PN compared to other two-component systems reported so far [23,25]. The metal
nitrosyl employed in this platform releases NO significantly faster than alternative sources [23],

−
and can match the relatively rapid release rate of O2 from the xanthine oxidase system. When

−
the flux ratio within the wells was adjusted to 1:2 (NO to O ), the absorption band at 370 nm
2
corresponding to 3-NT formation was absent in the electronic spectrum. This observation was

−
expected because it has been established that when O2 is in excess of NO, it reacts rapidly with

−
NO to form peroxynitrate (O NOO ) [35]. This anion rapidly decomposes to yield nitrite and
2
2
molecular oxygen, thus inhibiting nitration of tyrosine. This control study effectively
demonstrates the effectiveness of the platform to mimic in vivo scenarios where the flux of NO

−
and O2 can vary.
Because many NO donors are highly pH sensitive and only release optimal fluxes of NO
under strict conditions, their use is restricted to limited pH ranges [36]. This feature impedes the
utility of co-generative systems to study PN-dependent reactions under varied pH conditions. To
circumvent this complication flow reactors have been employed where one or both of the PN
precursors are flooded into a reaction vessel and a respective substrate is exposed to rapid bursts
of saturated solutions of NO gas and/or alkaline KO solutions [37]. In contrast, the light-
2
controlled PN-generating platform employed in the present study allowed us to study the effects
of pH on tyrosine nitration by PN without the use of flow reactor. When tyrosine was exposed to
PN within the prefabricated wells of the platform under varied pH conditions, diminution in 3-
NT formation was observed with increased acidity of the well milieu (pH = 7.4, 7.0, and 6.5,
Table 1). In general, the process of tyrosine nitration is mediated by peroxynitrous acid
−
(
ONOOH) and nitrosoperoxocarbonate anion (ONOOCO ) [1,2]. At physiological pH, PN is
2
−
−
stabilized in its anionic form (ONOO ) and reacts with CO to form ONOOCO [6,37].
2
2
1
5

ACCEPTED MANUSCRIPT
However, as the pH drops (characteristic of local sites of inflammation) towards the pK of PN
a
−
(6.8), the protonated form ONOOH predominates and inhibits the formation of ONOOCO2

(
Scheme 1) [2]. While both species are capable of inducing tyrosyl radical (TyrO ) formation to

−
initiate the generation of 3-NT, superior specificity of strong oxidant carbonate radical (CO )
3
−

generated from the homolytic cleavage of ONOOCO (compared to the hydroxyl radical (OH )
2
generated from ONOOH) accounts for diminished nitration of tyrosine within the wells.
To further explore the nitration of tyrosine by PN within a peptide frame we designed a model
peptide Y1 with a single tyrosine residue and examined the extent of its nitration relative to
nitration of free tyrosine. Exposure of Y1 to ~4 M per min flux of PN (under identical CO2
concentration and pH value as in the case of free tyrosine) promoted very similar extent of
nitration comparable to free tyrosine (percent nitration normalized to account for the
concentration difference between free tyrosine and Y1). As discussed above, the presence and
concentration of CO mediates PN-dependent nitration through the formation of intermediate
2
−
−
3

9 -1
ONOOCO that decomposes (homolytic) to yield CO and NO at a rate of ~10 s (Scheme
2
2
1
) [38]. Indeed, alteration of CO concentration within the wells of our platform resulted in
2
significant modulation of 3-NT formation with Y1 as the substrate. With a CO concentration of
2
~
100M, a modest increase in nitration was observed compared to wells where no NaHCO was
3
incorporated (Table 2). However, when the concentration of CO was approximately tripled, a
2
−
significant increase in nitration was observed (~4.5 fold). These results verify ONOOCO -
2
mediated nitration (characteristic of PN chemistry) of Y1 within the wells of our platform.
Scheme 1 here
1
6

ACCEPTED MANUSCRIPT
Following the successful nitration of tyrosine-containing peptide Y1 under tightly controlled
conditions, we sought out to observe the effect(s) of highly abundant antioxidant GSH on this
PN-dependent nitration process. The addition of physiological concentrations of GSH to the
wells of the platform where Y1 was exposed to PN resulted in a slight enhancement of nitration.
We assign this modest acceleration to the extended lifetime of NO in the presence of GSH within
the wells of our platform. The photorelease of NO from the Mn(NO)SG piece is fast compared

