Nitration of Tyrosine in the Mucin Glycoprotein of Edible Bird's Nest Changes Its Color from White to Red

Article abstract of DOI:10.1021/acs.jafc.8b01619

The edible bird's nest (EBN) of the swiftlet Aerodramus fuciphagus, a mucin glycoprotein, is usually white in color, but there also exist the more desirable red or "blood" EBN. The basis of the red color has been a puzzle for a long time. Here, we show that the nitration of the tyrosyl residue to the 3-nitrotyrosyl (3-NTyr) residue in the glycoprotein is the cause of the red color. Evidence for the 3-NTyr residue comes from (a) the quantitative analysis of 3-NTyr in EBN by enzyme-linked immunosorbent assay, (b) the ultraviolet-visible absorption spectra of red EBN as a function of pH being similar to 3-nitrotyrosine (3-NT), (c) the change in the color of red EBN from yellow at low pH to red at high pH just like 3-NT, and (d) strong Raman nitro bands at 1330 cm^-1 (symmetric -NO₂ stretch) and 825 cm^-1 (-NO₂ scissoring bend) for red EBN. The high concentrations of nitrite and nitrate in red EBN are also explained.

Full text of DOI:10.1021/acs.jafc.8b01619

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Food and Beverage Chemistry/Biochemistry
Nitration of tyrosine in the mucin glycoprotein of
edible bird’s nest changes its color from white to red
Eric Kian-Shiun Shim, and Soo-Ying Lee
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01619 • Publication Date (Web): 22 May 2018
Downloaded from http://pubs.acs.org on May 22, 2018
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Page 1 of 34
Journal of Agricultural and Food Chemistry
Nitration of tyrosine in the mucin glycoprotein of edible bird’s nest
changes its color from white to red
Eric KianꢀShiun Shim and SooꢀYing Lee*
Division of Chemistry & Biological Chemistry
School of Physical & Mathematical Sciences
Nanyang Technological University
21 Nanyang Link, Singapore 637371
Singapore
Author Information
Corresponding Author
*Eꢀmail: sooying@ntu.edu.sg. Phone: +65 93892112. Fax: +65 6515 9663
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ABSTRACT
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The edible bird’s nest (EBN) of the swiftlet Aerodramus fuciphagus, a mucin
glycoprotein, is usually white in color, but there also exist the more desirable red or ‘blood’
EBN. The basis of the red color has been a puzzle for a long time. Here, we show that the
nitration of the tyrosyl residue to the 3ꢀnitrotyrosyl (3ꢀNTyr) residue in the glycoprotein is the
cause of the red color. Evidences for the 3ꢀNTyr residue come from: (a) the quantitative
analysis of 3ꢀNTyr in EBN by ELISA, (b) the UVꢀVis absorption spectra of red EBN as a
function of pH being similar to 3ꢀnitrotyrosine (3ꢀNT), (c) the change in the color of red EBN
from yellow at low pH to red at high pH just like 3ꢀNT, and (d) strong Raman nitro bands at
1330 cmꢀ1 (symmetric –NO2 stretch) and 825 cmꢀ1 (–NO2 scissoring bend) for red EBN. The
high concentrations of nitrite and nitrate in red EBN are also explained.
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KEYWORDS
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red edible bird’s nest; mucin glycoprotein; nitration; 3ꢀnitrotyrosine; ELISA; UVꢀVisible
spectroscopy; Raman microspectroscopy
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1. INTRODUCTION
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Swiftlets of genus Aerodramus fuchiphagus, native to the IndoꢀPacific region, build
edible bird’s nest (EBN), also known as cubilose or nest cement, which is an unusual
amorphous, composite material of strands of mucin glycoprotein (ca. 90% by weight)
strengthened largely with its own bird feathers (ca. 10% by weight). The mucin glycoprotein
is secreted like saliva in copious amounts from a pair of sublingual glands under the tongue
of the swiftlet during breeding. In living systems, mucin glycoprotein is only present in tiny
amounts, but EBN is a material with the highest known concentration of mucin glycoprotein
and more than a million kg of EBN is harvested annually. The raw EBN is moistened and
cleaned of feathers, sand grains, etc., with tweezers, leaving behind strands of clean, white
glycoprotein to be used as food or medicine. When cooked, EBN is believed to have
exceptional nutritional and medicinal properties. It is one of the most expensive Asian food
delicacies today, and has been prescribed in traditional Chinese medicine for more than a
thousand years. Recent studies have shown that EBN can inhibit influenza virus infection,
stimulate the immune system, and promote epidermal growth.1
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The EBN mucin glycoprotein is a polymer with a protein backbone and many dendritic
carbohydrate chains attached to it. The proximate analysis of EBN revealed that it contains ca.
