As the major oxidized form is reversibly oxidized HSA, the proportion of reduced HSA [HSAred%] changes according to surrounding conditions: Keywords human serum albumin; mercaptoalbumin;
Trang 1serum albumin
A slight structural change impairs its ligand-binding and antioxidant functions
Asami Kawakami1,*, Kazuyuki Kubota2,*, Naoyuki Yamada2, Uno Tagami2, Kenji Takehana1,
Ichiro Sonaka1, Eiichiro Suzuki2and Kazuo Hirayama2
1 Pharmaceutical Research Laboratories, Ajinomoto Co Inc., Kawasaki, Japan
2 Institute of Life Science, Ajinomoto Co Inc., Kawasaki, Japan
Human serum albumin (HSA) is the most abundant
protein in plasma (40 mgÆmL)1 or 0.6 mm), and
accounts for 50–60% of total plasma protein (75–
80 mgÆmL)1) [1] HSA (66 kDa) is a single-chain
polypeptide of 585 residues, which has heterogeneity
as a result of post-translational nonenzymatic
modifi-cations such as oxidation and glycation Plasma HSA
is divided into two types depending on its redox
state: reduced HSA (HMA; human mercaptoalbumin)
and oxidized HSA (HNA; human
nonmercaptoalbu-min) Reduced HSA contains 17 disulfide bonds and
one free thiol group at Cys34 [2] Oxidized HSA is a
generic name for those proteins that have various modifications at Cys34 HSA is a mixture of reversi-bly and irreversireversi-bly oxidized HSA Reversireversi-bly oxid-ized HSA has mixed disulfide bonds with a thiol compound such as cysteine, homocysteine [3,4] or glutathione In irreversibly oxidized HSA, Cys34 is more highly oxidized to sulfenic acid (-SOH), sulfinic acid (-SO2H), sulfonic acid (-SO3H), or S-nitroso thiol (-SNO) [5,6] As the major oxidized form is reversibly oxidized HSA, the proportion of reduced HSA [HSA(red)%] changes according to surrounding conditions:
Keywords
human serum albumin; mercaptoalbumin;
nonmercaptoalbumin; ESI-TOFMS; oxidation
Correspondence
K Takehana, Pharmaceutical Research
Laboratories, Ajinomoto Co Inc., 1–1
Suzuki-cho, Kawasaki-ku, Kawasaki,
210–8681, Japan
Fax: +81 44 2105871
Tel: +81 44 2105822
E-mail: kenji_takehana@ajinomoto.com
*These authors contributed equally to this
work
(Received 26 April 2006, accepted 25 May
2006)
doi:10.1111/j.1742-4658.2006.05341.x
Human serum albumin (HSA) exists in both reduced and oxidized forms, and the percentage of oxidized albumin increases in several diseases How-ever, little is known regarding the pathophysiological significance of oxida-tion due to poor characterizaoxida-tion of the precise structural and funcoxida-tional properties of oxidized HSA Here, we characterize both the structural and functional differences between reduced and oxidized HSA Using LC-ESI-TOFMS and FTMS analysis, we determined that the major structural change in oxidized HSA in healthy human plasma is a disulfide-bonded cysteine at the thiol of Cys34 of reduced HSA Based on this structural information, we prepared standard samples of purified HSA, e.g nonoxi-dized (intact purified HSA which mainly exists in reduced form), mildly oxidized and highly oxidized HSA Using these standards, we demonstrated several differences in functional properties of HSA including protease susceptibility, ligand-binding affinity and antioxidant activity From these observations, we conclude that an increased level of oxidized HSA may impair HSA function in a number of pathological conditions
Abbreviations
HNA, nonmercarptoalbumin; HMA, mercarptoalbumin; HSA, human serum albumin; LC-ESI-TOFMS, liquid chromatography-electron spray ionization-time of flight.
