Introduction
Primary biological aerosols, or bioaerosols, including proteins, from
different sources and with distinct properties are known to influence
atmospheric cloud microphysics and public health (Lang-Yona et al., 2016;
D'Amato et al., 2007; Pummer et al., 2015). Bioaerosols represent a diverse
subset of atmospheric particulate matter that is directly emitted in form of
active or dead organisms, or fragments, like bacteria, fungal spores,
pollens, viruses and plant debris. Proteins are found ubiquitously in the
atmosphere as part of these airborne, typically coarse-sized biological
particles (diameter >2.5 µm), as well as in fine particulate
matter (diameter <2.5 µm) associated with a host of different
constituents such as polymers derived from biomaterials and proteins
dissolved in hydrometeors, mixed with fine dust and other particles (Miguel
et al., 1999; Riediker et al., 2000; Zhang and Anastasio, 2003). Proteins
contribute up to 5 % of particle mass in airborne particles (Franze
et al., 2003a; Staton et al., 2015; Menetrez et al., 2007) and are also found
at surfaces of soils and plants. Proteins can be nitrated and are then likely
to enhance allergic responses (Gruijthuijsen et al., 2006). Nitrogen dioxide
(⚫NO2) has emerged as an important biological reactant and
has been shown to be capable of electron (or H atom) abstraction from the
amino acid tyrosine (Tyr) to form TyrO⚫ in aqueous solutions
(tyrosine phenoxyl radical, also called tyrosyl radical; Prütz
et al., 1984, 1985; Alfassi, 1987; Houée-Lévin et al., 2015), which
subsequently can be nitrated by a second NO2 molecule. Shiraiwa
et al. (2012) observed nitration of protein aerosol, but not solely with
NO2 in the gas phase, and demonstrated that simultaneous O3
exposure of airborne proteins in dark conditions can significantly enhance
NO2 uptake and consequent protein nitration (3-nitrotyrosine
formation) by way of direct O3 mediated formation of the
TyrO⚫ intermediate. A connection between increased allergic
diseases and elevated environmental pollution, especially traffic-related air
pollution has been proposed (Ring et al., 2001). Tyrosine is one of the
photosensitive amino acids and it is subject of direct and indirect
photo-degradation under solar-simulated conditions (Boreen et al., 2008),
especially mediated by both UV-B (λ 280–320 nm) and
UV-A (λ 320–400 nm) radiation (Houee-Levin et al., 2015;
Bensasson et al., 1993). Direct light absorption or absorption by adjacent
endogenous or exogenous chromophores and subsequent energy transfer results
in an electronically excited state of tyrosine (for details see
Houée-Lévin et al., 2015, and references therein). If the triplet
state of tyrosine is generated, it can undergo electron transfer reactions
and deprotonation to yield TyrO⚫ (Fig. 1; Bensasson, 1993;
Davies, 1991; Berto et al., 2016). Regardless of how the tyrosyl radical is
generated, it can be nitrated by reaction with NO2, as well as
hydroxylated or dimerized (Shiraiwa et al., 2012; Reinmuth-Selzle et al.,
2014; Kampf et al., 2015).
With respect to atmospheric chemistry, Bejan et al. (2006) have shown that
photolysis of ortho-nitrophenols (as is the case for 3-nitrotyrosine) can
generate nitrous acid (HONO). HONO is of great interest for atmospheric
composition, as its photolysis forms OH radicals, which are the key oxidant for
degradation of most air pollutants in the troposphere (Levy, 1971). In the
lower atmosphere, up to 30 % of the primary OH radical production can be
attributed to photolysis of HONO, especially during the early morning when
other photochemical OH sources are still small (Reaction R1, Kleffmann
et al., 2005; Alicke et al., 2002; Ren et al., 2006; Su et al., 2008; Meusel
et al., 2016).
HONO⟶hvOH+NO(hν=300–405nm)
HONO can be directly emitted by combustion of fossil fuels (Kurtenbach
et al., 2001) or formed by gas-phase reactions of NO and OH (the backwards
reaction of Reaction R1) and heterogeneous reactions of NO2 on wet
surfaces according to Reaction (R2). On carbonaceous surfaces (soot, phenolic
compounds) HONO is formed via electron or H transfer reactions (Reactions R3
and R4–R6; Kalberer et al., 1999; Kleffmann et al., 1999; Gutzwiller et al.,
2002; Aubin and Abbatt, 2007; Han et al., 2013; Arens et al., 2001, 2002;
Ammann et al., 1998, 2005).
2NO2+H2O→HONO+HNO3NO2+{C-H}red→HONO+{C}oxArOH+NO2→ArO⚫+HONOArOH+H2O→ArO-+H3O+ArO-+NO2→NO2-+ArO⚫⟶H3O+HONO+H2O
Previous atmospheric measurements and modeling studies have shown unexpected
high HONO concentrations during daytime, which can also contribute to aerosol
formation through enhanced oxidation of precursor gases (Elshorbany et al.,
2014). Measured mixing ratios are typically about 1 order of magnitude
higher than simulated ones, and an additional source of
200–800 ppth-1 would be required to explain observed mixing
ratios (Kleffmann et al., 2005; Acker et al., 2006; Sörgel et al., 2011;
Li et al., 2012; Su et al., 2008; Elshorbany et al., 2012; Meusel et al.,
2016), indicating that estimates of daytime HONO sources are still under
debate. It was suggested that HONO arises from the photolysis of nitric acid
and nitrate or by heterogeneous photochemistry of NO2 on organic
substrates and soot (Zhou et al., 2001, 2002 and 2003; Villena et al., 2011;
Ramazan et al., 2004; George et al., 2005; Sosedova et al., 2011; Monge
et al., 2010; Han et al., 2016). Stemmler et al. (2006, 2007) found HONO
formation on light-activated humic acid, and field studies showed that HONO
formation correlates with aerosol surface area, NO2 and solar
radiation (Su et al., 2008; Reisinger, 2000; Costabile et al., 2010; Wong
et al., 2012; Sörgel et al., 2015) and is increased during foggy periods
(Notholt et al., 1992). Another proposed source of HONO is the soil, where it
has been found to be co-emitted with NO by soil biological activities (Oswald
et al., 2013; Su et al., 2011; Weber et al., 2015).