62% by weight of protein, made up of 17 types of amino acids, with the most abundant being
serine, valine, tyrosine, isoleucine, aspartic acid and asparagine, glutamic acid and glutamine,
and phenylalanine, and the distinct absence of tryptophan, cysteine, and proline.2-5 The
dendritic carbohydrates make up ca. 27% by weight and the major saccharides are sialic acid,
galactose, Nꢀacetylgalactosamine, Nꢀacetylglucosamine, fucose, and mannose.5, 6 The rest are
moisture (ca. 8%), ash/minerals (ca. 2%),7 and fat (< 1%).5
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There are several colors of EBN available in the market – white (Figure 1A shows a
natural white EBN), various shades of light yellow or golden, and red EBN (Figure 1B shows
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a natural red EBN from a birdhouse). The latter is often addressed as ‘blood’ nest due to its
color. Commercially, the more common cleaned, white EBN costs about US$3,000/kg today,
with the price fluctuating based on supply and demand, while the less common red EBN costs
several times more, as the red EBN is perceived to have superior nutritional and medicinal
properties over the white EBN. Today, the trade in EBN is a multiꢀbillion dollar industry
annually. Due to the lucrative return of red EBN, some EBN processors have used various
methods, including dyeing and fumigation with swiftlet ‘bird soil’ (feces), to convert the
white EBN into red ‘blood’ nest.8
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Contrary to a longꢀheld belief, red ‘blood’ EBN is a misnomer as it contains no
hemoglobin.9 In any case, we would not expect hemoglobin to be present in swiftlet saliva
unless the bird was injured or sick to regurgitate blood. Marcone found that: (a) the substance
giving the red color is neither water soluble nor lipid/solvent extractable, (b) white and red
EBN both have ovotransferrin, and red EBN has a relatively higher amounts of iron
compared to white EBN, and so it was suggested that an ovotransferrinꢀiron complex could
be the cause of the red color in red EBN.5 The red color is also not a structural coloration
(light interference effect) as both the white and red nests are made of similar amorphous
cementꢀlike material from the dried, clear liquid gel of mucin glycoprotein initially secreted
through the beak of the same species of swiftlet. The coloration appears after the initial white
EBN has been formed.
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Recently, But et al.8 showed that red EBN could be produced by fumigating white EBN
either with vapor from unwashed swiftlet bird soil (feces), simulating the environment in
some bird houses that accumulate the bird soil to produce colored nests, or with nitrous acid
(HNO2) vapor from a solution of sodium nitrite in 2% hydrochloric acid (Figure 1C shows a
red EBN prepared by HNO2 fumigation for 30 days). The similarity in the outcome is
because the vapor from the bird soil also contains HNO2 produced by bacterial
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decomposition of the protein/nitrogenꢀrich bird soil. Clearly, an ovotransferrinꢀiron complex
cannot be the cause of the red color in these experiments. In analogy to sodium nitrite
liberating nitric oxide (NO) and forming NOꢀmyoglobin to give the deep red color of cured
meat, it was conjectured that perhaps NO produced in the nitrous acid vapor equilibrium
could similarly bind to the glycoprotein of EBN to give a red color. However, But et al.8
noted that the chemistry of the color change from white to red EBN remains unknown.
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The xanthoproteic reaction, using a mixture of concentrated nitric and sulfuric acids, is a
standard biochemical test that gives a positive yellow to a deep red substance in proteins with
aromatic amino acids, e.g. tyrosine, phenylalanine or tryptophan, due to nitration of the
aromatic ring. Now, EBN is a glycoprotein containing substantial amounts of the aromatic
amino acids tyrosine and phenylalanine, and it reacts positively at room temperature, giving a
red color (Figure 1D shows a xanthoproteic reacted EBN). Clearly, the natural red EBN, the
HNO2 fumigated EBN, and the xanthoproteic reacted EBN in Figures 1BꢀD all have a similar
red color. It is known that nitration of phenylalanine by the xanthoproteic reaction requires
heat as the aromatic ring is not activated, but for tyrosine, it proceeds readily at room
temperature due to ring activation by the phenolic –OH group. So, the red color is most likely
due to the nitration of the glycoprotein tyrosine residues to give 3ꢀnitrotyrosyl (3ꢀNTyr)
residues. It remains for us to show that the 3ꢀNTyr residue is practically absent in the white
nest, but present significantly in the natural red EBN, the HNO2 fumigated EBN, and the
xanthoproteic reacted EBN, and 3ꢀNTyr is the cause of the red color.
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An antiꢀ3ꢀNTyr antibody enzymeꢀlinked immunosorbent assay (ELISA) kit can be used
to detect and quantitatively measure the 3ꢀNTyr in EBN. Further evidences for 3ꢀNTyr come
from: (a) the UVꢀVis absorption spectroscopy of solubilized red EBN showing pHꢀdependent
absorption spectra similar to that of free 3ꢀnitrotyrosine (3ꢀNT), (b) the change in the color of
red EBN strands from yellow at low pH to red at high pH, consistent with the UVꢀVis
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absorption spectra of red EBN and 3ꢀNT, and (c) Raman microspectroscopy showing the
presence of the nitro group in red EBN.