−
to the initial release of O2 from the XO/CATSG disk that requires diffusion of HX through the
sol-gel matrix in the larger well. Therefore in the first minute or so of the experiment when the
release rates are slightly unmatched, NO chemistry predominates and results in the nitrosylation
of GSH [39,40]. The resulting S-nitrosylated glutathione product (S-nitrosoglutathione, GSNO)
acts as a NO storage in the platform (much like in the body), extending the effective
concentration of this precursor within the wells. This hypothesis is supported by mass spectral
results of the well content where a peak corresponding to GSNO was observed (Fig. 3).
Extension of the lifetime of NO in the wells by this route allows for a higher concentration of PN
at the later part of the reaction and results in the moderately higher yield of nitrated product
under such conditions.
The observed enhancement in nitration of Y1 in the presence of thiol GSH (an intermolecular
effect) prompted us to design a series of peptides C1, CY1, CY2 and CY3 to study
intramolecular effects of thiols on the nitration of tyrosine within our platform. It has been
proposed that the relative proximity of thiols and tyrosine residues within peptide and proteins
can influence the extent of tyrosine nitration by PN and contribute to the general selectivity of
this dynamic process through an intramolecular radical transfer pathway [41,42]. The presence of
a cysteine residue spatially adjacent to a tyrosyl can act as a "radical sink," inhibiting nitration
1
7

ACCEPTED MANUSCRIPT

process of the tyrosyl radical by NO and enhancing oxidation and S-nitrosylation of the thiol
2
residue [43-46]. Interestingly, the sequence of radical-mediated modifications was indeed
observed within the wells of the platform where CY3 was exposed to PN. The extent of nitration
at the tyrosine residue within CY3, where a cysteine and tyrosine residue were placed directly
adjacent to each other, was significantly attenuated compared to the nitration observed in
peptides Y1, CY1, and CY2 (Table 3).
Mass spectral data collected from the CY3 study contained peaks corresponding to 16 Da
(
−O), 30 Da (−NO), and 46 Da (−NO ) mass additions to the parent peptide (Fig. 4). While the
2
3
0 Da and 46 Da mass additions (representing nitrosylation and nitration respectively) were
observed in all three peptide studies, the relative intensity of the peaks varied. In the mass spectra
of CY1 and CY2, the 46 Da mass addition was the predominant peak, whereas in CY3, the 30
Da mass addition was observed to be significantly greater than the nitration peak. The electronic
spectrum collected of the well content from the CY3 study also showed a significant diminution
of the absorbance band corresponding to the nitrated tyrosine (
= 370 nm), indicating that
the 46 Da mass addition observed in the mass spectrum was largely a result of S-nitration of the
cysteine residue within the peptide. The mass addition of 16 Da to the CY3 peptide is a unique
product that was not observed in CY1 and CY2 studies and likely corresponds to the oxidation
of the thiol group to a sulfenic acid (−SOH). It is important to note here that the extent of
nitration within peptide CY1 and CY2 (where the cysteine and tyrosine residues were 4 or 8
residues apart) were not significantly different (Table 3). Furthermore, the relative nitration of
CY1 and CY2 was similar to that observed when Y1 peptide was exposed to PN in the presence
of GSH (Table 3). Taken together, the distinct products induced within the CY3 peptide and the
1
8

ACCEPTED MANUSCRIPT
dramatic attenuation in the extent of tyrosine nitration supports the intramolecular radical
transfer process observed in other peptide and protein studies [47,48].
6
. Conclusion
The present work demonstrates the utility of the two-component PN-generating platform as a
convenient tool to study the reactions of PN with a wide range of substrates under controlled
conditions including changes in pH. Previously, poor nitration yields induced in situ generated
PN compared to yields generated upon exposure to authentic PN has been rationalized by Ingold
and coworkers to be a result of varied rates of radical formations by the respective sources [34].
A rapid rate of NO photorelease from Mn(NO)SG and careful matching of the flux ratio of NO

−
and O2 make this platform a valuable tool to study the effects of continuous exposure of a
substrate to PN. The efficient nitration of tyrosine and tyrosine-containing peptides by
physiologically relevant concentration of PN within the wells provide strong evidence in favor of
the utility of this platform in future studies on PN chemistry.
Abbreviations

−
CO3
carbonate radical
ESI-MS
GSH
electron spray ionization mass spectrometry
glutathione
GSNO
GSNO2
HOBt
HX
S-nitrosoglutathione
S-nitrated glutathione
hydroxybenzotriazole
hypoxanthine;
(Mn(NO)
[Mn(Papy )(NO)]ClO
3
4
Mn(NO)SG Mn(NO)-encapsulated sol-gel;
−
ONOOCO2
nitrosoperoxocarbonate anion;
1
9