Trang 2fHSA(red)% ¼ ½reduced HSA=ðreduced HSA
þ reversibly oxidized HSAÞ 100g HSA(red)% tends to be lower in patients with various
diseases or conditions such as hepatic disease [7],
dia-betes [8], renal disease [9], temporomandibular joint
disorders [10], aging [11], and tiredness or fatigue [12]
Although a large number of clinical studies have
reported changes of HSA(red)% in various clinical
conditions, little is known regarding its
pathophysio-logical significance
HSA has various functions, such as: (a) maintenance
of colloid osmotic pressure; (b) binding and transport
of a wide variety of metabolites including steroids,
fatty acids, bilirubin, tryptophan and hemin; (c)
sup-plying an amino acid source during times of
malnutri-tion; and (iv) acting as an antioxidant by radical
scavenging [13–19]
The goal of this study is to clarify the structure–
function relationship between reduced and oxidized
HSA in healthy human plasma, and to assess the
path-ophysiological significance of change in HSA(red)%
In this report, we determined an exact adduct bound
to Cys34 residue of oxidized HSA using mass
spectro-metry in order to prepare both reduced and oxidized
HSA as standard samples for functional studies Using
these, we evaluated several functional differences
between purified HSA samples with various states of
oxidation
Results
Analysis of the structure of oxidized albumin
purified from human plasma using
LC-ESI-TOFMS
Structural heterogeneity exists in plasma HSA, which
is a mixture of reduced, oxidized and glycated
albu-min We analyzed the purified HSA from healthy
human plasma by LC-ESI-TOFMS in order to
deter-mine structure based on mass information
The positive ionized albumin was observed with ions
distributed from [M + 66H]66+ to [M + 36H]36+
Figure 1A shows the ESI mass spectrum of albumin
purified by affinity chromatography Here, the eluted
fraction by affinity chromatography was defined as
purified albumin In the mass range (m⁄ z 1290–1320),
all observed peaks were [M + 51H]51+ ions These
peaks were named from lower mass in alphabetical
order: m⁄ z 1300.09 (peak a), 1301.55 (peak b), 1303.75
(peak c), 1306.10 (peak d) and 1306.94 (peak e),
respectively Because all observed peaks were
[M + 51H]51+ ions, the deconvoluted molecular
weights were 66 253.6 Da (peak a), 66 328.1 Da (peak b),
66 440.3 Da (peak c), 66 560.1 Da (peak d), and
66 603.2 Da (peak e), respectively In particular, peak
c was closest to the theoretical mass (66 437.2) calcula-ted from the known primary amino acid sequence of HSA after subtracting 34 Da due to 17 pairs of disul-fide bonds Therefore, we regarded peak c as reduced HSA, and the others were due to post-translational modification resulting in mass differences compared with peak c When plasma was incubated at 37C
to promote aerobic oxidation, we observed gradual increase in the intensity of peak d, while the intensity
of peak c was reciprocally decreased Thus, we regar-ded peak d as oxidized HSA From peak heights, we estimated that HSA(red)% of healthy human plasma pool is 78.5 Peak d was 119.8 Da heavier than reduced HSA (peak c), corresponding to being the Cys-adduct of HSA via an S–S bond We speculated peak a to be the N-terminal Asp-Ala truncated form
We also suggested peak b to be the C-terminal Leu truncated form and peak e to be glycated HSA Subsequently, we prepared highly oxidized HSA with Cys (HSA-Cys) as a standard Figure 1B shows the ESI mass spectrum of HSA-Cys Under the reac-tion condireac-tions, excess Cys⁄ cystine solution was added
to purified HSA of healthy human plasma pool, whose
Intensity
a b
c
d e d’
f g h
i j
m/z
A
B
C
Spectrum of HSA from fresh plasma, purified using a HiTrap Blue
added to purified HSA The ions correspond to the following: (a) Asp-Ala truncation from N-terminal of HSA, (b) Leu truncation from C-terminal of HSA, (c) HMA, (d) HSA-Cys, (d¢) the identical mass to peak d, (e) glycated HMA, (f) sulfonation after the cleavage of a disulfide bond in HSA-Cys, (g) glycated HSA-Cys, (h) HSA-Hcy, (i) sulfonation after the cleavage of a disulfide bond in HSA-Hcy, and (j) glycated HSA-Hcy.
Trang 3HSA(red)% value was originally 78.5 After removing
excess Cys⁄ cystine with a low molecular weight cut-off
ultrafilter membrane, the sample was applied to
LC-ESI-TOFMS in order to determine the structure
and purity of the HSA-Cys In this mass spectrum,
although the molecular-related ion of reduced HSA
was hardly observed, peak d¢ showed the most
signifi-cant intensity in the range of m⁄ z 1290–1320 The m ⁄
z-value of peak d¢ (Fig 1B) was identical to peak d
(Fig 1A) The HSA(red)% of HSA-Cys was only 5%,
therefore HSA-Cys accounted for 95% with the
excep-tion of the other peaks in the cysteinylated HSA
sam-ple solution The difference of relative molecular mass
of the peak d and the peak g (162.2 Da) and that of
peak c and peak e (162.9 Da) was consistent within
experimental error Therefore, peak g was probably
glycated HSA-Cys
Although the structure of peak f was unknown, it
could be due to a partially cleaved and irreversibly
oxidized S–S bond resulting in sulfenic acid (-SO3H)
This is supported by the mass difference between peak
d¢ and peak f (98.0 Da) corresponding to the mass of
six oxygen atoms
Figure 1C shows the ESI mass spectrum of the
pre-pared highly oxidized HSA with Hcy (HSA-Hcy), where
excess Hcy⁄ homocystine had been added to purified
HSA from healthy human plasma pool (HSA(red)%¼
78.5) After removing excess Hcy⁄ homocystine, the
sam-ple was analyzed by LC-ESI-TOFMS, as noted above
In this mass spectrum, the molecular-related ion of
reduced HSA was again hardly observed, and peak h
showed the most significant intensity in the range of m⁄ z
1290–1320 The molecular weight difference of peaks c