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2. MATERIALS AND METHODS
Edible bird’s nest (EBN) and other materials
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Raw white and natural red EBN samples produced by the species Aerodramus
fuciphagus were obtained from birdhouses from different geographical locations in Southꢀ
East Asia: Port Dickson (West Malaysia), Lahad Datu (East Malaysia), and Makassar
(Indonesia). All standard chemicals used were analytical grade. Ultrapure water (18.2 Mꢁ cm)
was prepared with Sartorius Arium Lab water purification system (Germany) and used for all
experiments. Sodium nitrite, silver nitrate, and citric acid were purchased from Sigmaꢀ
Aldrich Chemicals (St Louis, MO). Hydrochloric acid, sulfuric acid, and nitric acid were
purchased from Fisher Scientific Chemicals (Hampton, NH). Urea, disodium hydrogen
phosphate, sodium hydroxide, sodium carbonate, and sodium bicarbonate were purchased
from VWR chemicals (Radnor, PA). Lꢀtyrosine and Lꢀphenylalanine were purchased from
NowFoods (Bloomingdale, IL). 3ꢀNT ELISA Kit was purchased from Abcam (UK). Citric
acid and disodium hydrogen phosphate were used to make buffers from pH 4 to 8, and
sodium carbonate and sodium bicarbonate were used to make buffers from pH 9 to 10.
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Fumigation of white EBN by nitrous acid to produce red EBN
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EBN was fumigated with nitrous acid vapor to convert to red EBN.8 A solution of
sodium nitrite (200 mg) in 2% HCl (20 mL) was poured into a 250 mL glass beaker. A small
empty glass bottle was placed at the bottom of the beaker as support for a glass petri dish
containing the EBN so that it is above the fumigating solution. The glass beaker was then
sealed with a paraffin film and kept in the dark at room temperature, and the color change in
the EBN was monitored at regular intervals for up to 30 days, with a change of the NaNO2ꢀ
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HCl solution every 7 days, during which time the EBN changed color from white to yellow
to orange and finally to red.
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Xanthoproteic reaction to produce red EBN
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A whole piece of moistened raw white EBN (about 6 g) was dipped in a nitrating acid
mixture (500 mL; 69% nitric acid: 98% sulfuric acid: water (5: 4: 95 v/v)) for ca. 5 seconds,
removed and fanꢀdried for ca. 10 min at 25 ˚C and ca. 50% relative humidity, and the
dipping and drying was repeated for about 30 times, until the EBN turned yellow. The
dipping and drying helped to retain the shape of the EBN and to better control the rate of
nitration. Next, the dipping and drying was repeated for 30 times with water to rinse off
residual acid to stop the reaction. Finally, 1 mM sodium hydroxide solution was sprayed on
the surface of the nitrated EBN to neutralize any remaining acid, followed by fanꢀdrying for
ca. 10 min. The process was repeated several times, and the EBN turned red as shown in
Figure 1D.
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Synthesis of 3-nitrotyrosine (3-NT) and 4-nitrophenylalanine (4-NP)
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3ꢀNT was synthesized by reacting Lꢀtyrosine with an acid mixture of nitric acid and
sulfuric acid to generate the nitronium ion for electrophilic aromatic substitution.10 The
product was characterized by nuclear magnetic resonance (NMR) spectroscopy and liquid
1
chromatographyꢀmass spectroscopy (MS). H NMR spectra were recorded on a JOEL 400
(Japan) at 400 MHz and mass spectra were recorded on a Thermo Finnigan LCQ Fleet Mass
1
Spectrometer (San Jose, CA) using water as a solvent. H NMR (400 MHz, D2O) δ/ppm:
8.03 (s, 1H, aromatic proton), 7.57 (dd, 1H, J1 = 9 Hz, J2 = 2 Hz, aromatic proton), 7.17 (dd,
1H, J1 = 9 Hz, J2 = 2 Hz, aromatic proton), 4.22 (t, J = 8 Hz, 1H, ꢀCH), 3.26 (multiplet, 2H, ꢀ
CH2). LCꢀMS (ESI) m/z: [M + H]+ Calcd for C9H10N2O5 + H+, 227.19; found 226.88.
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4ꢀNP was synthesized from phenylalanine in the same way as 3ꢀNT by electrophilic
aromatic substitution of the nitronium ion,11 and subjected to reverseꢀphase highꢀperformance
liquid chromatography (RPꢀHPLC) for further purification. The mixture was injected onto
the Agilent ZORBAX SBꢀC18 column (9.4 x 250 mm, 5 µm) (Santa Clara, CA) and eluted
with eluent A (water: acetonitrile: trifluoroacetic acid = 95: 5: 0.1) and a linear gradient of
eluent B (water: acetonitrile: trifluoroacetic acid = 50: 50: 0.1) for 20 min, followed by a
washing and reꢀequilibration step. The injection volume was 400 µL at a flow rate of 3 mL
minꢀ1 and the UV absorbance of the eluents was monitored at 280 nm. The major product
1
eluted at 11.5 min was characterized by NMR and LCꢀMS. H NMR (D2O) δ/ppm: 8.20 (d,
2H, J = 9 Hz, aromatic proton), 7.51 (d, 2H, J = 8 Hz, aromatic proton), 4.40 (t, 1H, J = 7 Hz,
ꢀCH), 3.39 (multiplet, 2H, ꢀCH2). LCꢀMS (ESI) m/z: [M + H]+ Calcd for C9H10N2O4 + H+,
211.19; found 210.99.
