Red Blood Cells Protect Albumin from Cigarette Smoke–Induced Oxidation
et al. (2012) Red Blood Cells Protect Albumin from Cigarette Smoke-Induced Oxidation. PLoS
ONE 7(1): e29930. doi:10.1371/journal.pone.0029930
Red Blood Cells Protect Albumin from Cigarette Smoke- Induced Oxidation
Graziano Colombo 0
Ranieri Rossi 0
Nicoletta Gagliano 0
Nicola Portinaro 0
Marco Clerici 0
Andrea Annibal 0
Daniela Giustarini 0
Roberto Colombo 0
Aldo Milzani 0
Isabella Dalle-Donne 0
Peter Csermely, Semmelweis University, Hungary
0 1 Department of Biology, Universita` degli Studi di Milano , Milan , Italy , 2 Department of Evolutionary Biology, University of Siena , Siena , Italy , 3 Department of Human Morphology and Biomedical Sciences ''Citta` Studi'', Universita` degli Studi di Milano , Milan , Italy , 4 Department of Translational Medicine, Clinica Ortopedica e Traumatologica, Istituto Clinico Humanitas and Universita` degli Studi di Milano , Rozzano, Milan , Italy
Different studies reported the presence of oxidized (carbonylated) albumin in the extravascular pool, but not in the intravascular one of cigarette smokers. In this study we attempted to explain this apparent discrepancy exposing human serum albumin (HSA) to aqueous cigarette smoke extract (CSE). CSE induces HSA carbonylation and oxidation of the HSA Cys34 sulfhydryl group. An antioxidant action of glutathione, cysteine, and its synthetic derivative N-acetylcysteine was observed only at supra-physiological concentrations, suggesting that physiological (plasma) concentrations of glutathione and cysteine in the low micromolar range are ineffective in preventing cigarette smoke-induced oxidation of HSA. Differently, human erythrocytes resulted to be protective towards CSE-induced oxidation (carbonylation and thiol oxidation) of both HSA and total human plasma proteins.
Funding: This research was supported by PUR 2009 (Programma dellUniversita` per la Ricerca), University of Milan, and by Fondazione Ariel, Centro per le
Disabilita` Neuromotorie Infantili, Milan, Italy. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
Cigarette smoke (CS), an etiological factor for the development
of many tobacco-related diseases , is a complex mixture
containing more than 7000 different constituents, including
reactive oxygen and nitrogen species (ROS and RNS) . Further
ROS/RNS production mediated through inflammatory processes
may exacerbate those produced through direct exposure . One
pathway that may contribute to the untoward health effects of CS
is the systemic oxidants/antioxidants imbalance as reflected by
increased levels of products of oxidative stress and depleted levels
of antioxidants in plasma of smokers .
Oxidative damage induced by CS is caused by some
watersoluble oxidants that may undergo circulation through the body
fluids and produce sustained oxidative damage in different organ
systems . Human plasma antioxidants include the low
molecular mass aminothiols glutathione (GSH) and cysteine
(Cys-SH), which occur in the micromolar range . Their
concentrations in plasma are decreased in association with
cigarette smoking [9,10].
Protein thiol groups (PSH) are also essential in conferring
protection against oxidative stress [8,1113]. In plasma,
concentration of PSH is mostly due to the single free thiol (Cys34) of
albumin, a single-chain polypeptide with 17 disulfide bonds, which
reaches concentrations of more than 0.6 mM (mean
,43 mg?ml21, range 3552 mg?ml21) in healthy humans ,
accounting for ,60% of total protein in the plasma of healthy
people . As a consequence, the Cys34 sulfhydryl group of
human serum albumin (HSA) represents by far the largest fraction
(,80%) of all free thiols in plasma, thus conferring a major role in
serum antioxidant capacity to HSA, which therefore represents a
quantitatively important redox buffer as well as the main
scavenger of electrophiles of the blood .
Increased levels of variously oxidized proteins have been found
in blood and/or plasma of smokers, but none of those studies
reported the occurrence of oxidized albumin . Differently,
other studies showed that the major oxidized (carbonylated)
protein in the bronchoalveolar lavage fluid in smokers with a long
term history of smoking is albumin [24,25]. A recent study
determined that human lung parenchymal tissue from COPD
patients who were current smokers contained lower levels of total
HSA, but had proportionally greater levels of oxidized
(carbonylated) HSA, compared to patients with normal lung function. Lung
tissue from current smokers was also found to contain lower levels
of HSA, which was highly carbonylated compared to lung tissue
from ex-smokers and non-smokers .
We previously showed a,b-unsaturated aldehyde-induced
carbonylation in HSA exposed to whole-phase cigarette smoke
extract (CSE), a widely used model system for studying in vitro
effects of CS  and identified the amino acids forming
covalent adducts with acrolein and crotonaldehyde . We
investigated here the ability of some plasma low molecular mass
antioxidants (i.e., GSH and Cys-SH) and the synthetic cysteine
derivative N-acetylcysteine (NAC) to protect HSA from
CSEinduced oxidation. Furthermore, considering that carbonylated
HSA has been found in fluids and tissues of smokers other than
blood or plasma and also considering the preponderance of GSH
within erythrocytes (which contain ,3 mM GSH, i.e., one order
of magnitude higher than that present in plasma) besides several
other antioxidants (amongst which peroxiredoxins are
predominant; ), we asked whether red blood cells (RBCs) were
protective towards CSE-induced oxidation of both purified HSA
and human plasma proteins as a whole.
Materials and Methods
Human blood samples were obtained from healthy donors that
voluntarily went to the Analysis Laboratory for a routinary blood
check-up, after informed verbal consent. The verbal consent was
considered to be sufficient because the samples were handled
anonymously and were used only to isolate erythrocytes and
plasma proteins. The procedure was approved by the ethics
committee of the University of Milan.
Delipidized crystalline HSA (,99% agarose gel electrophoresis),
5,59-dithiobis(2-nitrobenzoic acid) (DTNB), N-ethylmaleimide
(NEM) and 2,4-dinitrophenylhydrazine (DNPH) were purchased
from Sigma-Aldrich (Milan, Italy). ECL Plus Western blotting
detection reagents were obtained from GE Healthcare (Milan,
Italy). EZ-Link Biotin-HPDP
(N-(6-(Biotinamido)hexyl)-39-(29-pyridyldithio)-propionamide) was obtained from Euroclone (Pero,
Milan, Italy). All other reagents were of analytical grade
(SigmaAldrich, Milan, Italy). The slot-blotter (Bio-Dot SF apparatus) was
obtained from Bio-Rad Laboratories (Hercules, CA, USA).