and h was 132.3 Da, corresponding to Hcy being
incor-porated by an S–S bond The HSA(red)% was only 9%,
therefore HSA-Hcy accounted for 91% with the
excep-tion of the other peaks in the homocysteinylated HSA
sample solution
The difference of relative molecular masses of peaks
h and j (161.7 Da) and those of peaks c and e
(162.9 Da) was again within experimental error,
sug-gesting that peak j was glycated HSA-Hcy Peak i
cor-responded to peak f, possibly due to sulfenic acid
formation, as described above If the thiol group at
Cys34 of reduced HSA was sulfenized (-SO3H), the
difference in relative molecular mass against reduced
HSA will be 49.0 Da due to the addition of three
oxy-gen atoms However, peaks with a relative molecular
mass difference of 49 Da were hardly observed on the
baseline level Calculated molecular weight and the
predicted structure of HSA corresponding to each
peak observed in the LC-ESI-TOFMS measurements
were listed in Table 1
The results of our ESI-TOFMS measurements showed that oxidized HSA in healthy human plasma showed mainly cysteine, and not homocysteine, adducts
The comparative FTMS measurement of peptide mixture derived from highly oxidized HSA standard (HSA-Cys) and purified HSA of healthy human plasma
From the result of ESI-TOFMS, we deduced that the main form of oxidized albumin in healthy human plasma is a cysteine adduct on reduced HSA In order
to prove exactly where cysteine bonds to reduced HSA, we digested the standard highly oxidized HSA
HSA(red)%¼ 9%] and the purified nonoxidized HSA [HSA(red)% ¼ 78.5] from healthy plasma with Lys-C The digested peptides were analyzed using FTMS FTMS has high performance in high mass resolution and accuracy If the precise mass is known, the exact composition formula of a low molecular weight com-pound can be determined
The peptide containing Cys34 generated by Lys-C enzyme reaction is from Ala21 to Lys40
cysteinylated through a S–S bond binding at Cys34, the mono-isotopic mass of the multiply-protonated
Table 1 Estimation of the various HSA structure based on
Peak
Observed mass
Molecular weight
Difference in mass from peak c
Estimated structure
of HSA
(N-terminal Asp-Ala)
(C-terminal Leu)
cleavage of
cleavage of
Trang 4molecule [M + nH]n+ was theoretically calculated to
be 2552.2682 (1+), 1286.6380 (2+) and 851.4279
(3+), respectively, from peptide sequence information
For the homocysteinylated peptide, these masses were
calculated to be 2566.2838 (1+), 1283.6458 (2+) and
856.0998 (3+)
Figure 2A,B shows FTMS spectra in the mass range
m⁄ z 851–858, which includes the peptides following
Lys-C digestion of the HSA-Hcy and HSA-Cys
stand-ards, respectively Both of the multiply-charged ions
were 3+ The mono-isotopic ions were observed at
m⁄ z 856.1109 (Fig 2A) and 851.4382 (Fig 2B),
respectively These values were within experimental
error of the theoretical mono-isotopic values (m⁄ z
856.0998, 851.4279) Accordingly, both HSA-Cys and
HSA-Hcy standards would be derived from a Cys and
a Hcy being incorporated into reduced HSA at Cys34
via an S–S bond, respectively
Figure 2C shows the FTMS mass spectrum (m⁄ z
851–858) of purified nonoxidized HSA using affinity
chromatography from healthy human plasma A
3+-charged mono-isotopic ion was observed at m⁄ z
851.4365 This is consistent with that of the digested
peptide from HSA-Cys standard Therefore, HSA-Cys
with cysteine incorporated at Cys34 via a disulfide
bond is the main form of purified HSA in healthy
human plasma
S-Cysteinylation affects susceptibility of albumin
to trypsin digestion
After we had determined the modification on Cys34
residue of oxidized albumin, we next examined
whether this affects its susceptibility to proteolysis We compared the proteolytic sensitivity of purified healthy
HSA(red)%¼ 8%] As shown in Fig 3A, both nonox-idized HSA and highly oxnonox-idized HSA (HSA-Cys) degraded in a time-dependent manner, but showed dif-ferent susceptibility to digestion by trypsin, with highly oxidized HSA being degraded far faster than nonoxi-dized HSA Figure 3B is the quantified results of each HSA band shown in Fig 3A After 8-h digestion, the remaining highly oxidized HSA was approximately one-half that of nonoxodized HSA As proteolysis pro-ceeded, new peptide bands appeared with smaller molecular weights below HSA (indicated by an open arrow in Fig 3A) This suggests that HSA is degraded specifically into certain large fragments
To identify the specific enzymatic cleavage site in HSA, we analyzed the N-terminal sequence of the
ALVLIAFQYLQQ34 CPFEGHFEDVK
Hcy
ALVLIAFQYLQQ34 CPFEGHFEDVK
Cys
A
856 4452
856 1109 856 7793
857 1134
856 4443 856 7782
856 1099
856 1083 856 4427
851 7714
852 1052
851 4365
852 4404
852 1066
851 7724
851 4382
B
C
Intensity
854
Fig 2 ESI-FTMS spectrum, identification of binding site of adduct
Spec-trum of the Lys-C digested peptide containing Cys34 from
HSA-Hcy conjugate (B) Spectrum of the Lys-C digested peptide
contains Cys34 from HSA-Cys conjugate (C) Spectrum of the
Lys-C digested peptides from purified HSA.
A
N H N H N H N H N H
64 (kDa)
B
Non-oxidized HSA Highly oxidized HSA
0 20 40 60 80 100 120
Non-oxidized HSA Highly oxidized HSA
Trypsin treated time (h) Fig 3 Susceptibility of reduced HSA and S-cysteinylated HSA to
HSA samples were treated as described in Experimental proce-dures for the indicated times Twelve micrograms of each protein were loaded into each well and electrophoresis was performed using a 12.5% polyacrylamide gel The filled arrow indicates the un-digested HSA and the open arrow indicates the major tryptic frag-ment of HSA N, nonoxidized HSA; H, highly oxidized HSA (B) Densitometric analysis of intensities of undigested HSA Changes
in intensities of HSA bands relative to the band of starting point of digestion are shown.