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Microscopic images of red EBN strands with change in pH
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Carl Zeiss Stemi 305 Trino Greenough system with Stand K EDU and spot illuminator K
LED (Germany) was used to obtain the microscopic images of EBN strands. The images
were captured and recorded with Microscopy Camera Axiocam 105 color and Carl Zeiss
ZEN Lite software. EBN strands were soaked in the respective buffer solutions for 10 min
and fan dried for 15 min at 25 ˚C and ca. 50% relative humidity, before the images were
taken.
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Solubilization and lyophilization of EBN
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About 0.1g of white or red EBN was frozen in liquid nitrogen, pulverized, and sieved
through 0.5 mm mesh. The EBN was rinsed with water (4 L) to remove any nitrate/nitrite in
the EBN. Subsequently, the residue was further soaked and rinsed with 0.05 M urea (200 mL)
to remove any residual nitrite, and finally rinsed with water (4 L) again. We will call this
cleaned EBN residue. It was then added to 1 M sodium hydroxide (5 mL) and stirred until the
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EBN dissolved (ca. 12 hours). The solubility of the EBN was about 20 g/L. The solution was
centrifuged and the supernatant was ultraꢀfiltered with Sartorius Vivaspin 20 ultrafiltration
tube (MWCO 3000 Da) using water as eluent to remove any excess sodium hydroxide and
salt present. The neutral EBN solution was lyophilized and stored at 4 ˚C. We will call the
product lyophilized EBN.
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Enzyme-linked immunosorbent analysis (ELISA)
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The ELISA was conducted using the commercially available 3ꢀNTyr sandwich ELISA
kit. A calibration curve utilizing 3ꢀNTyr labeled bovine serum albumin (BSA) in the working
range from 8 to 1000 ng mLꢀ1 was fitted using a 4ꢀparameter logistic algorithm in Origin Pro
9.0 (Northampton, MA). Lyophilized white or red EBN samples, silver nitrate, and sodium
nitrite were appropriately diluted for the analysis, and triplicate measurements were carried
out for each sample. 3ꢀNTyr labeled BSA and incubation buffer were used as the positive and
negative control, respectively. The absorption at 600 nm was recorded at 15 min after the
addition of the horseradish peroxidase enzyme (HRP) development solution with Tecan 96ꢀ
wells plate spectrophotometer (Switzerland). The concentrations of 3ꢀNTyr in the lyophilized
EBN samples were determined.
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UV-Visible spectroscopy
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A series of solutions (~1 g Lꢀ1) at different pH were made by adding the lyophilized
EBN, 4ꢀNP or 3ꢀNT into pH buffers with pH from 4 to 10. The pH of the solutions was
monitored with a Mettler Toledo benchtop pH meter (Switzerland). The absorption spectra of
the solutions, in FisherꢀScientific 1 cm pathꢀlength quartz cuvette cell, were then recorded
from 250 to 600 nm with a Shimadzu UVꢀ1800 UVꢀVis spectrophotometer (Japan).
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Raman microspectroscopy
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Cleaned white or red EBN residues were preꢀsoaked in 0.01 M sodium hydroxide to
deprotonate the 3ꢀNTyr residue so as to intensify the Raman bands of the nitro group, and fan
dried for ca. 12 hours at 25 ˚C and ca. 50% relative humidity before measurement. Raman
spectra of the EBN were collected with the Nanophoton Ramantouch microspectrometer
(Nanophoton Corporation, Osaka, Japan) using the 532 nm laser excitation and instrument
parameters (LU Plan Fluor 100 X, 600 gr mmꢀ1, 28 mW, 30 s, 24 ˚C).
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Pure 3ꢀNT (0.1 mg) was dissolved in 1 M sodium hydroxide (10 µL), deposited on a
silicon substrate, and the solvent was allowed to evaporate slowly in a covered petri dish for
ca.72 hours before the Raman measurement. The Raman spectrum of 3ꢀNT was collected
also using the 532 nm laser excitation, but with lower power (3 mW, 180 s, 24 ˚C) to
minimize fluorescence.
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Each sample was measured in triplicate over different spots and the average Raman
spectrum was baseline corrected with the Origin Pro 8.0 software.
