Functional hybrids of layered double hydroxides with hemin: Synergistic effect for peroxynitrite-scavenging activity

Article abstract of DOI:10.1039/c4ra08200a

Hemin has been successfully modified onto the surface of CuAl layered double hydroxide nanosheets by a simple coprecipitation process, which afforded a hemin modified CuAl layered double hydroxide (H-LDH) hybrid functional material that exhibited protective effects against the harmful ONOO^-. The obtained products were characterized by X-ray powder diffraction, scanning electron microscopy, transmission electron microscopy and Fourier transform infrared spectroscopy, which showed that the samples had hexagonal symmetry structure with a mean lateral size of 1 μm, on the surface of which were adsorbed round particles with a diameter of about 300 nm. A detailed inhibition study on ONOO^--mediated nitration reactions indicated that the interaction between hemin and LDHs results in a synergistic effect, which leads to an efficient reduction of ONOO^- to nitrate. The present study suggests that the H-LDHs had an efficient ONOO^- scavenging ability, and may well be a potent ONOO^- scavenger for protection of the cellular defense activity against ONOO^- involved diseases.

Full text of DOI:10.1039/c4ra08200a

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Functional hybrids of layered double hydroxides
with hemin: synergistic effect for peroxynitrite-
scavenging activity
Cite this: RSC Adv., 2014, 4, 44614
Fengmin Qiao, Weijie Shi, Jing Dong, Wei Lv* and Shiyun Ai*
Hemin has been successfully modified onto the surface of CuAl layered double hydroxide nanosheets by a
simple coprecipitation process, which afforded a hemin modified CuAl layered double hydroxide (H-LDH)
ꢀ
hybrid functional material that exhibited protective effects against the harmful ONOO . The obtained
products were characterized by X-ray powder diffraction, scanning electron microscopy, transmission
electron microscopy and Fourier transform infrared spectroscopy, which showed that the samples had
hexagonal symmetry structure with a mean lateral size of 1 mm, on the surface of which were adsorbed
ꢀ
round particles with a diameter of about 300 nm. A detailed inhibition study on ONOO -mediated
Received 6th August 2014
Accepted 11th September 2014
nitration reactions indicated that the interaction between hemin and LDHs results in a synergistic effect,
ꢀ
which leads to an efficient reduction of ONOO to nitrate. The present study suggests that the H-LDHs
DOI: 10.1039/c4ra08200a
ꢀ
ꢀ
had an efficient ONOO scavenging ability, and may well be a potent ONOO scavenger for protection
ꢀ
www.rsc.org/advances
of the cellular defense activity against ONOO involved diseases.
test tubes such as selenomethionine, selenocystine, and
ebselen.
Increasing attention has been paid to layered double
1
. Introduction
14,15
ꢀ
Peroxynitrite (ONOO ), a potent cytotoxic oxidant, is a product
formed by the reaction between nitric oxide (NO) and super- hydroxides (LDHs), also known as hydrotalcite-like compounds
ꢀ
). Stimulated macrophages, neutrophils, and anionic clays. They can be represented by a general formula
2
1
oxide radicals (cO
endothelial cells and Kupffer cells have been known to generate of [M 1ꢀxM x(OH)
2
+
3+
x+ nꢀ
xꢀ
2+
3+
2
2
] [A x/n] $mH O (M /M : divalent and
ꢀ
2
ꢀ
ONOO . ONOO is able to induce peroxidation of lipids, trivalent metal cations; x ¼ 0.2–0.33), which consist of octahe-
nꢀ
oxidation of sulydryl group, nitration of tyrosine residue in dral brucite-like host layers, charge-balancing anions (A ), and
3
4
16
protein, and cause DNA damage. Protein tyrosine residues are interlayer water molecules. This highly tunable intralayer
5,6
especially susceptible to peroxynitrite nitration. The stable composition, coupled with a wide possible choice of anionic
end-product, 3-nitrotyrosine, is considered to be a biomarker of moieties, affords large varieties of multifunctional LDH mate-
7
17
peroxynitrite-specic protein damage. 3-Nitrotyrosine is found rials for potential applications including anion exchangers,
18
19
in tissue specimens from several diseases, including cardio- adsorbents, catalysts, solid-state nanoreactors and molec-
21
20
vascular disorders, degenerative neurological disorders and ular sieves, polymer composites, and bioactive materials.
certain renal diseases. This suggests that peroxynitrite is Furthermore, the bio-compatibility and low toxicity of LDHs as
8
involved in the pathological processes of these diseases. Since green materials enables their ecological, biological and medic-
ꢀ
ꢀ
endogenous scavenging enzymes responsible for ONOO inac- inal applications. Recent reports demonstrate that LDH crystals
tivation are lacking, seeking scavengers specic to ONOO is of can be articially exfoliated into macromolecular nanosheets
2
2–24
considerable importance. Currently, there is an increasing through so chemical procedures.