Research-grade cigarettes (3R4F) were purchased from the
College of Agriculture c/o Kentucky Tobacco Research &
Development Center, University of Kentucky (USA).
Preparation of human mercaptalbumin (HSA-SH)
Delipidized HSA (12 mg/ml, 0.18 mM) was quantitatively
converted to mercaptalbumin (HSA-SH), in which the single thiol
of HSA is completely reduced, by treatment with 1.5 mM
dithiotreitol (DTT) in 50 mM potassium phosphate buffer (PBS),
pH 7.4, for 15 min, at room temperature. The excess of DTT was
then removed by exhaustive dialysis against 50 mM PBS, pH 7.4.
Preparation of whole cigarette smoke extract (CSE)
Whole phase CSE from Kentucky 3RF4 reference cigarettes
was prepared as previously described . Mainstream smoke
from one cigarette (10 puffs) was allowed to dissolve (for 10 s each
puff) in one ml of 50 mM PBS, pH 7.4. The resultant dark yellow
solution was defined as 100% whole phase CSE and was filtered
through a 0.22 mm Millipore filter (Bedford, MA, USA) to remove
bacteria and large particles. The pH of the whole phase CSE was
adjusted to 7.4 by addition of 2 M sodium hydroxide solution. To
ensure standardization between experiments and batches of CSE,
CSE preparations were uniformed by measurement of absorbance
at 340 nm. CSE was freshly prepared immediately before use for
each experiment and diluted to appropriate concentration with
50 mM PBS, pH 7.4.
Blood collection and isolation of erythrocytes and plasma
Human blood was obtained in the morning after 1012 h of
starving from the antecubital vein. K3EDTA was used as an
anticoagulant. Blood samples were partitioned into two groups:
one group was used for collecting RBCs and the other one was
used for collecting plasma proteins. RBCs were collected by
centrifugation at 10,000 g for 20 s and washed three times with
200 mM Na+/K+ phosphate buffered saline (PBS, pH 7.4)/NaCl
0.9% (w/v), 1:9 v/v ratio (PBS/NaCl), containing 5 mM glucose.
The washed RBCs were suspended in PBS/NaCl containing
5 mM glucose to a hematocrit value of 20%.
Plasma proteins were obtained by blood centrifugation at
1000 g for 10 min, at 4uC.
Exposure of HSA-SH to CSE
HSA-SH (molecular mass, 66.438 kDa) concentration was
determined by measuring the absorbance at 280 nm, using an
extinction coefficient of 39,800 M21cm21. In a typical
experiment, HSA-SH was diluted with 50 mM PBS, pH 7.4, to a final
concentration of 4 mg/ml (60 mM) and treated for 60 min, at
37uC, with various concentrations (1%, 4%, and 16%, v/v) of
aqueous CSE, with gentle rotary shaking. For experiments
performed with RBCs, HSA-SH solutions were exposed to 1
16% (v/v) CSE for 60 min, at 37uC, in the absence or presence of
2.5% (v/v) or 5% (v/v) erythrocytes (i.e., equal to about one tenth
of the mean human hematocrit value as in our experiments HSA
concentration too is equal to about one tenth of the mean plasma
HSA concentration) in 200 mM PBS/NaCl (see above),
containing 5 mM glucose. The removal of CSE was accomplished by
acetone precipitation: protein samples were mixed with three
volumes of 100% acetone, allowed to precipitate for 30 min at
220uC and then centrifuged at 10,000 g for 10 min, at 4uC. Pellet
were washed with 70% acetone and centrifuged at 10,000 g for
10 min, at 4uC. Finally, dried pellets were re-suspended in 50 mM
PBS, pH 7.4. All reported experiments were carried on in
Exposure of human plasma proteins to CSE
Plasma protein concentration was determined by Bradford
assay. Experiments with plasma proteins and/or RBCs were
performed at 37uC, at the protein concentration of 4 mg/ml (i.e.,
equal to one tenth of the mean HSA concentration) and with
washed RBCs re-suspended in PBS/NaCl containing 5 mM
glucose at a hematocrit of either 5% (v/v; i.e., equal to about
one tenth of the mean human hematocrit value) or 2.5% (v/v).
Plasma proteins, in the absence or presence of 2.5% or 5% (v/v)
RBCs, were exposed to various concentrations (1%, 4%, and 16%,
v/v) of CSE PBS/NaCl/glucose, at 37uC, in a water bath. After a
60-min incubation, RBCs were separated by centrifugation at
10,000 g for 20 s and supernatant with plasma proteins collected
in a new microcentrifuge tube. The removal of CSE was
accomplished by acetone precipitation as described above for
albumin exposure to CSE. Depending on experimental analysis,
protein pellet was alternatively resuspended in either PBS/NaCl
or PBS/NaCl containing 0.4 mM biotinHPDP for carbonyl
analysis or free SH determination, respectively.
Determination of HSA Cys34 free sulfhydryl group by the
Cysteine residues were quantified by the Ellman assay .
Following exhaustive dialysis against 50 mM PBS, pH 7.4, protein
samples (600 mg in 950 ml) were added with 50 ml of 3 mM DTNB
(prepared in 0.05 M phosphate buffer, pH 7.4) and incubated for
15 min at 25uC. The number of cysteines was determined by
measuring the increase in absorbance caused by the released TNB
anion upon reaction of a thiol with DTNB at 412 nm and using a
molar absorption coefficient of 14.15 mM21 cm21 . From the
increased absorbance in protein samples, the molar concentration
Determination of HSA Cys34 and plasma protein free
sulfhydryl group by biotinHPDP binding and Western
After treatment with various concentrations of CSE and CSE
removal by acetone precipitation as described above, pellets of
HSA samples (400 mg) or plasma proteins (400 mg) were
resuspendend, respectively, in 50 mM PBS, pH 7.4, or PBS/
NaCl containing 0.4 mM biotinHPDP (stock solution 4 mM in
90% dimethyl sulfoxide and 10% dimethylformamide) and mixed
by gently vortexing. The biotinylation reaction was carried out at
room temperature in the dark for 60 min, mixing by vortex every
15 min from the start of incubation. To remove biotin-HPDP
excess, biotin-HPDP-labeled protein samples were precipitated
with acetone as in the previous step and resuspendend with an
equal volume of 26 non-reducing SDS-PAGE sample buffer.