Trang 5digested peptide by Edman degradation The
N-ter-minal sequence read Glu-Thr-Tyr-Gly, and concluded
that one of the target sites of tryptic digestion was
located between Arg81 and Glu82 (data not shown)
The binding properties of reduced HSA and
oxidized HSA are different
One significant functional role of serum albumin is
lig-and binding HSA binds many endogenous lig-and
exo-genous small molecular compounds, including l-trp,
fatty acids, bilirubin, and drugs; HSA also plays an
important role in delivering these compounds to target
tissues Several specific binding sites of these ligands
on HSA have been identified, and the two major
ing sites are designated as sites I and II [20] The
bind-ing properties of these sites are strongly correlated to
the structure of HSA As the structural change caused
by S-cysteinylation affected proteolytic susceptibility,
there is a possibility that the binding properties of
reduced and oxidized HSA may also be different
Therefore, we investigated the relative binding
proper-ties of these two types of HSA We investigated the
binding of L(small)-Trp as an endogenous ligand
which binds to site II, and of cefazolin (site I-ligand)
and verapamil (site I and II-ligand) as exogenous
lig-ands All these ligands are known for their high
bind-ing efficiencies to HSA
The binding affinity of each compound to purified
HSA was evaluated by ultrafiltration The results are
expressed as unbound fraction (%) in Table 2 All the
values tended to be relatively high in our experiments
compared with their binding capacities to human
plasma in the literature for unknown reasons When
we compared the unbound fractions of nonoxodized
HSA [HSA(red)%¼ 73.0] with mildly oxidized HSA
[HSA(red)%¼ 55.4], l-Trp bound less strongly to
mildly oxidized HSA The same result was obtained
when cefazolin was used as a ligand While l-Trp and
cefazolin showed decreased affinity to mildly oxidized
HSA, verapamil binding to mildly oxidized HSA was
found to be slightly increased These results suggest that reduced HSA and oxidized HSA have different ligand-binding properties
The antioxidant property of albumin is impaired
in oxidized HSA HSA is the major antioxidant in blood due to its free thiol at Cys34 In this study, we investigated the poten-tial effect of oxidation on the antioxidant capacity of HSA by comparing the radical scavenging activities of HSA in various states of oxidation The hydroxyl radical scavenging activity of nonoxidized HSA and highly oxidized HSA-Cys (10 mgÆmL)1) was studied using ESR The typical 1 : 2 : 2 : 1 four-peak ESR spectrum of the hydroxyl radical was observed and is shown in Fig 4A Addition of HSA caused a decrease
in the ESR signal intensities While nonoxidized HSA [HSA(red)% ¼ 73.0%] quenched up to 68.7% of the hydroxyl radical signal, HSA-Cys [HSA(red)%¼ 8%] reduced it by only 54.4% compared with the control This suggests that the radical scavenging activity of reduced HSA is greater than that of HSA-Cys When
we used mildly oxidized HSA [HSA(red)% ¼ 54.4%], the signal decreased by 62.3% As shown in Fig 4B, the HSA(red)% and hydroxyl radical scavenging activ-ities for each sample show a high positive correlation
To eliminate the possibility that varying iron binding affinity of HSA decreased radical generation, we gener-ated the hydroxyl radical by a different reaction, UV photolysis of H2O2 The same results were obtained, showing that oxidized HSA had a decreased radical scavenging activity (Fig 4C) Therefore, we concluded that oxidation of HSA reduced its radical scavenging activity
Discussion Oxidation of HSA has been reported in numerous dis-eases Although oxidation has been suggested to be of particular pathophysiological relevance for various conditions, there is no direct proof that oxidation of HSA leads to aberrant alterations in its structural con-formation and its functional properties
In this study, in order to clarify the pathological consequences of oxidation, we identified an exact adduct and position of the modification of oxidized HSA from human plasma and characterized its
speci-fic functional properties by comparing among the purified HSA samples which had distinct HSA(red)% values
To distinguish the different functional properties of oxidized HSA, it was first necessary to prepare clearly
and oxidized HSA Values are expressed in unbound fraction (%).
All experiments were performed in duplicate and each CV% was
less than 1%.