In view of light-induced nitration of proteins and HONO formation by
photolysis of nitrophenols, light-enhanced production of HONO on protein
surfaces can be anticipated, which, to the best of our knowledge, has not
been studied before.
This work aims to provide insight into protein nitration, the atmospheric
stability of the nitrated protein and respective formation of HONO from
protein surfaces that were nitrated either offline in liquid phase prior to
the gas exchange measurements or online with instantaneous gas-phase
exposure to NO2, with particular emphasis on environmental parameters
like light intensity, relative humidity (RH) and NO2 concentrations.
Bovine serum albumin (BSA), a globular protein with a molecular mass of
66.5 kDa and 21 tyrosine residues per molecule, was chosen as
a well-defined model substance for proteins. Nitrated ovalbumin (OVA) was
used to study the light-induced degradation of proteins that were nitrated
prior to gas exchange measurements. This well-studied protein has a molecular
mass of 45 kDa and 10 tyrosine residues per molecule.
Materials and methods
Protein preparation and analysis
BSA (Cohn V fraction, lyophilized powder, ≥96 %; Sigma Aldrich, St. Louis, Missouri, USA) or nitrated OVA
was solved in pure water (18.2 MΩcm) and coated
onto the glass tube.
The nitration of OVA was described previously (Yang et al., 2010;
Zhang et al., 2011). Briefly, OVA (grade V, A5503-5G, Sigma Aldrich, Germany)
was dissolved in phosphate-buffered saline PBS (P4417-50TAB, Sigma Aldrich,
Germany) to a concentration of 10 mgml-1. 50 µL
tetranitromethane (TNM; T25003-5G, Sigma Aldrich, Germany) dissolved in
methanol 4 % (v/v) were added to a 2.5 mL aliquot of the OVA
solution and stirred for 180 min at room temperature. Please note
that TNM is toxic if swallowed, can cause skin, eye and respiration
irritation, is suspected to cause cancer and causes fires or explosions. Size
exclusion chromatography columns (PD-10 Sephadex G-25 M, 17-0851-01, GE
Healthcare, Germany) were used for cleanup. The eluate was dried in a freeze
dryer and stored in a refrigerator at 4 ∘C.
After the flow-tube experiments (see below) the proteins were extracted with
water from the tube and analyzed with liquid chromatography (HPLC-DAD;
Agilent Technologies 1200 series) according to Selzle et al. (2013). This
method provides a straightforward and efficient way to determine the
nitration of proteins. Briefly, a monomerically bound C18 column (Vydac
238TP, 250mm×2.1mm inner diameter,
5 µm particle size; Grace Vydac, Alltech) was used for
chromatographic separation. Eluents were 0.1 % (v/v) trifluoroacetic
acid in water (LiChrosolv) (eluent A) and acetonitrile (ROTISOLV HPLC
gradient grade, Carl Roth GmbH + Co. KG, Germany) (eluent B). Gradient
elution was performed at a flow rate of 200 µLmin-1.
ChemStation software (Rev. B.03.01, Agilent) was used for system control and
data analysis. For each chromatographic run, the solvent gradient started at
3 % B followed by a linear gradient to 90 % B within 15 min,
flushing back to 3 % B within 0.2 min and maintaining 3 % B
for additional 2.8 min. Column re-equilibration time was
5 min before the next run. Absorbance was monitored at wavelengths of
280 (tyrosine) and 357 nm (nitrotyrosine). The sample injection
volume was 10–30 µL. Each chromatographic run was repeated three
times. The protein nitration degree (ND), which is defined as the ratio of
nitrated tyrosine to all tyrosine residues, was determined by the method of
Selzle et al. (2013). Native and untreated BSA did not show any degree of
nitration.
Details on the different experiments, aims and experimental
conditions (coating, applied NO2 concentration, number of lights
switched on, relative humidity and time for each exposure step).
Coating density (number of
NO2
No. of lamps
RH
Time per step
monolayers NMLf, thickness)
(ppb)
(%)
(h)
(a) Light-induced decomposition of nitrated protein and HONO formation
1
Light and NO2
n-OVA 21.5±0.8 µgcm-2
0–20
0–1–3–7 VIS
50
1
dependency
(68 NMLf, 298.05 nm)
(b) Heterogeneous NO2 transformation on BSA
2
NO2 dependency
BSA 16.1±0.4 µgcm-2
0–20–40–60–100
7 VIS
50
0.5–1
(50 NMLf, 217.6 nm)
3
Light dependency
BSA 31.4±1.4 µgcm-2
20
0–1–3–7 VIS
50
0.5–1
(99 NMLf, 435.2 nm)
4
Coating thickness
BSA 16.1±0.4 µgcm-2
20
7 VIS
0.5–3
(50 NMLf, 217.6 nm),
22.5±0.8 µgcm-2
(71 NMLf, 310.8 nm),
31.4±1.4 µgcm-2
(99 NMLf, 435.2 nm)
5
RH dependency
BSA 17.5±0.4 µgcm-2
25
0–7 VIS
0–50–80
0.25–1
(55 NMLf, 241.7 nm)
6
Time effect
BSA 17.5±0.4 µgcm-2
100
7 VIS
75
20
7
Time effect
BSA 17.5±0.4 µgcm-2
100
4 VIS + 3 UV
75
20
NMLf numbers of monolayers in flat orientation.