Samples were then run on SDSPAGE on TrisHCl 10%
resolving gels and electroblotted on to Immobilon P polyvinylidene
difluoride (PVDF; Sigma-Aldrich, Milan, Italy) membrane or
stored at 220uC for later use. To detect HSA sulfhydryl groups/
Cys34 labeled with biotinHPDP, membranes were blocked for
1 h in 5% (w/v) non-fat dry milk in PBST [10 mM Na-phosphate,
pH 7.2, 0.9% (w/v) NaCl, 0.1% (v/v) Tween 20] and probed with
horseradish peroxidase (HRP)-conjugated streptavidin (1:5000
dilution) for 2 h in 5% (w/v) non-fat dry milk in PBST. After
washing in PBST, immunoreactive bands were detected by using
enhanced chemiluminescence. Protein bands were then visualized
by washing the membrane extensively in PBS and then staining
with Amido black.
Determination of HSA Cys34 free sulfhydryl group by
biotinHPDP binding and slot blot analysis
HSA samples were treated with CSE as described above.
Protein pellets were resuspendend in 50 mM PBS, pH 7.4 and
each sample type was incubated with DTT (02 mM) for 15 min
at room temperature. A series of control and CSE-treated HSA
samples was added with 20 mM of the thiol alkylating agent NEM
and incubated for 30 min at 50uC before DTT addition. Protein
precipitation and the biotinylation reaction were induced and
carried out, respectively, as described above. An Immobilon P
PVDF membrane was prepared by wetting it with 100% methanol
for 5 min and then soaking it in a 100% Tris-buffered saline
solution (TBS) (20 mM Tris-base, pH 7.5, 500 mM NaCl) for
15 min. Protein pellets were resuspended in PBS and 0.6 ml of
TBS-diluted albumin solutions (3 mg total protein) was applied to
each slot. After three washes with TBS, the membrane was probed
with HRP-conjugated streptavidin and developed by using
enhanced chemiluminescence as described above for the Western
Incubation with GSH, Cys-SH or N-acetylcysteine (NAC)
before HSA-SH exposure to CSE
In order to evaluate the possible effectiveness of plasma low
molecular mass aminothiols in preventing CSE-induced oxidation
of albumin Cys34, HSA-SH samples were pre-incubated for
30 min with increasing concentrations of GSH, i.e., 0.3, 3 or
30 mM, corresponding to [GSH]/[HSA] molar ratios of 0.005
(16, mean physiological molar ratio), 0.05 (106) or 0.5 (1006),
respectively, or Cys-SH, i.e., 1, 10 or 100 mM, corresponding to
[Cys-SH]/[HSA] molar ratios of 0.016 (16, mean physiological
molar ratio), 0.16 (106) or 1.6 (1006), respectively, before
exposure to 116% (v/v) CSE. Parallel samples were prepared
in which GSH or Cys-SH were added just before exposure to 1
16% (v/v) CSE.
Another set of HSA-SH samples were pre-incubated for 30 min
with 1, 10 or 100 mM NAC before exposure to 116% (v/v) CSE.
Parallel samples were prepared in which NAC was added just
before exposure to 116% (v/v) CSE.
Reduction of reversible sulfhydryl modifications
After treatment with CSE (see above), reduction of HSA
reversible sulfhydryl modifications was accomplished by treatment
with different concentrations of DTT in 50 mM PBS, pH 7.4, for
15 min at room temperature. Alternatively, HSA reversible
sulfhydryl modifications were reduced through prolonged (two
days) dialysis against a 100-fold volume of 50 mM PBS, pH 7.4,
added with 5 mM GSH, at 4uC. Buffer changes were
accomplished every 24 h.
Spectrophotometric determination of protein
Carbonyl groups formed on human plasma proteins were
quantified by adding an equal volume of 10 mM
2,4-dinitrophenylhydrazine (DNPH) in 2 M HCl to the different plasma
solutions containing 2 mg of total proteins; these were allowed
to stand in the dark at room temperature for 1 h, with vortexing
every 10 min. Samples were precipitated with TCA (20% final
concentration) and centrifuged at 13,000 g in a tabletop
microcentrifuge for 5 min, at room temperature. The supernatants were
discarded and the protein pellets were washed once more with
20% TCA and then washed at least three times with 1 ml portions
of ethanol/ethylacetate (1:1) to remove any free DNPH. The
protein samples were resuspended in 1 ml of 6 M guanidine
hydrochloride (dissolved in 20 mM phosphate buffer, pH 2.3) at
37uC, for 15 min, with vortex mixing. Carbonyl contents were
determined from the absorbance at 366 nm using a molar
absorption coefficient of 22,000 M21 cm21 .
Densitometric analysis was performed after scanning the
chemiluminescence films using Image J 1.40d (National Institutes
of Health, Bethesda, MD, USA).
Effect of CSE on HSA Cys34 free sulfhydryl group and on
the HSA structure as a whole
When HSA-SH solutions were incubated with increasing
concentrations (016%, v/v) of CSE, the number of sulfhydryl
groups, as determined by reaction with DTNB, progressively
decreased from 0.9060.04 mol -SH/mol albumin to 0 mol -SH/
mol albumin (Fig. 1). Cys34 thiol modification was further
established in a complementary experiment by using one of the
biotin-based tagging techniques, which have been applied with
success to monitor the posttranslational modification of protein
thiols by ROS and electrophilic compounds . The biotin
tag can be detected at a level of sensitivity in the picomole range
using immunoblotting with HRP-conjugated streptavidin . In
our case, the loss of the biotin signal is proportional to the degree
of protein thiol modification. The results of the biotin-based
tagging experiment, after separation by non-reducing SDS-PAGE
and Western blotting, are presented in Fig. 1, inset. The inset
upper panel depicts results obtained when the membrane was
assayed for free sulfhydryl groups using immunochemical
detection of biotin-HPDP with HRP-conjugated streptavidin. The
albumin exposed to CSE clearly exhibited a decrease in the Cys34
sulfhydryl group relative to HSA-SH. Albumin staining with
Amido black performed on the same membrane evidenced that
total protein loaded in each lane was the same (not shown). The
inset bar graph shows densitometric analysis of biotin-HPDP
Exposure of HSA to 116% (v/v) CSE did not result in large
protein damage such as fragmentation or aggregation due to
intermolecular dityrosine, as assessed by reducing SDS-PAGE (not
shown). Protein analysis by SDS-PAGE under non-reducing
conditions demonstrated that no intermolecular disulfide linkage
was elicited in HSA by CSE exposure (not shown).