HSA sample
HSA (red)%
Unbound fraction (%)
Trang 6defined and highly purified HSA samples Initially, we attempted to analyze the structure of native oxidized HSA from healthy human plasma Oxidized HSA has generally been regarded to have various modifications
on free thiol group at Cys34 There are a number of previous reports in which diverse attempts to identify the actual structures of oxidized albumin in detail have been made Sugio et al [21] determined the X-ray structures of HSA, derived from human pooled plasma
or from a Pichia pastoris expression system, at a reso-lution of 2.5 A˚ However, they were not able to investigate in detail the final differences in the electron density map around the sulfhydryl side chain of Cys34 Therefore, X-ray analysis has not been able to precisely observe the structure of oxidized HSA Bar-Or et al [22] showed profiles of both typical commercial albu-min preparations and normal healthy volunteer human serum albumin by LC-ESI-TOFMS measurement Sengupta et al [23] also showed that thiols (Cys and Hcy) disulfide-bonded to albumin-Cys34 could be removed by treatment with dithiothreitol to form albu-min-Cys34-SH by ESI-TOFMS In these studies, the structure of oxidized albumin was determined by MS However, it is impossible to determine the exact bind-ing site for thiols by measurement of the mass of the whole albumin molecule Additionally, Kleinova et al [24] showed that the structure of pharmaceutical-grade HSA were mainly oxidized HSA However, as intact albumin from human plasma was not used in their study, the true structure of physiologically oxidized HSA was never proven Moreover, reduced HSA was indirectly measured after alkylation with 4-vinylpyri-dine and subsequent tryptic digestion Therefore, for definitive analysis of albumin oxidation it is necessary
to show that the oxidized albumin has no thiol group and has conjugated to cysteine, homocysteine or gluta-thione through a disulfide bond at Cys34
In this study, to solve the structure of physiological oxidized HSA, we purified HSA from healthy human plasma We employed ESI-TOF-MS analysis of HSA
as a whole molecule and FTMS analysis of proteolytic digests By combining both results, we succeeded in revealing that the majority of oxidized HSA in healthy human plasma has only a single modification at Cys34, which is due to a disulfide bond to cysteine After the determination of the molecular structure
of oxidized HSA, we subsequently prepared standard forms of nonoxidized HSA and oxidized HSA, using purified HSA from healthy human plasma The identi-ties of prepared HSA samples were confirmed by LC-ESI-TOFMS (Fig 1) and FTMS analysis (Fig 2) Therefore, well characterized and highly purified oxid-ized HSA samples were used for functional analysis
Field (mT)
A
Nonoxidized HSA
0
10
20
30
40
50
60
70
Non-oxidized
HSA
Highly oxidized HSA
C
B
20
30
40
50
60
70
80
HSA(red)%
Fig 4 Scavenging effects of purified HSA with various HSA(red)%
on hydroxyl radical (A) ESR spectra of the spin adducts of
DMPO-OH Grey spectrum corresponds to the radical generated without
HSA and dotted spectrum corresponds to the one generated when
nonoxidized HSA was added to the reaction (B) Radical scavenging
activities are plotted against HSA(red)% of the samples (C) Radical
scavenging activities of nonoxidized and highly oxidized HSA
against the hydroxyl radical generated by UV photolysis Values are
Trang 7In order to examine if the localized modification at
Cys34 of oxidized HSA causes an overall
conforma-tional changes in albumin, we investigated the
differ-ence in the susceptibility of the two types of HSA to
tryptic proteolysis As shown in Fig 3, highly
S-cysteinylated oxidized HSA showed increased
suscepti-bility to tryptic proteolysis Consistent results were
reported by Glowacki et al [25] in their study that
compared the proteolytic susceptibility of HSA-Cys
with dithiothreitol-treated, highly reduced HSA These
findings suggest that HSA undergoes a conformational
change upon S-cysteinylation, thereby making the
clea-vage sites more accessible We identified the cleaclea-vage
site of tryptic digested HSA by Edman sequencing and
revealed that the region encompassing Glu82 in
domain I becomes more susceptible to proteolysis To
gain further insight into the molecular basis for the
effects of oxidation on conformational change,
molecu-lar modeling of HSA-Cys was performed using the
crystal structure of human serum albumin (RSC PDB
ID: 1BM0) The expected modeled structures of
HSA-Cys were subjected to molecular dynamics simulations
using insightii (Accelrys Inc., San Diego, CA, USA)
The conditions of calculation were as follows: force
field¼ discover3, run time ¼ 1000 fs, temperature ¼
298 K These calculations showed that the
conforma-tional changes induced by S-cysteinylation of HSA
were not large scale, but were localized to five regions
of the molecule (indicated as orange ribbons in Fig 5)
One of the five conformationally altered regions, a
loop between Thr79 and Leu85, which is located near
to the Cys34 residue in the three-dimensional structure,
may be the reason for increased susceptibility to
pro-teolysis, as it contains the specific site for tryptic
diges-tion, Glu82 Stewart et al [2] recently analyzed X-ray
structures of recombinant HSA and showed that the
sulfur of Cys34 is tethered to the hydroxyl oxygen of
Tyr84 by a hydrogen bond They speculated that the
formation of disulfide at Cys34 would lead to the loss
of the H-bond between Cys34 and Tyr84, thereby
resulting in a conformational change In fact, using
1H NMR study, they demonstrated that
S-cysteinyla-tion altered the conformaS-cysteinyla-tion and dynamics of the
entire domain I, and also the domain I⁄ II interface
All these results suggest that oxidation, a single
modifi-cation at Cys34, could result in a number of regional
conformational changes of HSA resulting in increased
susceptibility to proteolysis
The structure of the protein should be highly
associ-ated to its specific functional activities We attempted
to investigate whether the conformational change of
oxidized HSA affects certain functional properties of
albumin To elucidate the structure–function
relation-ship between reduced and oxidized HSA, we examined two functional properties, ligand-binding properties and antioxidant activities, using both purified nonoxi-dized and oxinonoxi-dized HSA samples
Ligand-binding properties are the most significant functions of HSA [26] HSA binds and transports numerous endogenous and exogenous compounds, and controls their solubility and toxicity in vivo Among the several endogenous ligands of albumin, we focused on
l-Trp, which circulates in plasma mostly bound to site
II on HSA [27] Because l-Trp is the precursor of sero-tonin, it is hypothesized that increased levels of free
l-Trp in plasma enhance serotonin synthesis and release at the brain Excessive serotonin secretion is observed in many clinical conditions and modulates numerous physiological and psychiatric systems In this study, highly oxidized HSA showed decreased l-Trp binding The binding site of l-Trp on HSA is reported
to be located at site II [28], which is distant from Cys34
in the three-dimensional structure However, a spatial correlation between these two regions has been implica-ted in the study by Muscaritoli et al [29] In an in vitro study, they reported that the level of free unbound
l-Trp increased in the presence of cisplatin, an anti-neoplastic drug that binds to HSA at Cys34 They suggested that cisplatin administration caused l-Trp
Fig 5 Molecular dynamics simulation of reduced HSA and HSA-Cys Blue ribbon represents reduced form of HSA and green ribbon represents S-cysteinylated oxidized form Five regions of HSA-Cys,
reduced HSA The Cys34 residue is shown in pink.