Flow system and setup: thin blue lines show the flow of the gas
mixture, which direction is indicated by the grey triangles of the mass flow
controllers (MFC). Nitrogen passes a heated water bath to humidify the gas
and a HONO scrubber to eliminate any HONO impurities of the NO2
supply. The overflow maintains a constant pressure through the reaction tube
and the detection unit. The dotted boxes (blue, green, orange) indicate the
three different parts: the gas supply, reaction unit and detection
unit.
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Coated-wall flow tube system
Figure 2 shows a flowchart of the setup of the experiment. NO2 was
provided in a gas bottle (1 ppm in N2, Carbagas AG,
Grümligen, Switzerland). NO2 was further diluted (mass flow
controller, MFC3) with humidified pure nitrogen to achieve NO2 mixing
ratios between 20 and 100 ppb. Impurities of HONO in the
NO2-gas cylinder were removed by means of a HONO scrubber. The
Na2CO3 trap was prepared by soaking 4 mm firebrick in
a saturated Na2CO3 in 50 % ethanol–water solution and drying
for 24 h. The impregnated firebrick granules were put into
a 0.8 cm inner diameter and 15 cm long glass tube, which was
closed by quartz wool plugs on both sides. A constant total flow
(1400 mLmin-1) was provided by means of another N2 mass
flow controller (MFC2) that compensated for changes in NO2 addition.
Different fractions of total surface areas (50, 70 and 100 %) of the
reaction tube (50cm×0.81cm i.d.) were coated with
2 mg BSA or nitrated OVA, respectively. Therefore 2 mg
protein was dissolved in 600 µL pure water, injected into the
tube and then gently dried in a low-humidity N2 flow (RH ∼30–40 %) with continuous rotation of the tube. The coated reaction tube
was exposed to the generated gas mixture and irradiated with either (i) one,
three
or seven visible (VIS) lights (400–700 nm; L 15 W/954, Lumilux de Luxe
daylight, Osram, Augsburg, Germany), which is 0, 23, 69 or
161 Wm-2, respectively; or (ii) four VIS and three UV lights
(340–400 nm; UV-A, TL-D 15 W/10, Philips, Hamburg, Germany).
An overview of the experiments performed during this study is shown in
Table 1. Light-induced decomposition of nitrated proteins was studied on OVA.
Instantaneous NO2 transformation and its light and RH dependence on
heterogeneous HONO formation were studied on BSA in short-term experiments.
Extended studies on BSA were performed to explore the persistence of the
surface reactivity and respective catalytic effects.
A commercial long-path absorption photometry instrument (LOPAP, QUMA) was
used for HONO analysis. The measurement technique was introduced by Heland
et al. (2001). This wet chemical analytical method has an unmatched low
detection limit of 3–5 ppt with high HONO collection efficiency
(≥ 99 %). HONO is continuously trapped in a stripping coil flushed
with an acidic solution of sulfanilamide. In a second reaction with
n-(1-naphthyl)ethylenediamine-dihydrochloride an azo dye is formed, whose
concentration is determined by absorption photometry in a long Teflon tubing.
LOPAP has two stripping coils in series to reduce known interferences. In the
first stripping coil HONO is quantitatively collected. Due to the acidic
stripping solution, interfering species are collected less efficiently but in
both channels. The true concentration of HONO is obtained by subtracting the
interferences quantified in the second channel from the total signal obtained
in the first channel. The accuracy of the HONO measurements was 10 %,
based on the uncertainties of liquid and gas flow, concentration of
calibration standard and regression of calibration.
The reagents were all high-purity-grade chemicals, i.e., hydrochloric acid
(37 %, ACS reagent, Sigma Aldrich, St. Louis, Missouri, USA),
sulfanilamide (for analysis, >99 %; Sigma Aldrich) and
N-(1-naphthyl)-ethylenediamine dihydrochloride (> 98 %; ACS reagent,
Fluka by Sigma Aldrich). For calibration Titrisol®
1000 mg NO2- (NaNO2 in H2O; Merck) was
diluted to 0.001 mgL-1 NO2-. For preparation of all
solutions and for cleaning of the absorption tubes 18 MΩ
H2O was used.
NOx concentrations were analyzed by means of a commercial
chemiluminescence detector from EcoPhysics (CLD 77 AM, Duernten,
Switzerland).
Results and discussion
BSA nitration and degradation
Nitrated proteins can trigger allergic response. The nitration of proteins
can be enhanced by O3 activation (in the dark). In the atmospheric
environment, about half the time sunlight is present. What happens with
irradiated proteins when exposed to NO2? Can they be nitrated
efficiently? To investigate the degree of protein nitration under illuminated
conditions, BSA coated on the reaction tube (17.5 µgcm-2)
was exposed to seven VIS lamps (40 % of a clear-sky irradiance for a solar
zenith of 48∘; Stemmler et al., 2006) and 100 ppb NO2
at 70 % RH. After 20 h the BSA ND (concentration of nitrated tyrosine residues divided by the total
concentration of tyrosine residues) investigated by means of the HPLC-DAD
method was (1.0±0.1) %, significantly higher than the ND of
untreated BSA (0 %). Introducing UV radiation (four VIS plus three UV lamps)
resulted in a slightly higher ND of (1.1±0.1) %. Note that no
intact protein (nitrated and non-nitrated) could be detected by HPLC-DAD
after another 20 h of irradiation without NO2, indicating
light-induced decomposition of proteins. However, the applied HPLC-DAD
technique only detects (nitro-)tyrosine residues in proteins and does not
provide information about protein fragments or single nitrated or
non-nitrated tyrosine residues. Hence, proteins might have been decomposed
while tyrosine remains in its nitrated form, not detectable by our analysis
method. Similarly, proteins (here OVA) that were nitrated with TNM in
aqueous phase prior to coating (21.5 µgcm-2) to an extent of
12.5 % also decomposed when illuminated about 6 h (one to seven VIS
lights; with and without 20 ppb NO2). Thus the nitration of
proteins by light and NO2 was confirmed, but with simultaneous
gradual decomposition of the proteins. Effects of UV irradiation
(240–340 nm) on proteins containing aromatic amino acids were
reviewed previously (Neves-Peterson et al., 2012). It was shown that triplet
state tryptophan and tyrosine can transfer electron to a nearby disulfide
bridge to form the tryptophan and tyrosine radical. The disulfide bridge
could break leading to conformational changes in the protein but not
necessarily resulting in inactivation of the protein. In strong UV light
(≈200 nm) the peptide bond could also break (Nikogosyan and
Görner, 1999).