In addition, through
ꢀ
interest in screening products for possible ONOO scavengers. static adsorption, functional molecules could be modied on
ꢀ
25,26
To develop powerful ONOO scavengers, many different the surface of the hydrotalcite piece.
The nanosheets are also
methods have been utilized. One example is to screen naturally- expected to be used as building blocks for the construction of
occurring compounds from herbal sources, including ascorbic various functional nanocomposites or nanostructures.
9
10
11
12,13
acid, avonoids, isoavonoids, ergothioneine
and poly-
Functionalization of LDH crystals with polar molecules
hydroxyphenols from fruits, wine, tea, and green vegetables. imparts the hydrophilicity effects, and thus enhances its dis-
ꢀ
27
While another method is to synthesize ONOO scavengers in persibility in polar solvents. Particularly, noncovalent func-
tionalization of LDH crystals with biomolecules make the LDH
crystals highly biocompatible with the conservation of intrinsic
properties of LDH crystals. Hemin, a well-known protopor-
phyrin found at the active sites of hemin proteins, plays a key
College of Chemistry and Material Science, Shandong Agricultural University, Taian,
28
2
5
71018, P. R. China. E-mail: ashy@sdau.edu.cn; Fax: +86 538 8242251; Tel: +86
38 8247660
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29,30
role in biochemical reactions and electron-transport chain.
native LDH was prepared by the same method without intro-
Accordingly, hemin was stabilized on LDHs sheets by non- ducing hemin solution.
covalent functionalization through p–p interactions to yield
The morphology and size distribution of the product were
hemin-functionalized CuAl-layered double hydroxides (H- studied by using a JEM-2010 transmission electron microscope
LDHs) hybrid microchips. Herein, we describe a coprecipita- (TEM, Japan) and a JSM-6610 scanning electron microscopy
tion method for the construction of H-LDHs microchips. We (SEM, Japan).The crystal phase was investigated by a Rigaku
ꢀ
also demonstrate, to our knowledge, for the rst time, that H- DLMAX-2550 V diffractometer (40 kV, Cu Ka (l ¼ 1.54056 A), 2q
ꢁ
ꢁ
LDHs microchips exhibit remarkable peroxynitrite-scavenging range 5–80 ; scan speed of 6 per min). Fourier transform
activities. The data support the conclusion that H-LDHs can infrared (FT-IR) spectra were obtained on a Thermo Nicolet-380
exhibit a biological function by acting as a peroxynitrite scav- IR spectrophotometer (USA).
enger. Scavenging activity has a wide range of practical appli-
cations. From the chemical and medicinal point of view, the
ability to catalyse isomerization and reduction of ONOO and
2
.3 Preparation of peroxynitrite
ꢀ
Peroxynitrite (PN) was prepared using a quenched-ow reactor
2
scavenging of cNO to reduce their toxicity is frequently used in
32
as described in the literature. A 20.00 mL solution containing
.6 M HCl and 0.7 M H O was rapidly mixed with an equal
the pathological processes of related disease.
0
2
2
volume of solution containing 0.6 M NaNO in ice-water bath,
2
the above reaction was quenched by 1.5 M NaOH with volume of
2. Experimental section
30.00 mL simultaneously. The excess H
2 2
O was eliminated by a
2.1 Reagents and apparatus
small amount of MnO2 powder. The resulting solution was

ltered and obtained light yellow ltrate, which could be stably
Copper nitrate (Cu(NO ) $3H O), Aluminium nitrate
3
2
2
ꢁ
stored at ꢀ20 C for several weeks. The concentration (2.41 mM)
of the peroxynitrite stock solution was determined by
measuring the absorbance at 302 nm using a molar extinction
(
Al(NO ) $9H O) were purchased from Aladdin (Shanghai,
3 2 2
China). Sodium hydroxide (NaOH), absolute ethyl alcohol
(CH
3
CH
), ammonium hydroxide (NH
) were purchased from Kay Tong Chemical
Reagents Co. Ltd (Tianjin, China). L-tyrosine(C ) and
hemin(C H ClFeN O ) were purchased from Shanghai Bio 2.4 Reaction of tyrosine with peroxynitrite
2
OH), hydrogen peroxide (H
2
O
2
), sodium nitrite
ꢀ
1
ꢀ1
coefficient of 1670 M cm at 302 nm using a UV-2450 Shi-
(
NaNO
2
3
$H
2
O), sodium hydrogen
33
madzu Vis-spectrometer.