Trang 8displacement from HSA and enhanced precursor
avail-ability for serotonin synthesis and release at the brain,
and that might contribute to the pathogenesis of
cisp-latin-induced emesis These findings indicate that the
state of Cys34 of HSA affects the l-Trp binding affinity
on the other side of the molecule As shown in Table 2,
our study also showed that S-cysteinylation at Cys34
decreased binding affinity towards l-Trp of HSA This
might also be related to increased levels of serotonin
and the frequent occurrence of some adverse
complica-tions of diseases which have an increased level of
oxid-ized HSA On the other hand, exogenous ligands of
albumin such as cefazolin and verapamil also showed
different binding affinities between nonoxidized HSA
and mildly oxidized HSA Mera et al [30] recently
demonstrated that purified albumin from hemodialysis
patients with decreased HSA(red)% showed reduced
drug-binding properties to warfarin and ketoprofen In
our experiment, mildly oxidized HSA displayed
bidirec-tional changes in binding properties dependent on
dif-ferent types of ligands These observations indicate that
structural changes of HSA caused by oxidative
modifi-cation on the thiol moiety of Cys34 affect the
drug-binding properties of this protein These phenomena
are important from a therapeutic point of view, as the
concentration of unbound free drugs in plasma has an
impact on pharmacokinetics, monitoring efficacy and
adverse effects These factors are essential in specifying
the patient’s therapeutic regimen Altered steady-state
plasma concentration of drugs is a clinical therapeutic
problem for the treatment of various inflammatory
dis-eases, especially in elderly patients It is assumed that
reduced HSA(red)%, together with hypoalbuminemia,
often found in these patients is responsible for this
problem
Antioxidant activity is also an important function of
albumin The function is believed to be ascribed to its
single exposed thiol group at Cys34 [31–33] Because
albumin accounts for most of the total plasma thiol
content (about 80%), it can act as a major antioxidant
in plasma or extracellular fluids where the amounts of
antioxidant enzymes are relatively small [34,35] In the
former article, Mera et al [30] reported that purified
albumin from hemodyalysis patients showed a
decreased ability to scavenge chemical synthetic DPPH
radicals In this study, we also demonstrated that
oxidized HSA has reduced scavenging ability against
highly reactive oxygen species, in this case, hydroxyl
radicals (Fig 4C) It is found that the degree of
hydroxyl radical scavenging activity of HSA is highly
correlated with HSA(red)% (Fig 4B). This
observa-tion confirms that the antioxidant activity of HSA, at
least in part, depends on the state of the thiol at
Cys34 As oxidized HSA has decreased antioxidant activity, decreased HSA(red)% not only reflects the oxidative shift of the redox state of the human body, but also may be a factor influencing the redox state of
a number of diseases
In conclusion, the present study demonstrated that oxidized HSA, primarily cysteinylated via a disulfide bond at Cys34, exhibits various differences in its biolo-gical properties relative to reduced HSA Although it
is still unknown whether highly oxidized HSAs from patients with a number of diseases have a similar structure, we suggest that reduced HSA(red)% may result in impaired function of HSA We suggest that there may be potential diagnostic and therapeutic benefits of measuring HSA(red)% in a variety of dis-ease conditions
Experimental procedures Plasma collection
Blood (160 mL) was collected using a vacuum tube collec-tion system, using heparin as an anticoagulant, from two healthy volunteers Plasma fractions of the two volunteers were isolated by centrifugation at 4C for 20 min (2000 g), and were mixed together The pooled plasma, which is used
as healthy human plasma in this study, was immediately frozen in liquid nitrogen prior to long-term storage at )80 C
Preparation of purified HSA with various states
of oxidation
We prepared purified albumin samples with various HSA(red)% from healthy human plasma
Non-oxidized HSA
Just after plasma collection, the intact purified albumin pre-pared by affinity chromatography using HiTrapTMBlue HP Column (Amersham Bioscience) was designated as ‘nonoxi-dized albumin’ HSA(red)% of this sample was high as 78.5% For functional assays (ligand-binding and antioxid-ant assay), the purified nonoxidized HSA was concentrated
by ultrafiltration, followed by incubation at 37C for 48 h After the treatments, HSA(red)% of the purified nonoxi-dized HSA slightly changed to 73.0
Mildly oxidized HSA
Mildly oxidized HSA was purified from the plasma which was incubated at 37C for 18 h to promote aerobic oxida-tion The HSA(red)% value of this sample was 54.4%