Reversibility of Cys34 oxidation
Oxidation of HSA Cys34 thiol is partially reversible in the
presence of 0.25 and 0.5 mM DTT and almost completely
reversible in the presence of 12 mM DTT. Fig. 2A shows the
detection of the Cys34 free sulfhydryl group in both control and
CSE-exposed HSA, in the presence of increasing concentrations of
DTT, by means of biotin-HPDP binding and slot-blot analysis
with streptavidin-HRP (strips a, c, e, g, i). The basal level of the
free sulfhydryl group at Cys34 in HSA-SH was easily detectable
(Fig. 2A, strip c, absence of DTT), whereas the thiol of Cys34
decreased after HSA exposure to 116% (v/v) CSE (Fig. 2A, strips
e, g, i, absence of DTT). Treatment of parallel samples with the
reducing agent DTT partially (0.250.5 mM DTT) or completely
(12 mM DTT) abolished Cys34 oxidation in HSA exposed to 1
16% CSE (Fig. 2A, strips c, e, g, i), as judged by the appearance of
the chemiluminescence signal related to biotinylation of the thiol
of Cys34, suggesting a mainly reversible oxidation of Cys34
induced by 116% CSE. Membrane strips marked with NEM
refer to HSA samples in which the Cys34 sulfhydryl group was
alkylated with 20 mM NEM at 50uC before DTT addition. In
these samples, no biotinylation signal was expected as the only
HSA free sulfhydryl group was covalently blocked with NEM.
Detection of biotin-HPDP binding in the sample treated with
2 mM DTT, and to a much lesser extent in that treated with
1 mM DTT, suggests that the highest DTT concentrations caused
cleavage of one or more out of the 17 albumin disulfide bond(s)
and, consequently, exposure of additional SH groups (Fig. 2A,
strip a). Such a drastic reductive effect of 12 mM DTT obviously
occurred in CSE-treated HSA samples too, therefore in such
samples a small (2 mM DTT) or minimal (1 mM DTT) part of the
biotinylation signal is due to exposure of additional SH groups
because of the breaking of one or more HSA disulfide bond(s).
Strips marked with b, d, f, h, and l depict the same slot blot
membrane strips after Amido black staining.
In order to avoid any undesired and uncontrolled breaking of
intramolecular disulfides within the HSA molecule, we performed
an additional experiment reducing HSA sulfhydryl modifications
through a prolonged (two days) dialysis against a 100-fold volume
of 50 mM PBS, pH 7.4, added with 5 mM GSH, showing that
GSH is able to almost completely revert Cys34 oxidations induced
by 116% (v/v) CSE (Fig. 2A, strips c, e, g, l plus GSH) except for
a minimal amount (about 56%) of, likely, irreversible oxidations
occurring in HSA samples treated with 16% CSE (Fig. 2B).
Preliminary mass spectrometry data reveal that about 6% of
Cys34 occurs as sulfinic acid in HSA samples exposed to 16% CSE
(our laboratory, unpublished data). Therefore, we can guess that
the great majority of oxidative modifications occurring at the
Cys34 SH group are reversible ones.
Effects of physiological and supra-physiological
concentrations of GSH and Cys-SH on CSE-induced Cys34
Plasma low molecular mass aminothiols such as GSH and
CysSH could potentially provide some protection against
CSEinduced oxidation of HSA Cys34 thiol group. Human plasma
concentrations of GSH and Cys-SH are in the range 25 mM and
810 mM, respectively [8,11,38]. Therefore, the possibility of
preventing CSE-induced oxidation of the Cys34 sulfhydryl group
was investigated by incubating HSA-SH solutions with increasing
concentrations of GSH (Fig. 3A) or Cys-SH (Fig. 3B) for 30 min,
before exposing protein samples to 116% (v/v) CSE.
Concentrations of GSH and Cys-SH were chosen so as to reproduce the
mean physiological blood molar ratios of aminothiols to HSA as
Figure 2. Reversibility of Cys34 oxidation as determined by biotinHPDP binding and slot blot analysis. (A) HSA samples (60 mM) were
treated with CSE and then incubated for 15 min with different concentrations of DTT as described under Materials and methods. The biotinylation
reaction was carried out as described under Materials and methods. A series of control and CSE-treated HSA samples was added with 20 mM NEM
and incubated for 30 min at 50uC before DTT addition (strips marked with NEM). In another series of control and CSE-treated HSA samples, reduction
of HSA sulfhydryl modifications was obtained through a prolonged dialysis against 50 mM PBS, pH 7.4, added with 5 mM GSH (strips marked with
GSH). After protein precipitation and resuspension in TBS, diluted protein solutions (3 mg total protein) were applied to each slot and the membrane
was probed with HRP-conjugated streptavidin and developed by using enhanced chemiluminescence (strips a, c, e, g, i) as described under Materials
and methods. Strips marked with b, d, f, h, l show the corresponding duplicate slot-blot stained for proteins with Amido black. (B) Graph shows
densitometric analysis of biotin-HPDP incorporation in HSA samples treated with CSE without further DTT, NEM or dialysis against GSH (filled circles)
and in HSA samples treated with CSE and then dialyzed against GSH (open circles). Data are presented as the mean 6 SD of three independent
well as molar ratios of one and two orders of magnitude higher.