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3. RESULTS AND DISCUSSION
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Fumigation of white EBN with nitrous acid vapor and by xanthoproteic reaction to
produce red EBN
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But et al.8 showed that red EBN can be formed by fumigating the white EBN with HNO2
vapor formed by the reaction between sodium nitrite and hydrochloric acid or the vapor from
‘bird soil’ collected from birdhouses. Figure 1 shows the natural white nest (A), natural red
nest (B), red nest produced by HNO2 fumigation (C), and red nest produced by the
xanthoproteic reaction (D).
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Figure 2 shows the red color development of a piece of raw white EBN when fumigated
with a mixture of sodium nitrite and hydrochloric acid over a period of 30 days (d). The white
EBN had turned yellow/golden after 1 d, orangeꢀred after 5 d and gradually a terracotta red
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after 30 d. This can be explained by the increasing concentration of 3ꢀNTyr residue in the
EBN as the fumigation time increases.
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In fumigation, sodium nitrite and hydrochloric acid react steadily to yield nitrous acid
vapor,
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NaNO2(aq) + HCl(aq) → HNO2(aq, g) + NaCl(aq) .
(1)
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The nitrous acid undergoes disproportionation to yield reactive nitrogen species (RNS), e.g.
•NO2 and •NO,12, 13
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2HNO 2
•NO2 + •NO + H2O .
(2)
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Besides reacting with the glycoprotein tyrosine, the radicals can also undergo the following
reactions,
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2•NO2
N2O4
(3)
(4)
N2O4 + 2H2O → NO3 + NO2 +2H3O+
.
‾
‾
In the presence of atmospheric O2, the oxidation of •NO can also occur,14
4•NO + O2 + 6H2O → 4NO2 + 4H3O+
.
(5)
‾
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The EBN in the birdhouse is thus exposed to an atmosphere containing •NO2, •NO, NO2 , and
‾
NO3 .
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In analogy to protein tyrosine nitration in living systems, the reaction between the RNS
and protein tyrosine can occur by a radical mechanism as shown in Scheme 1. The one
electron oxidant •NO2 can undergo a slow reaction with the glycoprotein tyrosine to give the
tyrosyl radical, and a further fast reaction with •NO2 gives 3ꢀNTyr.15, 16 The tyrosyl radical
can also react (slowly) with •NO to form the intermediate 3ꢀnitrosotyrosyl residue which is
rapidly oxidized to 3ꢀNTyr, e.g. by HNO3.13, 15, 17 The intermediate 3ꢀnitrosotyrosyl residue
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can be trapped by the addition of Hg(II) to give a red pigment as in Millon’s test for
tyrosine.18
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The natural red EBN, Figure 1B, came from a birdhouse. In birdhouses, there’s swiftlet
bird soil on the floor. The bird soil is rich in protein and nitrogenous compounds because the
swiftlets feed on a high protein diet of aerial insects. Aerobic bacteria can feed on the bird
soil and its metabolism produces nitrous acid and nitric acid vapors. If the concentration of
nitrous acid reaches a sufficient level in the largely enclosed birdhouse, it will be absorbed by
the white EBN initially built by the swiftlet on the walls and ceiling of the birdhouse, and
reacts by Scheme 1 first to a yellow/golden color, then to red, and finally to a terracotta red as
the concentration of 3ꢀNTyr increases in the EBN, just as in Figure 2. Thus, even the white
nest can have a small amount of 3ꢀNTyr. Equations (4) and (5), and also nitric acid vapor
from the bird soil, explains why red EBN would have a significant amount of nitrite and
nitrate. This makes red EBN an unsafe food or health supplement, if not properly cleaned.
The toxicity of high concentrations of nitrite and nitrate (found in thousands of ppm in red
EBN) was what led China to put a ban on the sale of both white and red EBNs at one time.19
The nitrate and nitrite in EBN, being water soluble, can be rinsed away, and this is carried out
before analyzing for 3ꢀNTyr to avoid interference.
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In the xanthoproteic reaction of EBN with a mixture of nitric and sulfuric acid to give red
EBN, significant amounts of 3ꢀNTyr is formed by Scheme 2. By conjugating with sulfuric
+
acid, nitric acid produces a nitronium ion (NO2 ), which is the active species in electrophilic
aromatic nitration,
+
ꢀ
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HNO3(aq) + 2H2SO4(aq)
NO2 (aq) + H3O+(aq) + 2HSO4 (aq) .
(6)
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The nitronium ion reacts with the aromatic ring to form a nitrotyrosyl arenium ion, and
10,
subsequently the H+ is eliminated by a nucleophile, H2O, and a neutral 3ꢀNTyr is formed.
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Determination of 3-NTyr in white and red EBN with ELISA
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An antiꢀ3ꢀNTyr ELISA kit was used to detect and quantify the 3ꢀNTyr in natural white
EBN, natural red EBN, HNO2 fumigated EBN and xanthoproteic reacted EBN. The
concentrations of 3ꢀNTyr detected and the degree of protein tyrosine nitration are shown in
Table 1.