Trang 9Highly oxidized HSA
Highly oxidized albumin was prepared by artificial
incor-poration of cysteine or homocysteine into reduced albumin
Cysteinylated HSA (HSA-Cys) and homocysteinylated
HSA (HSA-Hcy) were prepared as follows Purified reduced
HSA at a concentration of 4 mgÆmL)1 (0.06 mm) was
trea-ted with a 50-fold molar excess of l-cysteine⁄ cystine by
mixing 80 mL of 4 mgÆmL)1 HSA, 72 mL of 3 mm
cys-teine, and 8 mL of 3 mm cystine All solutions were in
0.1 m calcium carbonate⁄ hydrogen carbonate buffer
(pH 10.0), and the mixture was incubated at 37C for
48 h Next, low molecular weight compounds were removed
by ultrafiltration through an Ultrafree-3000 Da membrane
(Millipore, Billerica, MA, USA) at 4C The HSA(red)%
value of this sample was 5–9% HSA-Hcy was prepared by
exactly the same procedure except that dl-homocysteine
was used in place of l-cysteine, and homocystine was used
in place of cystine LC-ESI-TOFMS was used to determine
the purity of HSA-Cys and HSA-Hcy The additive
reac-tion resulted in high yield and HSA(red)% value of each
sample was <10% We also confirmed the remaining
l-cys-teine⁄ cystine or homocysteine ⁄ homocystine in the standard
solutions were minute using LC-MS⁄ MS measurement
LC-ESI-TOFMS measurement
Each plasma and albumin preparation was analyzed by
HPLC (LC-Packings, Amsterdam, Netherlands) coupled to
ESI-TOFMS (Bruker Daltonics Inc., Billerica, MA, USA)
Plasma or purified HSA samples was diluted to
0.4 mgÆmL)1 (0.06 mm) The diluted sample was filtered
with a 0.1 lm cut-off membrane filter microfilter tube
Sub-sequently, aliquots of 100 lL of filtered sample were
trans-ferred to a sample vial Two microliters of each sample was
injected into the pre-column (100 mm· i.d 200 lm, length
packed with monolith C18; GL Science Inc., Tokyo,
Japan) Albumin was eluted using a 20-min linear gradient
method by 75 : 25 water⁄ acetonitrile containing 0.1%
for-mic acid (solution A) to 10 : 90 water⁄ acetonitrile
contain-ing 0.1% formic acid (solution B) All albumin samples
were desalted and concentrated by a column switching
method
The eluted albumin from the main column was
intro-duced into ESI-TOFMS (microTOF; Bruker Daltonics
Inc.) In all experiments, spectra were acquired over the
range m⁄ z 50–3000 The observed mass spectrums were
averaged from 30 scans of the albumin ion The averaged
data were smoothed using a Gauss algorithm and baseline
subtracted using the microtof software
To determine HSA(red)% of the samples, we focused on
the mass range of m⁄ z 1250–1480 to identify certain
signa-ture peaks Eight pairs (reduced and oxidized) of albumin
ions charged from 47+ to 54+ were obtained in this
range HSA(red)% was calculated independently for each
identically charged ion using the following formula: HSA(red)%¼ {(peak height of reduced HSA) ⁄ [(peak height
of reduced HSA) + (peak height of oxidized HSA)]}· 100 The average of the eight values was assumed as HSA(red)% of the sample
Enzymatic digestion for FTMS measurement
Lyophilized lysylendopeptidase (20 lg per vial; mass spectr-ometry grade; Wako Pure Chemical Industries, Osaka, Japan) was dissolved in 2 mL of 0.1 m ammonium acetate buffer (pH 6.0) This lysylendopeptidase solution was diluted 2.4 times with 0.1 m ammonium acetate buffer (pH 6.0) Purified fresh nonoxidized HSA and highly oxid-ized HSA-Cys solution buffers were changed to 0.1 m ammonium acetate by membrane filtration using 3000-Da cut-off filters One hundred microliters of 30 lm purified nonoxidized HSA or HSA-Cys standards were mixed with
100 lL of 0.15 mm lysylendopeptidase, and incubated at
37C for 8 h The enzymatic digestion was inactivated with the 200 lL of 0.1% formic acid
Digested peptides measurement by FTMS
We used a Bruker Daltonics Apex II FT-MS equipped with
a Bruker-Magnex actively shielded superconducting magnet operating at 7.0 T and a nano ESI Sources with Microme-talTip stainless emitter (Eisho-metal Co Ltd, Tokyo Japan) The digested sample was diluted 10-fold with 0.1% formic acid in 50% acetonitrile⁄ water (v ⁄ v) For syringe infusion experiments, the digested albumin samples were individually 100-fold diluted and infused into the ESI source at a flow rate of 200 nLÆmin)1 MS data were acquired in the positive ionized mode over an m⁄ z range of 400–2000 Data acquisi-tion was performed using with the xmass software (Bruker Daltonics) We focused on the peptide containing Cys34 in the peptide mixture produced by enzyme digestion
Tryptic digestion of HSA
Purified nonoxodized HSA (300 lg) and highly oxidized HSA (HSA-Cys) (2 mgÆmL)1) were submitted to trypsin digestion by incubating the samples at the final substrate to trypsin (86 units, Wako) ratio of 25 : 1 in NaCl⁄ Piat 37C for 8 h Aliquots from each digested sample were collected
at 0, 0.5, 1, 2, 4, and 8 h after the beginning of proteolysis