Physiological concentrations (relative to that of HSA) of both GSH
(molar ratio [GSH]/[HSA] = 0.005, where [GSH] = 0.3 mM)
(Fig. 3A, triangles) and Cys-SH (molar ratio [Cys-SH]/
[HSA] = 0.016, where [Cys-SH] = 1 mM) (Fig. 3B, triangles)
produced no protection. Physiological plasma concentrations of
other antioxidants including ascorbic acid, methionine, and uric
acid were also ineffective (data not shown). In contrast, both GSH
and Cys-SH partially prevented CSE-induced oxidation of the
Cys34 thiol at supra-physiological concentrations relative to that of
HSA (molar ratio [GSH]/[HSA] = 0.050.5, where [GSH] = 3
30 mM, and molar ratio [Cys-SH]/[HSA] = 0.161.6, where
[CysSH] = 10100 mM). Supra-physiological amounts (330 mM) of
GSH produced, respectively, minimal (less than 10%) or moderate
(about 30%) protection against oxidation of the Cys34 thiol
induced by CSE (Fig. 3A). Supra-physiological (relative to that of
HSA) concentrations of Cys-SH, namely, 10 mM and 100 mM,
produced a slightly higher protection of about 10 and 3040%,
respectively (Fig. 3B). The highest concentration of both
aminothiols afforded the greatest protection towards HSA exposed
to 14% CSE.
As a 30-min pre-incubation with the thiols could promote
auto-oxidation, in particular of Cys, we performed analogous
experiments with parallel samples in which GSH or Cys-SH
were added just before exposure to CSE, obtaining results
Effects of pharmacological NAC concentrations on
CSEinduced Cys34 oxidation
The potential protective effect of NAC was also considered.
The diverse pharmacological applications of NAC are inherent in
the multifaceted chemical properties of its constituent, cysteinyl
thiol, which enable NAC to act as a nucleophile as well as a
scavenger of ROS . A series of HSA-SH samples were
incubated with 1, 10 or 100 mM NAC (concentrations that equate
to those found in plasma after tolerable oral NAC dosing
(,800 mg/m2/day) and enhancing intracellular GSH levels,
while maintaining a low side-effect profile ) for 30 min before
exposure to 116% (v/v) CSE in order to investigate the
possibility of preventing CSE-induced oxidation of Cys34
sulfhydryl group by means of NAC supplementation. Fig. 3C
shows that the lowest concentration of NAC (1 mM) produced
negligible protection (about 5%) of the HSA thiol. Higher NAC
concentrations (10100 mM) induced higher protection, reducing
oxidation of Cys34 SH group by about 20 and 35%, respectively
An eventual NAC auto-oxidation occurring during the 30-min
pre-incubation was checked performing analogous experiments
with parallel HSA-SH samples in which NAC was added just
before exposure to CSE. The obtained results were similar to those
obtained after pre-incubation with NAC (Fig. 3F).
Prevention of CSE-induced Cys34 oxidation by means of
supra-physiological concentrations of GSH, Cys-SH, or
All the three antioxidants strongly suppressed oxidation of the
sulfhydryl group of Cys34 elicited by HSA exposure to 16% CSE
only at exaggeratedly high concentrations of each aminothiol, i.e.,
at molar ratios of aminothiols to HSA of hundreds-fold (i.e., 106,
256, 506, 1006, 2006, 4006, 8006) compared with the
physiological plasma ratios (Fig. 4). For example, a 50% protection
against CSE-induced oxidation of Cys34 thiol group was afforded
by 120 mM GSH, corresponding to a [GSH]/[HSA] molar
ratio = 2 (i.e., 4006).
Human RBCs protect the Cys34 sulfhydryl group of HSA
against CSE oxidizing effects
Previous studies carried out to determine the occurrence of
oxidative modifications in plasma proteins of smokers did not
specifically evidence HSA oxidation, indeed the contrary, as
fibrinogen was the only oxidized (carbonylated) protein found in
human plasma of smokers . Consequently, also considering
the preponderance of RBCs in blood as well as their high GSH
content, we asked whether human RBCs were protective against
CSE-induced thiol oxidation of HSA Cys34. HSA-SH solutions
were exposed to 116% (v/v) CSE in the absence or presence of
2.5% (v/v) or 5% (v/v) erythrocytes. After a 60-min incubation,
samples were centrifuged and HSA solutions were recovered from
the supernatants. The extent of CSE-induced oxidation of HSA
Cys34 free sulfhydryl group was then evaluated by tagging SH
groups with biotin-HPDP, followed by Western blot analysis with
streptavidin-HRP and enhanced chemiluminescence, after protein
separation by non-reducing SDS-PAGE (Fig. 5). As shown in
Fig. 5A, HSA exposed to CSE in the presence of RBCs clearly
exhibited an increase in the reduced form of Cys34 as compared
with the protein alone exposed to CSE. The related graph shows
densitometric analysis of biotin-HPDP incorporation (Fig. 5B).