Franze et al. (2005) analyzed a variety of natural samples (road dust, window
dust and particulate matter PM2.5) collected in the metropolitan
area of Munich, containing 0.08–21 gkg-1 proteins, and revealed
equivalent degrees of nitration (EDN, concentration of nitrated protein
divided by concentration of all proteins) between 0.01 and 0.1 % only.
Such low nitration degree is in line with light-induced decomposition of
(nitrated) proteins. In contrast, an EDN up to 10 % (average
5 %) was found for BSA and birch pollen extract exposed to Munich
ambient air for 2 weeks under dark conditions, with daily mean NO2
(O3) concentration of 17–50 ppb (7–43 ppb) in the
same study, possibly suggesting the deficiency of decomposition without being
irradiated. BSA and OVA loaded on syringe filters and exposed to
200 ppb NO2 / O3 for 6 days under dark conditions
were nitrated to 6 and 8 %, respectively (Yang et al., 2010).
Reinmuth-Selzle et al. (2014) found similar ND for major birch pollen
allergen Bet v 1 loaded on syringe filters exposed to 80–470 ppb
NO2 and O3. When exposed for 3–72 h to
NO2 / O3 at RH < 92 % the ND was 2–4 %,
while at condensing conditions (RH >98 %) the ND increased to 6 %
after less than 1 day (19 h). The ND of Bet v 1 was considerably
increased to 22 % for proteins solved in the aqueous phase
(0.16 mgmL-1) when bubbling with a 120 ppb
NO2 / O3 gas mixture for a similar period of time
(17 h). Shiraiwa et al. (2012) performed kinetic modeling and found
that maximum 30 % (conservative upper limit) of N uptake on BSA could be
explained by NO3 or N2O5, which are generated by the
reaction of NO2 and O3, while overall nitration was
governed by an indirect mechanism in which a radical intermediate was formed
by the reaction of BSA with ozone, which then reacted with NO2. On
NaCl surface N uptake was dominated by NO3 and N2O5.
Furthermore, NO3 radicals, which in this study could be formed by
photolysis of NO2 (> 410 nm, disproportionation of excited
NO2), are not stable under the light conditions applied
(400–700 nm) (Johnston et al., 1996). Therefore, in the present
study reactions with NO3 were neglected. Photolysis of NO2
forming NO (< 400 nm) can also be neglected (Gardner et al., 1987;
Roehl et al., 1994). A photolysis frequency for NO2 of up to 5×10-4 s-1 under similar experimental light conditions
was determined by Stemmler et al., 2007. Other nitration methods
investigated by Reinmuth-Selzle et al. (2014), e.g., nitration of Bet v 1
with peroxynitrite (ONOO-, formed by reaction of NO with
O2-) or TNM, lead to ND between 10 and 72 % depending on
reaction time, reagent concentration and temperature. Similarly, high NDs of
45–50 % were obtained by aqueous-phase TNM nitration of BSA and OVA by
Yang et al. (2010).
HONO formation
HONO formation from nitrated proteins
To study HONO emission from nitrated proteins, OVA was nitrated with TNM (see
Sect. 2.1) in liquid phase. The nitrated OVA (2 mg;
ND =12.5 %) was coated onto the reaction tube and exposed to VIS
lights under either pure nitrogen flow or 20 ppb NO2 gas.
Strong HONO emissions were found. A high correlation between HONO emission
and light intensity was observed (50 % RH; Fig. 3). Initially, we did not
apply NO2. Thus the observed HONO formation (up to 950 ppt)
originated from decomposing nitrated proteins rather than from heterogeneous
conversion of NO2. However, when exposed to 20 ppb of
NO2 in dark conditions, HONO formation increased 4-fold
(50–200 ppt) and about 2-fold with seven VIS lamps turned on
(950–1800 ppt). After 7 h of flow tube experiments
(4.5 h irradiation with varying light intensities (0, 1, 3, 7
lights) + 2.5 h irradiation/20 ppb
NO2 (7, 3, 0 lights)), no intact
protein was found according to the analysis of HPLC-DAD.
Light-enhanced HONO formation from TNM-nitrated proteins (n-OVA: ND
12.5 %, coating 21.5 µgcm-2). Black squares indicate
HONO formation via decomposition from nitrated proteins (without NO2)
while red squares indicate additional HONO formation via heterogeneous
NO2 conversion (20 ppb NO2) at 50 % RH (HONO is
scaled to the HONO concentration measured without NO2 and no light
([HONO]lights; NO2/[HONO]dark;
NO2=0)).
![]()
Light-induced HONO formation on BSA. (a) HONO formation
under alternating dark and light conditions on BSA surface
(22.5 µgcm-2); yellow shaded areas indicate periods in which
seven VIS lamps were switched on (RH =50 %, NO2=20 ppb).