carbonate (NaHCO
3
3 3
H11NO
3
4
32
4 4
Science & Technology Co. Ltd (Shanghai, China). Phosphatic
buffer solution (100 mM, pH value range from 5.5 to 11.5) were
used in this work and double distilled deionized water was
applied throughout the experiment. All chemicals used in this
work were of analytical grade and used as received without
further purication.
ꢀ
The reaction between tyrosine and ONOO was investigated.
4
ꢀ
00 mL increasing nal concentration of ONOO (from 0.08 mM
to 2.41 mM) was reacted with 400 mL tyrosine (0.86 mM) in pre-
blended solution containing 400 mL phosphate buffer (100 mM,
pH 7.5) and 200 mL Sodium hydrogen carbonate (NaHCO , 0.14
3
M) at room temperature for 10 min, followed by a spectropho-
tometric scan with a UV-2450 Shimadzu Vis-spectrometer from
3
00 to 600 nm.
2
.2 Preparation of hemin-functionalized CuAl-layered
To test the inuence of temperature and pH on tyrosine
double hydroxides
nitration, peroxynitrite (2.41 mM) was added to 0.86 mM tyro-
Hemin-functionalized CuAl-layered double hydroxides (H- sine in phosphate buffer (100 mM) at a range of temperatures
ꢁ
LDHs) with different content of hemin were prepared by the (0–55 C) or a range of values of pH (5.5–11.5) for 10 min
31
coprecipitation method adapted for enzyme solution. Typi- incubation, the absorbance at 426 nm was determined.
cally, hemin (1 mg) was rstly dissolved in 20 mL ethanol by the
addition of 80 mL NH
3
to get dark reddish-brown solution. A
2
.5 Investigation of the peroxynitrite-scavenging activities of
2
+
mixed aqueous solution of Cu(NO ) and Al(NO ) , with a Cu /
3 2 3 3
H-LDHs
3
+
Al molar ratio r ¼ 2 and a total concentration of metallic
cations of 100 mM, was introduced into the above dark reddish-
brown solution. The pH was maintained constant at a value of
The peroxynitrite-scavenging activity of the H-LDHs (hemin
content 20 mg) was evaluated on the basis of the decomposition
of peroxynitrite. The scavenger was added into ONOO stock
solution, the reaction process of equivalent H-LDHs, LDHs and
hemin with 400 mL ONOO were monitored in timescan mode
ꢀ
9
.0 during all the coprecipitation by the simultaneous addition
of a 2 M NaOH solution. This solution was purged by a nitrogen
gas ow to expel air/O . Aer aged at room temperature with
ꢀ
2
at 302 nm using a UV-2450 Shimadzu Vis-spectrometer.
stirring for 6 h, the sample was ltered, washed with water, and
air-dried for physical characterization. Samples with different
content of hemin (5, 10, 15, 20, 25, 30 mg) were prepared by the
same method. For the sake of convenience in writing, H-LDHs
2.6 Inhibition of peroxynitrite-mediated tyrosine nitration
by H-LDHs
with different contents of hemin (1, 5, 10, 15, 20, 25, 30 mg) The peroxynitrite-scavenging activity of the H-LDHs was evalu-
were aliased as H-LDH (1), H-LDH (2), H-LDH (3), H-LDH (4), ated based on the inhibition of peroxynitrite-induced nitration
H-LDH (5), H-LDH (6), H-LDH (7), respectively. For comparison, of free tyrosine. The nitration of tyrosine was performed as
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previously reported. Briey, We employed a mixture containing
L-tyrosine (0.86 mM), sodium hydrogen carbonate (NaHCO ,
3
ꢀ
0
.14 M) and ONOO (2.41 mM) in sodium phosphate buffer
(
100 mM, pH 7.5) without and with increasing content of hemin
ꢁ
in H-LDHs at 35 C. It was incubated for 10 min before
recording absorbance. The formation of 3-nitrotyrosine was
monitored at the wavelength 426 nm using UV/vis spectroscopy.