ACCEPTED MANUSCRIPT
−
PN, ONOO
ONOOH
PBS
peroxynitrite;
peroxynitrous acid;
phosphate buffer saline;
superoxide

−
O2
TyrO

tyrosyl radical;
XO/CATSG xanthine oxidase/catalase-containing sol gel;
-NT 3-nitrotyrosine.
3
Acknowledgements
This research was supported by a grant from the NSF (DMR-1105296). TdBM, RIP, and SOI
acknowledge support from the IMSD grant GM-58903.
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2
3

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FIGURE LEGENDS

−
Fig. 1. The designed platform comprised of a O -generating sol-gel layer at the bottom of a
2
well and NO-releasing sol-gel disc on top. It releases PN upon addition of HX and exposure of
the plate to low power light.
Fig. 2. (Left) Mass spectrum of the well content collected after Y1 was exposed to PN for 30
3
+
3+
min. The two peaks highlighted in red correlate to the [M+3H] and [M+Na] of the nitrated
3
+
Y1 peptide. Peak at 506 m/z corresponds to the [M+3H] peak of the unmodified parent peptide.
3
+
The peak at 514 m/z represents [M+Na] of the parent peptide Y1. (Right) Electronic
absorption spectrum of the well content. The absorbance band (max= 370 nm) highlighted in red
corresponds to the absorption band of the nitrated tyrosine residue within Y1.
Fig. 3. Partial mass spectrum collected from the well content where Y1 was exposed to PN in
the presence of GSH, highlighting the region with peaks corresponding to GSH (307 m/z,
+
+
+
[
M+H] , 330 m/z, [M+Na] ) and its nitrosylation adduct GSNO (360 m/z, [M+Na] ).
2
4

ACCEPTED MANUSCRIPT
Fig. 4. (Left) Mass spectrum of the well content collected after CY3 was exposed to PN for 30
min. The mass spectral data showed mass additions of 16 Da, 30 Da, and 46 Da corresponding to
possible peaks 745 m/z (green), 749 m/z (blue), and 755 m/z (red), respectively. The second red-
labeled peak at 762 m/z corresponds to the addition of a nitro group and pairing of the resulting
CY3 peptide with Na. (Right) Structures representative of the potential products associated with
the observed additions in the mass spectrum.
Fig. 5. Electronic absorption spectrum of CY1 peptide in the presence of NO (blue dashed line),

−
O2 (green dotted line) and PN (red solid line)
Scheme 1. The reaction and decomposition rates of critical primary and secondary
radicals mediated by PN
2
5

ACCEPTED MANUSCRIPT
FIGURE 1
FIGURE 2
506
5
14
0
0
0
0
0
.4
.3
.2
.1
.0
5
29
5
21
500
520
540
560
300
350
400
450
500
m/z
Wavelength (nm)
2
6

ACCEPTED MANUSCRIPT
FIGURE 3
330
3
07
360
3
00
320
340
360
m/z
FIGURE 4
CY3
M+3H]
OH
OH
3
+
[
O
H
7
39
2
SNO
CH2
S
3
+
[
M+3H]
CH2
CH2
CH2
7
49
H
H
H
H
H
N
H
N CH
C
O
N
C
C N
O
N CH
C
O
C
H
C N
O
H
1
1
2
3
+
3+
[
M+3H]
[M+Na]
62
OH
OH
7
NO2
O2N
7
45
3
S
SH
3
+
[
M+3H]
CH2
CH2
CH2
CH2
H
H
H
H
H
H
7
55
N CH
C
O
N
C
C N
O
N CH
C
N
C
C N
H
H
O
O
3
7
40
760
m/z
2
7

ACCEPTED MANUSCRIPT
FIGURE 5.
SCHEME 1
4
-1 -1
k= 4.6 x 10 M s
k= 6.5 x 108 s-1
k= 0.63 s-1
[37]
ONOO
CO2
ONOOCO2
[
21]
ONOOCO2
ONOOH
CO3
NO3
NO2
[
37]
H
k= 0.27 s-1 [37]
OH
NO2
2
8

ACCEPTED MANUSCRIPT
Pictogram
Synopsis for Graphical Abstract
Highly efficient nitration of tyrosine in model peptides has been achieved under varied pH, CO2
and thiol concentrations within the wells of a peroxynitrite-generating platform controlled by
light. Placement of a neighboring cysteine residue strongly attenuates the effects of peroxynitrite
on tyrosine.
2
9