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The concentrations of 3ꢀNTyr in red EBNs are hundreds to thousands of times higher
than in white EBN. In the natural white EBN, the concentration of 3ꢀNTyr is very low 2.5 ±
1.2 ppm, while natural red EBN and HNO2 fumigated EBN contained (9.84 ± 1.24)x102 ppm
and (2.53 ± 0.45)x103 ppm of 3ꢀNTyr, respectively. It was observed that a higher degree of
nitration could be achieved if the fumigation time was lengthened or the surface area of the
EBN was increased, e.g. by pulverization. The xanthoproteic reaction gave a more effective
nitration of the glycoprotein tyrosine, and the red EBN produced had an even higher
concentration of 3ꢀNTyr, (8.28 ± 1.64)x103 ppm. The EBN tyrosyl nitration degree in Table
1, ranging from <0.0034% for natural white EBN to about 7.6% for the xanthoproteic reacted
EBN, was calculated based on 87.2 mg tyrosine per g of EBN.5
273
Color change in the red EBN strands as a function of pH
274
275
276
277
278
279
280
281
282
A color change as a function of pH, like an indicator, was observed in strands of red
EBNs when they were soaked in the respective buffers and airꢀdried. Figure 3 shows the
microscopic images where a color change from light yellow/golden to a deep red can be
observed when the buffer changed from acidic to alkaline pH. The natural red EBN and
HNO2 fumigated EBN were pale yellow from pH 2 to 5, then an orange hue developed from
pH 6 to 9, and finally a deep red was seen from pH 10 to 13. The color intensity of natural
red EBN is lower than HNO2 fumigated EBN due to the lower concentration of the 3ꢀNTyr
chromophore. A similar color change (not shown) occurred with strands of the xanthoproteic
reacted red EBN.
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284
UV-Vis absorption spectra of 4-nitrophenylalanine and 3-nitrotyrosine as a function of
pH
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290
Figure 4A shows the absorption spectra of 4ꢀnitrophenylalanine (4ꢀNP) as a function of
pH from 4 to 10. It absorbs in the UV region and would not show any visible color. The
absorption bands are similar with a maximum at 280 nm for pH 4ꢀ8, and a maximum at 290
nm for pH 9ꢀ10. There would be a gradual change in the absorption band from one form to
another at around pH 9, due to the change from protonated to deprotonated amine on the
amino acid as phenylalanine has pKa2≈9.1.
291
292
293
294
295
296
297
298
In the UVꢀVis absorption spectra of 3ꢀNT, the hydroxyl (ꢀOH) auxochrome in 3ꢀNT,
conjugated with the nitro (ꢀNO2) group chromophore, when deprotonated (to ꢀO‾) will give a
bathochromic shift of the absorption. The absorption spectra of 3ꢀNT (Figure 4B) in acidic
solutions, with –OH intact, show an absorption maximum at 358 nm. As the pH is increased,
the absorption at 358 nm gradually decreases, and the absorption with a 427 nm maximum
for the deprotonated phenolate ꢀO‾ form of 3ꢀNT increases. The color change due to the
phenolꢀphenolate reversible equilibrium and the presence of an isosbestic point at 380 nm
makes 3ꢀNT, with a pKa of 7.1, an indicator.21, 22
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300
301
302
303
304
The color change of 3ꢀNT solutions from light yellow to red as the pH is changed from 2
to 13, is shown in Figure 4C. Clearly, the color change with pH for red EBN strands in Figure
3 is similar. It suggests that the chromophore present in red EBN is likely to be 3ꢀNT. The
phenylalanine in EBN is much less readily nitrated compared to tyrosine, and even if it is
nitrated, its absorption is in the UV region and will not show any visible color change with
pH.
305
UV-Vis absorption spectra of white and red EBN as a function of pH
306
307
To ascertain that the absorbing chromophore in red EBN is 3ꢀNTyr, the absorption
spectra of solubilized white and red EBNs were investigated. Figure 5 shows the absorption
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spectra of white EBN as a function of pH. The absorption falls in the UV region and is nearly
pH independent. The absorption band at 280 nm is due to the πꢀπ* transition of the aromatic
amino acids – tyrosine and phenylalanine – in EBN.23
309
310
311
312
313
314
315
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317
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319
320
321
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324
Figures 6AꢀC show the absorption spectra as a function of pH for the three types of red
EBNs – natural, HNO2 fumigated, and xanthoproteic reacted. The corresponding difference
spectra between the red and white EBNs, Figures 6DꢀF, would reveal the absorbing
chromophore. They each show a bathochromic shift with a clear resemblance to the pHꢀ
dependent absorption spectra for 3ꢀNT (Figure 4B), with absorption maxima at ca. 360 nm in
acidic medium, shifting to ca. 430 nm in basic medium, and an isosbestic point at ca. 380 nm.