The protective effect of RBCs against CSE-induced HSA Cys34
thiol oxidation was also observed when an analogous experiment
was carried out with human plasma proteins instead of the only
HSA (Fig. 6). Human plasma diluted 1:10 with PBS/NaCl was
incubated with 116% (v/v) CSE in the absence or presence of
2.5% (v/v) or 5% (v/v) erythrocytes. After a 60-min incubation at
37uC, samples were processed as described above. The results of
the biotin-based tagging experiment, after separation by
nonreducing SDS-PAGE and Western blotting with
HRP-streptavidin, are presented in Fig. 6A. Each band represents a protein
containing one or more reduced or reversibly oxidized protein
thiol. Increasing band intensity indicates increasing amount of
reduced protein thiols due to the concurrent presence of RBCs in
the incubation mixture with CSE. This is clearly evident for HSA
(arrow) as well as for a few other plasma proteins. The related
graph shows densitometric analysis of biotin-HPDP incorporation
in HSA Cys34 (Fig. 6B). However, Fig. 6A also shows very strong
signals of higher molecular weight than albumin. This is in
apparent conflict with albumin being the most abundant (,80%)
thiol in human plasma . Therefore, we performed a series
of additional experiments aimed at explaining such an apparent
contradiction (Fig. 7). As the very strong signals of higher
molecular weight than albumin shown in Fig. 6A could be due
to a non-specific binding of biotin-HPDP, we repeated the
experiment with control plasma proteins in the absence of RBCs,
diluting human plasma to 1:10 with PBS/NaCl and varying either
the time of incubation with biotin-HPDP (Fig. 7A) or its
concentration (Fig. 7B), whereas all the other experimental
conditions were the same as those of the experiment shown in
Fig. 6. In both cases, the chemiluminescence signal from proteins
of higher molecular weight than albumin remained markedly
higher than that from HSA (Fig. 7A,B). Finally, we performed a
series of experiments with or without NEM in order to verify the
eventuality of a non-specific binding of biotin-HPDP to sites other
than protein sulfhydryl groups (Fig. 7C). In particular, plasma
proteins (4 mg/ml, i.e., human plasma was diluted 1:10 with PBS/
NaCl) were incubated for 30 min at 50uC with 20 mM NEM,
precipitated with three volumes of 100% acetone and protein
pellets were resuspended with PBS/NaCl containing 0.4 mM
biotinHPDP (for further details, see Materials and Methods)
(Fig. 7C, lanes 1); plasma proteins (4 mg/ml) were incubated with
0.4 mM biotin instead of biotin-HPDP (the biotinylation reaction
was performed as described under Materials and Methods)
(Fig. 7C, lanes 2); plasma proteins (4 mg/ml) were incubated for
30 min at 50uC with 20 mM NEM before starting the
biotinylation reaction with 0.4 mM biotinHPDP (for further details, see
Figure 5. RBCs protect the Cys34 sulfhydryl group of HSA against CSE oxidizing effects. HSA-SH solutions (60 mM) were exposed to 1
16% (v/v) CSE in the absence (filled circles) or presence of 2.5% (v/v; open circles) or 5% (v/v; open squares) erythrocytes in 200 mM PBS/NaCl,
containing 5 mM glucose. After a 60-min incubation at 37uC, samples were centrifuged and HSA solutions were recovered from the supernatants. (A)
The extent of CSE-induced oxidation of HSA Cys34 free sulfhydryl group was then evaluated by biotin-HPDP, as described under Materials and
methods, followed by Western blot analysis with streptavidin-HRP and enhanced chemiluminescence, after protein separation by non-reducing
SDSPAGE. (B) Graph shows densitometric analysis of the HSA protein band, corresponding to biotin-HPDP incorporation. Data are presented as the mean
6 SD of three independent determinations.
Materials and Methods) (Fig. 7C, lanes 3); plasma proteins (4 mg/
ml) were incubated for 30 min at 50uC with 20 mM NEM but
were not subject to the biotinylation reaction (Fig. 7C, lanes 4);
plasma proteins (4 mg/ml) were not subject to the biotinylation
reaction (Fig. 7C, lanes 5). After non-reducing SDS-PAGE, the
Western blot was developed as described under Materials and
Methods. The chemiluminescence signal clearly evident in plasma
protein samples alkylated with NEM, which covalently binds to
sulfhydryl groups, and further biotinylated (Fig. 7C, lanes 1 and 3)
suggests that biotin-HPDH binds to some plasma proteins in a
non-specific way, i.e., non dependent on the HPDP mojety
interaction with SH groups.
Human RBCs protect human plasma proteins against
CSE-induced protein carbonylation
To determine whether erythrocytes protected human plasma
proteins also against CSE-induced carbonylation, human plasma
was diluted 1:10 with PBS/NaCl and protein solutions were
exposed to 116% (v/v) CSE in the absence or presence of 2.5%
(v/v) or 5% (v/v) erythrocytes (i.e., equal to about one tenth of the
mean human hematocrit value as in our experiments plasma
protein concentration too is equal to about one tenth of the mean
in vivo concentration) in PBS/NaCl containing 5 mM glucose.
After a 60-min incubation at 37uC, samples were centrifuged at
10,000 g for 20 s, at room temperature and plasma proteins
recovered from the supernatants were processed for determining
the extent of protein carbonylation as described in the Materials
and methods section. Fig. 8 shows that human RBCs effectively
protect plasma proteins from CSE-induced protein carbonylation.
In plasma of healthy young adults, 7080% of total HSA
contains the fully reduced sulfhydryl group of Cys34; ,25% of the
Cys34 forms a mixed disulfide with low molecular mass
aminothiols, generating S-thiolated albumin; a small fraction (2
5%) of albumin Cys34 is more highly oxidized to the sulfinic
(HSA-SO2H) or sulfonic acid (HSA-SO3H) form, which cannot
usually be reversed with DTT and can cause loss of protein
function [13,41,42]. Albumin antioxidant role is mostly attributed
to its sole cysteine thiol, that of Cys34, which accounts for the bulk
of free thiols in plasma , and its ability to scavenge various
oxidizing species [13,17,41,43]. Although HSA Cys34 does not
react particularly fast with oxidants, HSA-SH can be considered
an important plasma scavenger and a key element of antioxidant
defenses due to its very high concentration [13,17,18,4145]. The
oxidized forms of albumin are picked up in the circulation as they
are not present in albumin secreted from the liver cells .
The purpose of the present study was twofold. Firstly, because
some low molecular mass plasma antioxidants have been proposed
to have a protective effect against exposure to CS , we
examined the protective effects of some plasma antioxidants and
the synthetic aminothiol NAC against CSE-induced Cys34
oxidation in vitro. Secondly, given the high GSH concentration
(,3 mM) within erythrocytes as well as the great number of RBCs
in human blood, where no oxidized form of HSA has been
detected in smokers , we asked whether RBCs were
protective towards CSE-induced oxidation of both HSA and total
human plasma proteins.