2.7 Investigation of the H-LDHs's cyclic utilization
In order to explore whether the product H-LDHs can be reused
for the scavenging of peroxynitrite, the same test was repeated
for ve times. Briey, the inhibition of peroxynitrite-mediated
tyrosine nitration by H-LDHs (1.2 mg) was performed as previ-
ously reported. Soon aerwards, the reaction solution still
containing H-LDHs was centrifuged and washed several times
with decarbonated water, and nally dried in air at room
temperature for the next use. In each time, the formation of 3-
nitrotyrosine was monitored at the wavelength 426 nm using
UV/vis spectroscopy to test the H-LDHs' inhibition
performance.
Fig. 1 (a) TEM; (b) SEM; (c) EDS images of hemin modified CuAl-LDHs
nanocomposite.
2.8 PN scavenging activity in PN-mediated protein (BSA)
nitration
In terms of bovine serum albumin (BSA), the nitration was
performed by the addition of PN (2.41 mM) to BSA (0.29 mM) in
ꢁ
1
00 mM phosphate buffer of pH 7.5 at 35 C. The reaction
mixture was incubated for 10 min. Similarly, the reactions of
BSA with PN were performed in the absence and presence of H-
LDHs. The formation of 3-nitrotyrosine was monitored at the
wavelength 426 nm using UV/vis spectroscopy.
3. Results and discussions
3.1 Characterization of the H-LDHs
The non-covalently graed hemin on LDHs was characterized
by various methods. Fig. 1a shows TEM images of H-LDHs
sample. As can be seen, the sample consisted of irregular and
thin hexagonal platelets with a mean lateral size as large as 1
mm and a thickness of about 30 nm. In addition, on the surface
of LDHs, round particles with a diameter of about 300 nm, are
also observed. The TEM (Fig. 1a) and SEM images (Fig. 1b) of H-
LDHs hybrid showed aky features with folded morphologies
and round particles absorbed on surface. As is known, the
native LDHs displays uniform and thin hexagonal platelets,
whereas the presence of biomolecules in the reactive medium
Fig. 2 FT-IR spectra of (a) CuAl LDHs, (b) heme modified H-LDHs, (c)
heme.
structures. A broad absorption in the low-frequency region (593
ꢀ1
and 876 cm ) may be assigned to M–O (M:Cu/Al) stretching
and M–OH bending vibrations in the octahedral hydroxyl
sheets. Aer treatment with hemin (spectrum b), a broad band
ꢀ1
centered at 3408 cm replaces the sharp band of the OH
stretching mode, which is usually interpreted as stretching
modes of OH groups with hydrogen bonding and of interlayer
34–36
leads to quite different sample morphology.
It seems that
the modication of the LDHs surface by the covering of hemin
induces such aggregation of platelets strongly associated with
the biomolecules. As determined by EDS (Fig. 1c), the
predominant elements in the H-LDHs samples were Cu, Al and
O in various compounds. Lesser amounts of the elements Fe, N
and C were also observed.
ꢀ1
hemin molecules. In particular, the bands at 876, 593 cm
ꢀ1
sharpens strongly and the bands at 1386 cm are markedly
enlarged. Obviously, these changes are due to the formation of
hydrogen bonding, p–p interaction between LDHs sheets and
hemin. For the loading of hemin, the broadening and enlarging
of nitrate can be explained by the coverage of the LDH surface
As shown in Fig. 2, the interactions between hemin and CuAl
LDHs were investigated by FT-IR. Spectrum a shows a sharp
by hemin, while when the surface is not saturated by the hemin,
2ꢀ
the NO
3
anions ensure the charge compensation of the
ꢀ
1
band at 3408 cm , attributed to the OH stretching mode,
which is characteristic of free OH groups in brucite-like
lattice. The vibration bands of the conned heme are at the
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Fig. 4 a) Nitration of free L-tyrosine with increasing concentration of
peroxynitrite and (b) linear fitting. Dependency of the PN-mediated
nitration of free L-tyrosine on Temperature (c) and pH (d). Experiments
Fig. 3 XRD spectra of heme modified H-LDHs.
ꢀ
were carried out using 2.41 mM ONOO in 400 mL sodium phosphate
buffer (100 mM) contains 0.86 mM L-tyrosine, 0.14 M sodium
same wavelength position than those of the free enzyme, sug- hydrogen carbonate.
gesting that the molecular structure of the heme seems to be
preserved aer being absorbed onto the inorganic materials.