The absorptivity ε in Figures 6AꢀC is given in terms of the concentration (g Lꢀ1) of EBN, and
then was scaled using the concentration of 3ꢀNTyr determined by ELISA in Table 1 for the
respective red EBNs to give the molar absorptivity of 3ꢀNTyr in EBN in Figures 6DꢀF. It
corresponds well to the molar absorptivity of pure 3ꢀNT at the 427 nm absorption maximum,
within the margin of error of the determination of 3ꢀNTyr concentration by ELISA. The
nitration could also produce a small amount of 4ꢀNP residue on the glycoprotein and this has
not been subtracted out in Figures 6DꢀF; it would raise the difference absorption spectra only
in the 290ꢀ350 nm region slightly, but would not affect the major band at around 427 nm.
325
Raman spectra showing the nitro group in red EBN
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328
329
330
331
Raman microspectroscopy with 532 nm laser excitation was used to confirm the presence
of the nitro group in red EBN. The Raman spectra of white EBN, natural red EBN, HNO2
fumigated EBN, xanthoproteic reacted EBN, and pure 3ꢀNT are shown in Figures 7AꢀE,
respectively. The two rectangular boxes in Figures 7BꢀD frame the spectral regions where the
red EBNs differ significantly from the white EBN. The red EBNs show a strong Raman band
at 1330 cmꢀ1 which is due to the symmetric –NO2 stretch, and a strong Raman band at 825
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333
cmꢀ1 which is due to the –NO2 scissoring bend.24, 25 These two –NO2 Raman bands are also
present in the Raman spectrum of 3ꢀNT, Figure 7E.
334
EBN as a scavenger for reactive nitrogen species
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336
337
338
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340
341
In summary, our findings, based on the color change with pH, UVꢀVis spectra as a
function of pH, and Raman microspectroscopy on red EBN, provide conclusive evidence for
the nitration of tyrosine residues in the mucin glycoprotein of EBN to yield 3ꢀNTyr which
gives rise to the red color. The presence of 3ꢀNTyr in red nest is a postꢀtranslational
modification. The 3ꢀNTyr pigment, with a pKa≈7.1, behaves like an indicator and exhibits a
color change from yellow in acid to red in alkali, with the color change occurring around pH
7.
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347
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349
350
351
Generally, protein nitration by RNS is a complex process. It has been suggested that
protein nitration is dependent on the proximity of the protein tyrosine to the nitration source,
but not all tyrosine residues in a protein are equally susceptible to nitration. There is no
specific amino acid consensus sequence or specific protein primary structure that determines
the site of tyrosine nitration.26, 27 From our results, it was found that EBN is quite readily
nitrated. The high concentration of asparagine/aspartic acid (60.1 mg gꢀ1), glutamine/glutamic
acid (48.9 mg gꢀ1), and glycine (21.1 mg gꢀ1) residues5 in EBN might be responsible for this,
as it has been noted that the acidic amino acids (glutamic acid or aspartic acid) or turnꢀ
inducing amino acids (proline and glycine) in the vicinity of the targeted tyrosine are the
characteristic features observed for nitrated proteins.28
352
353
354
355
356
In vivo, a high concentration of RNS can induce pathological effects on our body, and the
proteinꢀbound tyrosine in our organs and tissues react with RNS to form 3ꢀNTyr.29, 30 The
levels of protein nitration is clinically viewed as a marker of oxidative or nitrative stress.31, 32
For this reason, a food material like EBN that is able to mop up RNS may help to alleviate
diseaseꢀrelated nitrative stress.33-36 This could be one of the new benefits in the prescription
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of EBN in traditional Chinese medicine. White EBN, where >99% of the tyrosine residues
are not nitrated would therefore be more beneficial than the colored yellow, golden, orange or
red EBNs where a portion of the tyrosine residues would have already been spent, i.e.
converted to 3ꢀNTyr.
358
359
360
361
362
363
364
365
Preliminary results show that EBN is also a very strong antioxidant, with higher
antioxidant capacity than black garlic, ginseng or goji berry, and white EBN is a stronger
antioxidant compared to red EBN. Part of the antioxidant capacity of EBN arises from the
protein bound tyrosine, so tyrosine plays multiple roles of mopping up RNS as well as free
radicals and oxidants. This work is ongoing.
366
ABBREVIATIONS USED
367
368
369
370
371
EBN, Edible bird’s nest; RNS, reactive nitrogen species; 3ꢀNT, pure 3ꢀnitrotyrosine; 4ꢀNP, 4ꢀ
nitrophenylalanine; 3ꢀNTyr, 3ꢀnitrotyrosine residue in the glycoprotein; ELISA, Enzymeꢀ
linked immunosorbent analysis; NMR, nuclear magnetic resonance; LCꢀMS, liquid
chromatographyꢀmass spectroscopy; ESI, electrospray ionization; RPꢀHPLC, reversedꢀphase
highꢀperformance liquid chromatography; HRP, horseradish peroxidase
372
FUNDING SOURCES
373
374
We gratefully acknowledge the financial support of the School of Physical and Mathematical
Sciences, Nanyang Technological University, for consumables and instrument time.