When HSA-SH solutions were incubated with increasing
concentrations (016% v/v) of CSE, the Cys34 thiol pool
progressively decreased, as determined by both DTNB assay
(Fig. 1) and biotin-HPDP binding revealed by Western blotting
analysis (Fig. 1, inset). The observed diminution in the level of
Cys34 SH groups was not ascribable to formation of
intermolecular disulfide bridges. Thiol oxidation induced by exposure of
HSA-SH to 116% CSE was essentially reversible, as judged by
the almost complete recovery of biotin-HPDP binding when
CSE-treated albumin samples were either further incubated in the
presence of 1 mM DTT (though a negligible amount of the
biotinylation signal may be due to the opening of a few
intramolecular disulfides; see lane 2) (Fig. 2A) or dialyzed against
PBS added with 5 mM GSH (Fig. 2A, strips c, e, g, l plus GSH),
except for a minimal amount (about 56%) of, likely, irreversible
oxidations occurring in HSA samples treated with 16% CSE
(Fig. 2B). Therefore, we can guess that the great majority of
oxidative modifications occurring at the Cys34 SH group are
The possibility of preventing CSE-induced oxidation of albumin
Cys34 by physiological low molecular mass aminothiols was
investigated by incubating HSA-SH with increasing
concentrations of GSH or Cys-SH before protein exposure to CSE. Our
results suggest that plasma concentrations of GSH and Cys-SH are
ineffective in preventing CSinduced oxidation of HSA Cys34
(Fig. 3). Differently, the redox status of the Cys34 thiol group is
partially preserved by incubation with GSH (Fig. 3A) or Cys-SH
(Fig. 3B) at supra-physiological concentrations before exposure to
CSE. The synthetic antioxidant NAC, whose use includes the
treatment of a variety of diseases sharing alterations of the redox
status and GSH depletion as well as dietary supplementation, is an
analogue and precursor of Lcysteine and GSH, which exhibits
the ability to scavenge ROS and could significantly increase the
GSH level in the plasma . NAC at the lowest concentration
produced very limited protection against CSE-induced Cys34
oxidation, whereas the antioxidant preventive effect against HSA
thiol oxidation was more pronounced when using higher
concentrations of NAC (Fig. 3C). At the highest CSE
concentration (16% v/v), the strong oxidation of the Cys34 sulfhydryl group
was partially prevented only in the presence of extremely high (not
physiological) concentrations of GSH, Cys-SH or NAC, the latter
exerting the most effective antioxidant effect (Fig. 4). A marked
protective effect against the CSE-induced oxidation of HSA Cys34
thiol was exerted by human RBCs, whose antioxidant action
was effective not only when HSA alone was exposed to CSE (Fig. 5)
but also when total human plasma proteins were exposed to the
oxidizing insult (Fig. 6). In addition, the RBC antioxidant action
was not limited to protecting protein thiols (Figs. 5 and 6) but
resulted to be effective also in protecting total plasma proteins
from CSE-induced carbonylation (Fig. 8).
This study suggests the possibility that GSH, Cys-SH, and NAC
at exceedingly supra-physiological concentrations could provide
some benefit in preventing or reducing oxidative modifications of
HSA Cys34 in smokers, also considering that smoking alters
plasma thiol homeostasis, causing an oxidation in both the plasma
GSH/GSSG redox and the Cys-SH/cystine redox and
significantly decreasing the plasma cysteine pool size . However, it
seems highly improbable that such exaggerate concentrations of
those aminothiols may be reached in human plasma by means of
nutritional supplementation and/or pharmacological interventions
designed to improve plasma thiol homeostasis in smokers. In
addition, a recent study involving 156 daily smokers showed that
participants who believed that they were taking a dietary
supplement smoked more cigarettes than did controls. This study
suggests that smokers use of dietary supplements may create
illusory invulnerability, which, in turn, reduces the self-regulation
of smoking .
By contrast, our results provide evidence that plasma
(physiological) concentrations of GSH and Cys-SH were absolutely
ineffective as well as other antioxidants, i.e., ascorbic acid,
methionine, and uric acid, which are strong reducing agents and
potent antioxidants that act together in circulation, at plasma
(physiological) concentrations were similarly ineffective to prevent
the CSEinduced thiol oxidation and carbonylation of HSA. As a
whole, the role of antioxidants in preventing smoke-associated
diseases remains controversial .
Several studies have shown that RBCs are important as
biological carriers of GSH, the major antioxidant in erythrocytes,
where its concentration is approximately 3 mM, by de novo
synthesis and as such appear to provide an important detoxifying
system within the circulation [55,56]. This could at least in part
explain why oxidized/carbonylated HSA has been found within
extravascular fluids (e.g., in the brochoalveolar lavage fluid) and
parenchymal lung tissue in cigarette smokers  but not
within blood circulation . This could also partly justify the
protective action shown by RBCs against CSE-induced oxidation
of both HSA and total plasma proteins (Figures 5, 6, and 8),
although such a protective effect cannot be due only to GSH, as
suggested by the only ,50% protection shown by GSH at low
millimolar concentrations, i.e., at a concentration eight
hundredfold compared with the physiological plasma ratio and comparable
to that found within RBCs (Figure 4). It can be hypothesized that
the antioxidant protection afforded by RBCs against CSE-induced
oxidation may also be due to their other rich antioxidant systems
In erythrocytes the major antioxidant is GSH, which protects
important proteins such as spectrin, the oxidation of which can
lead to increased membrane stiffness. GSH not only supports
antioxidant defense, but is also an important sulfhydryl buffer,
maintaining SH groups in hemoglobin and enzymes in the
reduced state . Compared with other somatic cells,
erythrocytes are exposed to oxidative stress from a wide variety of sources;
therefore, RBCs are well equipped with many antioxidants,
besides GSH. Despite their lack of mitochondria, as well as that of
nucleus, ribosomes, and every other intracellular organelle, which
are lost when the RBC emerges from the bone marrow, ROS are
continuously produced within human RBCs due to the high O2
tension in arterial blood and their abundant heme Fe content
within the O2-carrier hemoglobin, which is able to initiate a wide
array of free radical reactions . Erythrocytes transport large
amount of O2 and nitric oxide (NON) over their lifespan resulting in
oxidative stress [60,61]. Various factors can lead to the generation
of oxidizing species in erythrocytes: evidence indicates that many
physiological and pathological conditions such as aging,
inflammation, eryptosis (apoptosis-like process of RBCs, also known as
erythroptosis) develop through ROS and/or RNS action. A major
source of ROS in erythrocytes is hemoglobin, which undergoes
auto-oxidation to produce O2N2. Since the intraerythrocytic
concentration of oxygenated hemoglobin is 5 mM, even a small
rate of auto-oxidation can produce substantial levels of ROS .
As a consequence, RBCs have potent antioxidant protection
consisting of both enzymatic and non-enzymatic systems (the latter
not limited to GSH) that modify highly ROS/RNS into
substantially less reactive intermediates [57,63]. Under
physiological conditions, RBCs exert a scavenging activity towards ROS/
RNS often over-produced in morbidity states, e.g., in inflamed
tissues, or when they experience hyperglycemic conditions after a
meal . Indeed, RBCs are equipped with a very efficient
antioxidant machinery that ensures a reducing environment to
maintain both a functional spectrin-based skeleton-membrane
interaction and hemoglobin in a Fe2+-active form. Thus, under
physiological conditions, RBCs serve the important function of
circulating scrubbers: as ROS/RNS scavenging devices,
RBCs can improve organisms antioxidant defenses.