Fig. 3 showed the X-ray powder diffraction (XRD) pattern of
H-LDHs composites, and the XRD pattern showed that they
have a hexagonal symmetry, the same as reported in the liter-
showed the impact of pH on the tyrosine nitration. As shown,
under acid conditions, the content of 3-nitrotyrosine in the
reaction products is less. Once nitrotyrosine was formed at pH
37
ature. The characteristic diffraction peaks of H-LDHs still
appeared aer interaction, revealing that the process of hemin
adsorption did not do damage to the interlayer structure of
LDHs. They all had a good crystallinity. The new peaks appeared
might be caused by the coordinate bond which formed aer
hemin was adsorbed on the surface of LDHs. Obviously, the
presence of the (012) and (110) diffraction lines evidence the
formation of the layered structure. The basal spacing of LDHs
was around 0.7 nm before adsorption and tiny changes
7
.5, the absorbance was maximal at the 426 nm. Then, with the
increasing pH of solution, the content of 3-nitrotyrosine
decreased dramatically. ONOO could stay stable under alka-
line conditions, while the content of 3-nitrotyrosine decreased,
this phenomenon implies that tyrosine nitration is not the
result of direct reaction between tyrosine and ONOO . By
contrast, under acid and neutral conditions, ONOO was
protonated into ONOOH, therefore reaction between tyrosine
and ONOOH could induce nitration of tyrosine.
ꢀ
ꢀ
ꢀ
37
appeared on it aer adsorption. The changes of the basal
spacing on LDHs before and aer adsorption may due to the
2
loss of H O molecules inside or on the surface of LDHs. It can 3.3 Investigation of the peroxynitrite-scavenging activities of
be concluded that hemin was not intercalated into the inter- H-LDHs
layer of LDHs but just was adsorbed on the surface of them.
ꢀ
To determine the mechanism of ONOO scavenging activity for
ꢀ
H-LDHs, the reaction process of scavengers with ONOO was
investigated. The spectrum changes at 426 nm were monitored
spectrophotometrically to ascertain whether H-LDHs undergoes
3.2 The reaction of tyrosine with PN and conditions
optimization
ꢀ
Tyrosine undergoes the nitration process to form 3-nitro- a scavenging nitration process when reacting with ONOO (8.42
ꢀ
tyrosine when it is exposed to ONOO at pH 7, showing a peak mM). As shown in Fig. 5, compared with system (a) in which the
at 426 nm. In the present study, it was found that exposure of peroxynitrite exists on its own at the start, the absorbance of
ꢀ
ꢀ
tyrosine (0.86 mM) to increasing concentrations of ONOO (8– ONOO in system (b), (c) and (d) decreased in turn. Obviously,
2
41 mM) resulted in an increased production of 3-nitrotyrosine the decomposition rate of peroxynitrite of system (d) in which
ꢀ
(Fig. 4a) with a linear dependence in the linear range of ONOO
the peroxynitrite was mixed with H-LDHs is far faster than that
ꢀ
from 8 to 120 mM. Nitration of tyrosine can be easily detected by of system (b) and (c), which indicates that the ONOO scav-
color change from colorless to the characteristic yellow color enging activity of hemin is increased by about 10-fold upon
ꢀ
when tyrosine and ONOO are mixed.
functionalization with LDHs. Treatment of LDHs with hemin
The temperature dependence of the conversion yield of afforded a functional hemin-modied (H-LDHs) hybrid mate-
tyrosine into 3-nitrotyrosine with peroxynitrite was studied in rial that exhibited remarkable protective effects against the
ꢁ
the range of 0–55 C. The yield of 3-nitrotyrosine offers potentially harmful peroxynitrite.
upgrading rstly and descending latter tendency with the
maximal absorbance at 35 C, which is approximately equal to with hemin in exhibiting the ONOO scavenging activity. This is
These observations suggest that LDHs may act synergistically
ꢁ
ꢀ
the normal temperature of human body (Fig. 4c). Fig. 4d probably due to the p donor property of LDHs to the Fe center of
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cNO
2
, and then nitrotyrosine is produced by a coupling of
5
radicals Tyr-Oc and cNO . These observations suggest that the
2
p–p interactions between the LDHs and hemin play a signi-
cant role in the observed scavenging effect.