375
376
CONFLICTS OF INTEREST
The authors declare no conflicts of interest.
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FIGURE CAPTIONS
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Figure 1. A. White EBN; B. Natural red EBN; C. HNO2 fumigated EBN; and D.
Xanthoproteic (HNO3+H2SO4) reacted EBN.
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478
479
Figure 2. Development of the red color in EBN by HNO2 fumigation from 0 – 30 days (d).
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481
Figure 3. Color change in strands of natural red EBN (top panel) and HNO2 fumigated EBN
(bottom panel) as pH is varied from 2 to 13, observed under the microscope.
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483
484
485
486
Figure 4. UVꢀVis absorption spectra of A. 4ꢀnitrophenylalanine, and B. 3ꢀnitrotyrosine – pH
10 (black), pH 9 (red), pH 8 (orange), pH 7 (green), pH 6 (blue), pH 5 (indigo) and pH 4
(purple); and C. Color change of 3ꢀnitrotyrosine solution (5 g Lꢀ1) from yellow to red with pH
from 2 to 13.
487
488
489
490
Figure 5. pH independent UVꢀVis absorption spectra of solubilized white EBN – pH 10
(black), pH 9 (red), pH 8 (orange), pH 7 (green), pH 6 (blue), pH 5 (indigo) and pH 4
(purple).
491
492
493
494
495
Figure 6. pH dependent UVꢀVis absorption spectra (A ꢀ C) of A. natural red EBN (top panel),
B. HNO2 fumigated EBN (middle panel), and C. xanthoproteic reacted EBN (bottom panel)
and the corresponding difference spectra (D ꢀ F) between red and white EBN – pH 10 (black),
pH 9 (red), pH 8 (orange), pH 7 (green), pH 6 (blue), pH 5 (indigo) and pH 4 (purple). In (Aꢀ
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C), the absorption spectra in the region between 320ꢀ600 nm have been scaled up by an
appropriate factor, as indicated, for visualization.
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498
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500
501
502
503
504
Figure 7. Comparison of Raman spectra of A. raw white EBN (black), B. natural red EBN
(red), C. HNO2 fumigated EBN (orange), D. xanthoproteic reacted EBN (green), and E. 3ꢀ
nitrotyrosine (blue). The boxes over the red EBNs show: (a) the 1330 cmꢀ1 Raman band
which is due to the symmetric –NO2 stretch, and (b) the 825 cmꢀ1 Raman band which is due
to the –NO2 scissoring bend. These bands are also present in 3ꢀnitrotyrosine.
505
506
507
508
Scheme 1. The reactions between the protein tyrosine residues and reactive nitrogen species ꢀ
•NO2 and •NO – to produce 3ꢀNTyr during HNO2 fumigation. The illustration is not to scale
as the mucin glycoprotein is a 3D giant macromolecule in comparison to the tyrosine residue.
For the sake of clarity, only one tyrosine residue is shown.
509
+
510
511
Scheme 2. Electrophilic aromatic substitution reaction by the nitronium ion, NO2 , on the
protein tyrosine residues in the xanthoproteic reaction to produce 3ꢀNTyr.
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TABLES
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515
Table 1. Concentrations of 3ꢀnitrotyrosine residue found in white and red EBNs by ELISA
test.
EBN tyrosyl nitration
sample
[3-nitrotyrosine] (ppm)
degree (%)
< 0.0034
natural white EBN
natural red EBN
2.5 ± 1.2
(9.84 ± 1.24)x102
0.90 ± 0.11
EBN fumigated with NaNO2/HCl
for 30 days
(2.53 ± 0.45)x103
(8.28 ± 1.64)x103
2.32 ± 0.41
7.60 ± 1.50
xanthoproteic reacted EBN
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GRAPHIC FOR TABLE OF CONTENTS
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Figure 2. Development of the red color in EBN by HNO2 fumigation from 0 – 30 days (d).
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Figure 3. Color change in strands of natural red EBN (top panel) and HNO2 fumigated EBN (bottom panel) as
pH is varied from 2 to 13, observed under the microscope.
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Figure 4. UV-Vis absorption spectra of A. 4-nitrophenylalanine, and B. 3-nitrotyrosine – pH 10 (black), pH 9
(red), pH 8 (orange), pH 7 (green), pH 6 (blue), pH 5 (indigo) and pH 4 (purple); and C. Color change of 3-
nitrotyrosine solution (5 g L-1) from yellow to orange-red with pH from 2 to 13.
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Figure 5. pH independent UV-Vis absorption spectra of solubilized white EBN – pH 10 (black), pH 9 (red),
pH 8 (orange), pH 7 (green), pH 6 (blue), pH 5 (indigo) and pH 4 (purple).
533x381mm (96 x 96 DPI)
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