(b) Dependency of HONO formation on radiation intensity at
20 ppb NO2 and 50 % RH
(BSA =31.4 µgcm-2). The experiment started with seven VIS
lights switched on, sequentially decreasing the number of lights (red
symbols, nominated 1–4), prior to applying the initial irradiance again (blue
symbol, 5). HONO was scaled to the HONO concentration in darkness
([HONO]lights/[HONO]dark). Error bars indicate SD of
20–30 min measurements; SD of point 5 covers 2.75 h
measurement.
![]()
As proteins can efficiently be nitrated by O3 and NO2 in
polluted air (Franze et al., 2005; Shiraiwa et al., 2012; Reinmuth-Selzle
et al., 2014), the emission of HONO from light-induced decomposing nitrated
proteins could play an important role in the HONO budget. As proteins are
nitrated at their tyrosine residues (at the ortho position to the OH group on
the aromatic ring) the underlying mechanism of this HONO formation should be
very similar to the HONO formation by photolysis of ortho-nitrophenols
described by Bejan et al. (2006). This starts with a photo-induced hydrogen
transfer from the OH group to the vicinal NO2 group (Fig. 1), which
leads to an excited intermediate from which HONO is eliminated subsequently.
Light dependency
To investigate HONO formation on unmodified BSA coating
(31.4 µgcm-2) dependent on light conditions, the
radiation intensity (number of VIS lamps) was changed under otherwise
constant conditions of exposure at 20 ppb NO2 and 50 %
RH. Decreasing light intensity revealed a linearly decreasing trend in HONO
formation from about 1000 to 140 ppt (red symbols in Fig. 4). After
re-illumination to the initial high light intensity the HONO formation was
reduced by 32 % (blue symbol in Fig. 4). Stemmler et al. (2006) and
Sosedova et al. (2011) also observed a similar saturation of HONO formation
on humic, tannic and gentisic acid at higher light intensities. Stemmler
et al. (2006) argued that surface sites activated for NO2
heterogeneous conversion by light (Reaction R3) would become de-activated by
competition with photo-induced oxidants (X∗, Reactions R7–R8), e.g.,
primary chromophores or electron donors are oxidized by surface*, which is in
line with the observed decomposition of the native protein presented above.
surface⟶hvsurface∗⟶NO2HONO+surfaceoxX⟶hvX∗⟶surface∗surface–X
In other studies the NO2 uptake coefficient on soot,
mineral dust, humic acid and other solid organic compounds similarly
increased at increasing light intensities (George et al., 2005; Stemmler
et al., 2007; Ndour et al., 2008; Monge et al., 2010; Han et al., 2016;
Brigante et al., 2008). Note that the HONO yield (ratio of HONO formed to
NO2 lost) was found to be constant at light intensities in the range
of 60–200 Wm-2 in the work of Han et al. (2016) but has shown
a linear dependence on light for nitrated phenols (Bejan et al., 2006).
Comparison of HONO formation dependency on NO2 at different
organic surfaces. HONO concentrations are scaled to the HONO concentration at
20 ppb NO2
([HONO]NO2/[HONO]NO2=20ppb).
The red squares indicate BSA coating (16 µgcm-2) at
161 Wm-2 and 50 % RH (this study). Blue triangles pointing
up are humic acid coating (8 µgcm-2) at
162 Wm-2 and 20 % RH (Stemmler et al., 2006), while the blue
triangles pointing down are the humic acid aerosol with 100 nm diameter
and a surface of 0.151 m2m-3 at 26 % RH and
1×1017 photonscm-2s-1 (Stemmler et al., 2007).
The black circles are gentisic acid coating (160–200 µgcm-2)
at 40–45 % RH and light intensity similar to that in the humic acid aerosol
study (Sosedova et al., 2011). Green diamonds are ortho-nitrophenol in gas
phase (ppm level) illuminated with UV/VIS light. Dotted lines are exponential
fittings of the measured data points and are meant to guide the eyes.
![]()
<inline-formula><mml:math id="M277" display="inline"><mml:mrow class="chem"><mml:msub><mml:mi mathvariant="normal">NO</mml:mi><mml:mn mathvariant="normal">2</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula> dependency
At about 50 % relative humidity and high illumination intensities (seven VIS
lamps, ∼161 Wm-2), heterogeneous formation of HONO
strongly correlated with the applied NO2 concentration (Fig. 5). On
a BSA surface of about 16.1 µgcm-2 (Table 1) the produced
HONO concentration increased from 56 ppt at 20 ppb
NO2 to 160 ppt at 100 ppb NO2. Only at
a threshold NO2 level well above those typically observed in natural
environments (≫ 150 ppb) did this increasing trend slow down to
some extent, indicative of saturation of active surface sites. A similar
pattern of NO2 dependence was also observed for light-induced HONO
formation from humic acid (Stemmler et al., 2006) and phenolic compounds like
gentisic and tannic acid (Sosedova et al., 2011) or polycyclic aromatic
hydrocarbons (Brigante et al., 2008) and for heterogeneous NO2
conversion on soot under dark conditions (Stadler and Rossi, 2000; Salgado
and Rossi, 2002; Arens et al., 2001).
For better comparison of the different studies the HONO concentration
measured at different NO2 concentrations was scaled to the HONO
concentration at 20 ppb NO2
([HONO]NO2/[HONO]NO2=20ppb) in
Fig. 5, as variable absolute amounts of HONO were found in different studies
and matrices. A cease of the NO2 dependency on heterogeneous HONO
formation can be assessed for most of the studies at NO2
concentrations ≥200 ppb. A very similar correlation (up to
40 ppb NO2) was observed when NO2 was applied
additionally during the gas-phase photolysis of nitrophenols (Fig. 5; Bejan
et al., 2006). Even though the matrix (nitrophenols) and conditions
(illuminated) of the latter is comparable to the experiment presented here,
for BSA no clear indication of saturation was found up to 160 ppb of
NO2, pointing to a highly reactive surface of BSA for NO2
under illuminated conditions. As shown with Reactions (R7) and (R8), the
concentration dependence depends on the competing channel (Reaction R8);
therefore, this is strongly matrix dependent, both in terms of chemical and
physical properties.