3.4 Inhibition of peroxynitrite-mediated tyrosine nitration
by H-LDHs
In the present study, when free hemin or LDHs was tested as a
scavenger of PN in PN-mediated nitration of free L-tyrosine,
little scavenging activity was observed (Fig. 4). Peroxynitrite
(8.42 mM) produced approximately a 50-fold increase in nitro-
tyrosine compared to degraded peroxynitrite (Fig. 6). Interest-
ingly, when H-LDHs was employed as a scavenger, incubation of
ꢀ
H-LDHs with tyrosine prior to the addition of ONOO resulted
Fig. 5 Time course of peroxynitrite decomposition without (a) and in the decrease and even disappearance of the nitrotyrosine
with equivalent weight of LDHs (b), Heme (c),and H-LDHs (d). Perox- peak at 426 nm, implying that H-LDHs suppressed the forma-
ynitrite (8.42 mM) was mixed with scavengers at room temperature,
and the reaction was followed at 302 nm.
tion of nitrotyrosine. To evaluate the potency of H-LDHs to
block nitration, a full weight/weight hemin/LDHs ratio-
response histogram for the inhibition of nitrotyrosine forma-
tion was generated. This histogram revealed that H-LDHs was a
potent inhibitor of nitration. As is presented intuitively, the
extent of this inhibition was dose-dependent. With the
increasing of the content of hemin adsorbed on the LDHs
surface during the coprecipitation route, the PN-scavenging
capacity enhanced successively. As mentioned earlier, syner-
gistic effect of H-LDHs plays a major role in scavenging of PN in
these potentially harmful PN-mediated reactions.
hemin through cationic p interactions. As described for this
ꢀ
III
mechanism (Scheme 1), ONOO reacts with the Fe center in
III
the H-LDHs to generate Fe –OONO. The homolysis of the O–O
III
bond in Fe –OONO leads to the formation of caged radical
IV
Fe ]OcNO
2
intermediate. The LDHs layers may help to
and decrease their escape, which result
increase the local cNO
in the recombination of Fe ]O and cNO
reconstruction of Fe ]O and cNO generates the Fe -nitrato.
2
The break of the Fe–O in the Fe –ONO regenerates the Fe
2
center with an elimination of nitrate (NO3 ). The major effect of
2
IV
2
. Therefore, the
IV
III
III
III
3.5 Recycling properties of H-LDHs for scavenging PN
ꢀ
In summary, the H-LDHs has been used several times as a
scavenger in PN-mediated nitration of free L-tyrosine. A simple
recycling procedure has been developed and has led to the reuse
of the scavenging peroxynitrite up to four times without
LDHs is to minimize the cNO escape reaction that is usually
observed for free hemin. It should be noted that the rapid
diffusion of cNO from the hemin site is the reason for the
2
nitration of free tyrosine and tyrosine residues in protein. As
2
reported that nitrotyrosine is formed via a radical reaction of
tyrosine with cOH and cNO
caged radical pair of ONOOH. During this reaction, Tyr-Oc is
rst formed through the reaction of tyrosine with either cOH or
2
that have escaped from a solvent-

Fig. 6 PN scavenging activity in PN-mediated nitration of L-tyrosine in
the presence of various concentrations of H-LDHs. Experiments were
carried out in 400 mL sodium phosphate buffer (100 mM, pH 7.5)
contains 0.86 mM L-tyrosine, 0.14 M Sodium hydrogen carbonate, the
Scheme 1 Proposed mechanism for the isomerization and reduction above solution were mixed with PN treated in the absence and pres-
ꢀ
of ONOO and scavenging or cNO
2
by H-LDHs hybrid nanosheets.
ence of increasing concentration of scavengers.
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ꢀ
ability, which may well be a potent ONOO scavenger for the
ꢀ
protection of the cellular defense activity against the ONOO
involved diseases.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (no. 21375079, 21105056) and Project of
Development of Science and Technology of Shandong Province,
China (no. 2013GZX20109).
Fig. 7 (a) Recycling properties of H-LDHs for scavenging PN in PN-
mediated nitration of free L-tyrosine; (b) Effect of H-LDHs with per-
oxynitrite-mediated nitration of BSA. BSA (1 mM) was incubated
without (the red line) or with (the blue line) H-LDHs prior to the
addition of ONOO . Each mixed solution was incubated at 35 C with
shaking for 10 min and scanned between 300 and 600 nm with
spectrophotometric analysis. The spectrum of the peak displayed at
Notes and references
ꢀ
ꢁ
1
2
3
4
5
6
7
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2
(
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