2· SDS ⁄ PAGE sample buffer and denatured for 10 min at
95C Twelve micrograms of HSA per lane were subjected
to SDS⁄ PAGE on 12.5% polyacrylamide gels for 30 min at constant voltage (200 V) Protein bands were visualized by Coomasie brilliant blue staining The gel image was captured using ImageMaster LabScan 5.0 (Amersham Biosciences,
GE Healthcare, Piscataway, NJ, USA) and protein bands
Trang 10corresponding to HSA were quantified using imagequant
5.1 software (Molecular Dynamics, Sunnyvale, CA, USA)
Ligand binding experiments
L-Trp binding property
(30 mgÆmL)1) and highly oxidized HSA (HSA-Cys,
30 mgÆmL)1) in NaCl⁄ Pi(pH 7.3) to give a final
concentra-tion of 100 lm The unbound ligand fracconcentra-tions were
separ-ated using ultrafiltration membranes in Centricon YM100
devices (Amicon⁄ Millipore Inc., Bedford, MA, USA) by
centrifugation (3000 g, 5 min, 37C) Fifty-microliter
aliqu-ots of the initial (before ultrafiltration), filtrate, and apical
(not ultrafiltered fraction) fractions were mixed with the
same amount of 8% (w⁄ v) trichloroacetic acid solution,
respectively Protein-free supernatants were separated by
centrifugation at 3000 g for 5 min l-Trp concentration of
each fraction was determined by LC-MS analysis
Drug-binding properties
The binding of cefazolin (5 lm) and verapamil (5 lm) to
HSA (30 mgÆmL)1) with two oxidative states in NaCl⁄ Pi
(pH 7.3, 37C) was examined The ultrafiltration method
was the same as in the l-Trp binding experiment
Fifty-microliter aliquots of each fraction was mixed with 300 lL
acetonitrile and left at room temperature for 5 min
Pro-tein-free supernatants were obtained by centrifugation at
3000 g for 5 min and the solvents were removed by
evapor-ation Samples were dissolved in the eluent for the
follow-ing LC-MS analysis The concentration of each compound
was determined according to the standard curve made by
LC-MS analysis
Calculation of the unbound fraction (%)
The unbound fraction (%) of each ligand was calculated as
follows:
Unbound fraction (%)¼ [ligand concentration in filtrate ⁄
the initial ligand concentration (before ultrafiltration)]· 100
Recovery was validated by the following calculation:
Recovery (%)¼ {[(ligand concentration in filtrate) ·
50+ (ligand concentration in apical fraction· 400)] ⁄ (the
initial ligand concentration· 450)} · 100
ESR spectroscopy
ESR spectra were obtained using a JES-FR80S
spectro-meter (JEOL, Tokyo, Japan) with a manganese marker at
room temperature Hydroxyl radicals were generated by the
two following different methods and trapped by
5,5-dimethyl-1-pyrroline-N-oxide (DMPO) (Sigma-Aldrich,
St Louis, MO, USA)
Fenton–reaction
A mixture of 5 mm DMPO, 0.025 mm FeSO4, 0.1 mm diethylenetriaminepentaacetic acid (DETAPAC), 0.5 mm
H2O2, and purified HSA samples with various states of oxi-dation (10 ngÆmL)1) in NaCl⁄ Pi(pH 7.3) was transferred to
a quartz flat cell and placed in the cavity of the ESR spectro-meter for the measurement The conditions of ESR measure-ment were as follows: center field 335.5 mT, microwave power 1 mW, modulation frequency 100 Hz, modulation width 0.1 mT, receiver gain 79, sweep width 10 mT, time constant 0.03 s, and sweep time 1 min
UV photolysis
A mixture of 88 mm DMPO and 0.3% H2O2 in NaCl⁄ Pi
(pH 7.3) with or without HSA samples (10 ngÆmL)1) was transferred to a quartz flat cell and irradiated for 8 s under
UV lamp at 230–430 nm in the cavity of the ESR spectro-meter The following measurement was conducted under the conditions as follows: center field 335.5 mT, microwave power 3 mW, modulation frequency 100 Hz, modulation width 0.063 mT, receiver gain 250, sweep width 5 mT, time constant 0.03 s, and sweep time 1 min
Calculation of the radical scavenging activity (%)
The scavenging effects of HSA on hydroxyl radicals were determined by the following calculation:
Radical scavenging activity (%)¼ [(hPBS )hHSA)⁄
hPBS]· 100%
Here, hPBSand hHSAare the ESR signal intensities without and with HSA, respectively The intensities of the signals were normalized to that of manganese as an internal control
Acknowledgements
We would like to give our special thanks to Dr Seiichi Era, Department of Physiology and Biophysics, Gifu University Graduate School of Medicine, and Drs Hisataka Moriwaki and Hideki Fukushima, the First Department of Internal Medicine, Gifu University Graduate School of Medicine, for numerous discus-sions and helpful suggestions We are grateful to Dr Masaichi-Chang-il Lee, Clinical Care Medicine Divi-sion of Pharmacology, Kanagawa Dental College, for valuable advice on ESR analysis We also thank Dr Itsuya Tanabe and other researchers in Ajinomoto Co., Inc., for technical advice and helpful discussions
References
1 Peters TJ (1995) All about albumin: Biochemistry, Genet-ics, and Medical Applications Academic, New York