Impact of coating thickness
Strong differences in HONO concentrations were found for experiments with
different coating thicknesses applying otherwise similar conditions
(20 ppb of NO2, seven VIS lamps and 50 % RH). While only
55 ppt of HONO concentration was observed for a shallow homogeneous
coating of 16.1 µgcm-2 (217.6 nm thickness, see
below) applied on the whole length of the tube, up to 2 ppb was
found for a thick (more uneven) coating of 31.44 µgcm-2
(435.2 nm thickness) covering only 50 % of the tube (Fig. 6).
Potential explanations are that thicker coating leads to (1) more bulk
reactions producing HONO or (2) different morphologies, e.g., higher
effective reaction surfaces. Exposing (20 %) different coated surface
areas in the flow tube, potentially introduced bias comparing different data
sets. Emitted HONO might be re-adsorbed differently by proteins and glass
surface. However, as the protein is slightly acidic, a low uptake efficiency
of HONO by BSA can be anticipated, which should not differ too much from the
uncovered glass tube surface (Syomin and Finlayson-Pitts, 2003).
Accordingly, NO2 uptake on glass is assumed to be significantly lower
than on proteins. A strong increase in NO2 uptake coefficients with
increasing coating thickness was also observed for humic acid coatings (Han
et al., 2016). However, they found an upper threshold value of
2 µgcm-2 of cover load (20 nm absolute thickness,
assuming a humic acid density of 1 gcm-3), above which uptake
coefficients were found to be constant. The authors also proposed that
NO2 can diffuse deeper into the coating and below
2 µgcm-2 the full cover depth would react with NO2,
respectively.
HONO formation on three different BSA coating thicknesses, exposed
to 20 ppb of NO2 under illuminated conditions (seven VIS lamps).
The HONO concentrations were scaled to reaction tube coverage (black:
100 % of reaction tube was covered with BSA; light blue: 70 % of tube
was covered; red: 50 % of tube was covered with BSA). The middle thick
coating (22.46 µgcm-2) was replicated and studied with
different reaction times (cyan and blue triangle). Solid lines (with circles
or triangles) present continuous measurements; when those are interrupted,
other conditions (e.g., light intensity, NO2 concentration) prevailed.
Dotted lines show interpolations and are meant to guide the eyes. Arrows
indicate the intervals in which the shown decay rates were determined. Error
bars indicates SDs from 10 to 20 measuring points (5–10 min).
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For proteins the number of molecules per monolayer depends on their
orientation and respective layer thickness can vary accordingly. One (dry,
crystalline) BSA molecule has a volume of about 154 nm3 (Bujacz,
2012). In a flat orientation (4.4 nm layer height and a projecting
area of 35 nm2molecule-1)
3.64×1014 molecules (40.5 µg;
0.32 µgcm-2) of BSA are needed to form one complete
monolayer in the flow tube (i.d. of 0.81, 50 cm length, 100 %
surface coating). Hence, the thinnest BSA coating applied in the experiment
(16.1 µgcm-2) would consist of 50 monolayers, revealing
a total coating thickness of 217.6 nm, and the thickest BSA coating
(31 µgcm-2) would have 99 monolayers and an absolute
thickness of 435.1 nm. At the other extreme (non-flat) orientation,
more BSA molecules are needed to sustain one monolayer. With
21.7 nm2 of projected area of one molecule and 7.1 nm
monolayer height, 5.86×1014 molecules of BSA are needed to
form one complete monolayer in the flow tube. The coatings would consist of
between 31 (thinnest) and 61 (thickest) monolayers of BSA. With a flat
orientation 1–2 % (number or weight) of BSA molecules would build the
uppermost surface monolayer, whereas in an upright molecule orientation
1.6–3.3 % would be in direct contact with surface ambient air.
In the crystalline form several molecules of water stick tightly to BSA. As
BSA is highly hygroscopic, more water molecules are adsorbed at higher
relative humidity. At 35 % RH BSA is deliquesced (Mikhailov et al.,
2004). Therefore the above described number of monolayers and the absolute
layer thickness are a lower bound estimate.
In conclusion, the thickness dependence on HONO formation is extremely
complex. Activation and photolysis of nitrated Tyr occurs throughout the BSA
layer. The heterogeneous reaction of NO2 may or may be not limited to
the surface depending on solubility and diffusivity of NO2. Also the
release of HONO may be limited by diffusion. The observed dependence on the
coating thickness suggests the involvement of the bulk reactions, but the
reactions can happen in both surface and bulk phase.
Dependency of relative humidity on HONO formation. 25 ppb
NO2 was applied on BSA surface (17.5 µgcm-2) either
in darkness (blue triangle) or at seven VIS lights (red star). HONO was scaled to
HONO concentrations in darkness under dry conditions
([HONO]lights on–off;
RH/[HONO]dark; RH=0). Dotted lines are meant to guide
the eyes.
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RH dependency
The dependence of HONO emission on relative humidity is shown in Fig. 7. Here
about 25 ppb of NO2 was applied to a (not nitrated) BSA-coated flow tube (17.5 µgcm-2) both in dark and illuminated
conditions (seven VIS lights). HONO formation scaled with relative humidity.
Kleffmann et al. (1999) proposed that higher humidity inhibits the
self-reaction of HONO (2HONO(s,g)→NO2+NO+H2O), which leads to higher HONO yield from
heterogeneous NO2 conversion.