In detail, human erythrocytes are well equipped with
nonenzymatic antioxidants such as GSH, thioredoxin, vitamin C, and
vitamin E. Furthermore, compared with other cell types, RBCs
exhibit high activities of the most important antioxidant enzymes,
including superoxide dismutase, thioredoxin/thioredoxin
reductase system, peroxiredoxin, catalase, glutathione peroxidase,
glutathione reductase, plasma membrane oxidoreductases, to
reduce extracellular oxidants and, finally, the methemoglobin
reductase/NADH/glycolysis system to maintain hemoglobin in a
Fe2+-active form .
Peroxiredoxins (Prxs) constitute a family of ubiquitous
thioldependent, homodimeric peroxidases that reduce hydrogen
peroxide (H2O2) and alkyl hydroperoxides to water and alcohol,
respectively. They rely on a conserved Cys residue to catalyze
peroxide reduction . The catalytic cycle involves the reduction
of oxidized Prx by thioredoxin and reduction capacity of NADPH
via NADPH -thioredoxin reductase [65,66]. When Prx2 (a typical
2-Cys Prx) reacts with peroxide, the peroxidatic cysteine at the
active site on one subunit is oxidized to a sulfenic acid. A second
conserved cysteine at the C-terminal end of the other subunit (the
resolving cysteine) then reacts with the sulfenic acid to form a
disulfide bridge. Reduction of the disulfide by thioredoxin
regenerates Prx2 and completes the cycle. Thioredoxin is in turn
regenerated by thioredoxin reductase, with reducing equivalents
derived from NADPH . An intriguing feature of mammalian
2-Cys Prxs is that, in the presence of high levels of peroxide, the
peroxidatic Cys becomes overoxidized to the sulfinic or sulfonic
acid form . This abolishes the enzymes peroxidase activity,
although overoxidized Prx can be slowly reverted to the reduced
state by sulfiredoxin or sestrins [68,69]. It has been suggested that
overoxidation allows intracellular accumulation of H2O2, which
can then function as a signal transducer for various pathways .
Erythrocyte Prx2 is the third most abundant erythrocyte protein
(,250 mM in the cytosol, equivalent to ,15 million copies per
cell)  after hemoglobin and carbonic anhydrase and reacts
with peroxides with rate constants six-eight orders of magnitude
faster than GSH . Erythrocytes also possess Prx1, Prx3 and
Prx6, although in lesser amounts than Prx2. Erythrocyte Prxs also
contribute as peroxynitrite scavengers in the circulation, being also
able to act as peroxynitrite reductases catalytically very efficiently
. Prx2 is able to protect hemoglobin from exogenous oxidation
, and is thought to remove hydroperoxides at the erythrocyte
membrane . In erythrocytes, Prx2 is peculiarly resistant to
overoxidation (i.e., oxidation of the cysteine thiol to a sulfinic/
sulfonic acid). Furthermore, erythrocyte Prx2 is extremely efficient
at scavenging H2O2 non-catalytically and competes effectively
with catalase and glutathione peroxidase to scavenge low levels of
H2O2: it is remarkably sensitive to reversible oxidation by H2O2
concentrations in the low micromolar range. However, recycling
of the oxidized dimer occurs very slowly [71,73]. Although it does
not act as a classical antioxidant enzyme, its high concentration
and substrate sensitivity enable it to handle low H2O2
concentrations efficiently without the need for recycling. This large excess of
Prx2 over its substrate suggests that Prx2 does not function in the
erythrocyte as a classical erythrocyte antioxidant enzyme, but as a
very effective H2O2 scavenging protein [71,73]. These unique
redox properties may account for its non-redundant role in
erythrocyte defense against oxidative stress.
Altogether this powerful antioxidant machinery makes the RBC
a highly efficient antioxidant system not yet fully appreciated.
Moreover, specialized mechanisms have been evolved to repair
and eventually remove damaged proteins as well as damaged
lipids. RBCs, in fact, being devoid of protein synthesis, must be
equipped with several mechanisms, not yet completely clarified, to
counteract cell alterations induced by ROS/RNS or, alternatively,
to signal irreversibly damaged cells to the reticulo-endothelial
system for their removal .
In conclusion, given the importance of cigarette smoking as a
risk factor for numerous diseases and the pathophysiological role
played by oxidative stress in these illnesses, quitting smoking
represents an irreplaceable preventive strategy against CS-induced
oxidative stress and oxidative damage. Smoking cessation is
followed by symptom improvement and by a marked increase in
plasma concentrations of vitamins A, C, E, uric acid, total thiols,
and carotenoids, and substantially improves plasma resistance
towards oxidative challenge , although the oxidant burden in
the airways continues for months . However, endeavours in
identifying new and more efficacious antioxidants as a therapeutic
strategy should continue. Under this point of view, it could be
useful to study in detail the specific RBC antioxidant systems
exerting protection against CS-induced oxidative damage to
plasma proteins, in order to verify the possibility of potentiating
them in smokers. The mobility of RBCs makes them an
antioxidant not only for their local environment, but also an
oxidant scavenger throughout the circulation . Erythrocytes
efficient intracellular antioxidant machinery, coupled with their
high blood concentration, renders RBCs an effective sink of
reactive species . Indeed, RBCs can act as scavengers for
plasma hydrogen peroxide and superoxide anion  as well as of
nitric oxide radical in circulation, because of their high
concentration (,9 mM) of hemoglobin . Therefore, the
scavenging ability of RBCs could benefit not only the blood per
se, but more importantly, the entire organism. Thus, researchers
planning to investigate the effect of CS on the blood redox status
could take into account the biological peculiarities of RBCs.
The authors are grateful to Prof. Edgardo DAngelo, Dr. Barbara Ponzini
and all the personnel at the Analysis Laboratory, Department of Human
Physiology, University of Milan, for their invaluable support in providing
Conceived and designed the experiments: GC RR AM IDD. Performed
the experiments: GC MC AA. Analyzed the data: GC RR MC AA AM
IDD. Contributed reagents/materials/analysis tools: NG NP DG RC.
Wrote the paper: AM IDD.
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