Extended measurements (20 h) of light-enhanced HONO
formation on BSA (three coatings of 17.5 µgcm-2) at 80 %
RH, 100 ppb NO2. HONO formation under VIS light is shown in
red and orange, under UV/VIS light in blue. HONO decay rates
(ppth-1) are shown with time periods (in brackets) in which they
were calculated, suggesting a stable HONO formation after 4 h. Right:
magnification of the first 2 h. Straight lines (black, grey, light and dark
blue) show the slopes of which d[HONO]/dt were used in the kinetic
studies.
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The RH dependence of HONO formation on proteins is different to other
surfaces. For example, no influence of RH has been observed for dark
heterogeneous HONO formation on soot particles sampled on filters (Arens
et al., 2001). No impact of humidity on NO2 uptake coefficients on
pyrene was detected (Brigante et al., 2008). For HONO formation on tannic
acid coatings (both at dark and irradiated conditions) a linear but
relatively weak dependence has been reported between 10 and 60 % RH,
while below 10 % and above 60 % RH the correlation between HONO
formation and RH was much stronger (Sosedova et al., 2011). Similar results
were obtained for anthrarobin coatings by Arens et al. (2002). This type of
dependence of HONO formation on phenolic surfaces on RH equals the HONO
formation on glass, following the BET water uptake isotherm of water on polar
surfaces (Finnlayson-Pitts et al., 2003; Summer et al., 2004). For humic acid
surfaces the NO2 uptake coefficients also weakly increased below
20 % RH and were found to be constant between 20 and 60 % (Stemmler
et al., 2007).
While on solid matter chemical reactions are essentially confined to the
surface rather than in the bulk, proteins can adopt an amorphous solid or
semisolid state, influencing the rate of heterogeneous reactions and
multiphase processes. Molecular diffusion in the non-solid phase affects the
gas uptake and respective chemical transformation. Shiraiwa et al. (2011)
could show that the ozonolysis of amorphous protein is kinetically limited by
bulk diffusion. The reactive gas uptake exhibits a pronounced increase with
relative humidity, which can be explained by a decrease of viscosity and
increase of diffusivity, as the uptake of water transforms the amorphous
organic matrix from a glassy to a semisolid state (moisture-induced phase
transition). The viscosity and diffusivity of proteins depend strongly on the
ambient relative humidity because water can act as a plasticizer and increase
the mobility of the protein matrix (for details see Shiraiwa et al., 2011, and
references therein). Shiraiwa et al. (2011) further showed that the BSA phase
changes from solid through semisolid to viscous liquid as RH increases, while
trace gas diffusion coefficients increased about 10 orders of magnitude. This
way, characteristic times for heterogeneous reaction rates can decrease from
seconds to days as the rate of diffusion in semisolid phases can decrease by
multiple orders of magnitude in response to both low temperature (not
investigated in here) and/or low relative humidity. Accordingly, we propose
that HONO formation rate depends on the condensed-phase diffusion
coefficients of NO2 diffusing into the protein bulk, HONO released
from the bulk and mobility of excited intermediates.
Long-term exposure with <inline-formula><mml:math id="M367" display="inline"><mml:mrow class="chem"><mml:msub><mml:mi mathvariant="normal">NO</mml:mi><mml:mn mathvariant="normal">2</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula> under irradiated conditions
To study long-term effects of irradiation on HONO formation from proteins,
flow tubes were coated with 2 mg BSA (17.5±0.4 µgcm-2; 90 % of total length) and exposed to
100 ppb NO2, at 80 % RH at illuminated conditions for
a time period of up to 20 h (Fig. 8). Samples illuminated with VIS
light only (red and orange colored lines in Fig. 8) showed persistent HONO
emissions over the whole measurement period. For unknown reasons, and even
though the observed HONO concentrations were within the expected range with
regard to the applied NO2 concentrations, RH and cover
characteristics, one sample (orange in Fig. 8) showed a sharp short-term
increase in the initial phase followed by respective decrease, not in line
with all other samples (compare Fig. 6). However, after 4 h both VIS
irradiated samples showed virtually constant HONO emissions (-3.8 and
+1.6 ppth-1, respectively). The sample illuminated with UV and VIS
light (three UV and four VIS lamps) showed a sustained sharp increase in the first
4 h, followed by persistent and very stable (decay rate as low as
-0.5 ppth-1) HONO emissions at an about 3-fold higher level
compared to samples irradiated with VIS only. HONO formation by photolysis of
(adsorbed) HNO3 is assumed to be insignificant in this study. With
N2 as carrier gas, gas-phase reactions of NO2 do not produce
HNO3. Even when small amounts of HNO3 would be formed by
unknown heterogeneous reactions, photolysis of HNO3 is only
significant at wavelengths <350 nm, which is close to the lowest
limit of the UV wavelength applied in this study. Likewise, the respective
photolysis frequency recently proposed by Laufs and Kleffmann (2016) of about
2.4×10-7 s-1 is very low.
Integrating the 20 h experiments, 9.23×1015
(4.6 ppbh, VISa), 1.53×1016 (7.7 ppbh, VISb)
and 4.01×1016 (20 ppbh, UV/VIS) molecules of HONO were
produced. This means between 7.7×1013 and
3.3×1014 molecules of HONO per cm2 of BSA
geometric surface were formed. With respect to the different experimental
conditions concerning cover thickness, RH, and NO2 concentrations,
this is in a similar order of magnitude as found for humic acid
(2×1015 moleculescm-2 in 13 h) by Stemmler
et al. (2006).
If BSA acts like a catalytic surface as in a Langmuir–Hinshelwood reaction
each BSA molecule can react several times with NO2 to heterogeneously
form HONO. As described in 3.1, BSA nitration is in competition with
NO2 surface reactions and only a limited number of
NO2molecules could react with BSA forming HONO via nitration of
proteins and subsequent decomposition of nitrated proteins. A BSA molecule
contains 21 tyrosine residues, which could react with NO2. However,
even a strong nitration agent such as TNM is not capable of nitrating all
tyrosine residues and a mean ND of 19 % was found (Peterson et al., 2001;
Yang et al., 2010); i.e., four tyrosine residues of one BSA molecule can be
nitrated to form HONO. As 2 mg of BSA was applied for each flow tube
coating, a total of 1.8×1016 protein molecules can be inferred. In
20 h of irradiating with VIS light 13–22 % of the accessible Tyr
residues (four Tyr per BSA molecule) would have been
reacted. Irradiating with additional UV lights at least 56 % of the
tyrosine residues would have been nitrated and decomposed. However, as
NO2 is a much weaker nitrating agent and nitration of only one
tyrosine residue is probable (ND of BSA with O3 / NO2
6 %; Yang et al., 2010) up to 85 % BSA molecules would have been
reacted when irradiated with VIS lights and even more HONO molecules as
coated BSA molecules would have been generated under UV/VIS light conditions.
Other amino acids of the protein like tryptophan or phenylalanine might also
be nitrated but without formation of HONO (Goeschen et al., 2011). Hence,
a contribution of heterogeneous conversion of NO2 can be anticipated.
Kinetic studies
The experimental results (especially the stability over a long time) indicate
that the formation of HONO from NO2 on protein surfaces likely
underlies the Langmuir–Hinshelwood mechanism in which the protein would act
as a catalytic surface (Fig. 9). The first step is the fast, reversible
physical adsorption of NO2 (k1) and water followed by the slow
conversion into HONO.
There are two possible processes for the HONO formation. HONO is formed by
heterogeneous NO2 conversion (k2) but also via nitration and
decomposition of nitrated proteins (k4, k5). The final step of the
mechanism is the release of the generated HONO into the air. Since proteins
are in general slightly acidic, the desorption of HONO (k3) should be
fairly fast. Pseudo-first-order kinetics are assumed for the reaction of
NO2 to HONO (Stemmler et al., 2007) and the reaction can be described
as follows (Eq. 1).
d[HONO]gdt=keff⋅[NO2]g,
with keff the effective pseudo-first-order rate constant (for more
detailed information check the Supplement).
In this study, neither HONO nor NO2 photolysis is considered, as the
overlap of the applied UV/VIS or VIS range (340–700 or 400–700 nm)
and the HONO and NO2 photolysis spectrum (< 400 nm) is
low. Furthermore, the applied light intensity is lower compared to clear-sky
irradiance and the respective UV light is partly absorbed by the reaction
tube although quartz glass was used (transmission ∼ 90 %) and the
photolysis frequency would decrease down to 10-4 s-1. Hence,
the photolysis is assumed to be not significant.
Schematic illustration of the underlying
Langmuir–Hinshelwood mechanism of light-induced HONO formation on protein
surface. Reaction constants for NO2 uptake, direct NO2
conversion, protein nitration, HONO formation from decomposing nitrated
proteins and HONO release are indicated by k1, k2, k4,
k5, and k3.
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In the first 5–10 min of the long-term experiments, HONO increased
(Fig. 8 – zoomed in range). This slope was taken as
d[HONO]g/dt in Eq. (6). Effective rate
constants between 1.48×10-6 s-1 (VISa) and
7.40×10-6 s-1 (VISb) were calculated. When irradiating
with VIS light only, the concentration of HONO was either constant or
decreased for 2 h after this first 10 min. When irradiating
with additional UV light, the HONO signal showed an enhancement in two steps.
In the first 10 min it was strongly increasing
(1327 ppth-1) and then in the next hour it increased less with
170 ppth-1 prior to stabilization. Therefore two rate constants
of 4.10×10-6 and 5.2×10-7 s-1 were obtained,
respectively.
Reactive uptake coefficients for NO2 were calculated according to Li
et al. (2016). For both irradiation types the uptake coefficient γ was
in the range of 7×10-6 at the very beginning of each experiment.
After a few minutes they decreased to a mean of 1×10-7. The
calculated keff values and uptake coefficient are in the same
range and match the NO2 uptake coefficients on irradiated humic acid
surfaces (coatings) and aerosols obtained by Stemmler et al. (2006/07) which
were in between 2×10-6 and 2×10-5 (coatings) and
1×10-6 and 6×10-6 (aerosols), depending on NO2
concentrations and light intensities. Similar NO2 uptake coefficients
on humic acid were observed by Han et al. (2016). George et al. (2005)
reported about a 2-fold increased NO2 uptake coefficients for
irradiated organic substrates (benzophenone, catechol, anthracene) compared
to dark conditions, in the order of (0.6–5)×10-6.
NO2 uptake coefficients on gentisic acid and tannic acid were in
between (3.3–4.8)×10-7 (Sosedova et al., 2011), still
higher than on fresh soot or dust (about 1×10-7; Monge
et al., 2010; Ndour et al., 2008). The NO2 uptake coefficients on BSA
in the presence of O3 (1×10-5, for 26 ppb NO2
and 20 ppb O3) published by Shiraiwa et al. (2012) were
somewhat higher than the values calculated here without O3 but with
light.
It was not possible to extract a set of parameters for a Langmuir–Hinshelwood
mechanism (like Langmuir equilibrium constant, surface accommodation
coefficient or second-order rate constant) from the presented data. The
saturating behavior of photochemical HONO production may be due to either the
adsorbed precursor on the surface or due to a photochemical competition
process, which also leads to a Lindemann–Hinshelwood type kinetic expression
(Minero, 1999).