Báo cáo khoa học: Xác định và mô tả đặc điểm albumin huyết thanh người bị oxy hóa - Thay đổi cấu trúc nhỏ làm suy giảm chức năng liên kết phối tử và chống oxy hóa

Identification and characterization of oxidized human

serum albumin

A slight structural change impairs its ligand-binding and antioxidant

functions

Asami Kawakami

*, Kazuyuki Kubota

*, Naoyuki Yamada

, Uno Tagami

, Kenji Takehana

Ichiro Sonaka

, Eiichiro Suzuki

and Kazuo Hirayama

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

or 0.6 mm), and

accounts for 50–60% of total plasma protein (75–

80 mgÆmL

) [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 irreversibly oxidized HSA. Reversibly 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 (-SO

H), sulfonic acid (-SO

H), 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 characterization of the precise structural and functional

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.

3346 FEBS Journal 273 (2006) 3346–3357 ª2006 The Authors Journal compilation ª2006 FEBS

fHSA(red)% ¼½reduced HSA=ðreduced HSA

þreversibly 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⁄z1290–1320),

all observed peaks were [M + 51H]

51+

ions. These

peaks were named from lower mass in alphabetical

order: m⁄z1300.09 (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 37 C

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 conditions, excess Cys ⁄cystine solution was added

to puriﬁed HSA of healthy human plasma pool, whose

Intensity

d’

1290 1295 1300 1305 1310 13201315

Fig. 1. [M + 51H]

51+

ion from ESI-TOFMS spectra of HSA. (A)

Spectrum of HSA from fresh plasma, purified using a HiTrap Blue

HP column. (B) Spectrum of excess Cys ⁄cystine solution added to

purified HSA. (C) Spectrum of excess Hcy ⁄homocystine solution

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.

A. Kawakami et al. Characterization of oxidized human serum albumin

FEBS Journal 273 (2006) 3346–3357 ª2006 The Authors Journal compilation ª2006 FEBS 3347

HSA(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⁄z1290–1320. 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 (-SO

H).

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 (-SO

H), 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-Cys; HSA(red)% ¼5% and HSA-Hcy;

HSA(red)% ¼9%] and the puriﬁed nonoxidized HSA

[HSA(red)% ¼78.5] 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 (ALVLIA-

FAQYLQQCPFEDHVK). When the peptide was

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 LC-

ESI-TOFMS information. All observed masses between m⁄z1290

and 1320 were [M + 51H]

51+

ions in LC-ESI-TOFMS spectrum.

Peak

Observed

mass

[M + 51H]

51+

Molecular

weight

Difference in

mass from

peak c

Estimated

structure

of HSA

a 1300.09 66253.6 )186.7 Deficient form

(N-terminal Asp-Ala)

b 1301.55 66328.1 )112.2 Deficient form

(C-terminal Leu)

c 1303.75 66440.3 0 Reduced HSA

d 1306.10 66560.1 119.8 HSA-Cys

e 1306.95 66603.2 162.9 Glycated HSA

f 1308.02 66658.1 217.8 A disulfide bond

cleavage of

HSA-Cys ﬁ-SO

+HO

S-

g 1309.28 66722.3 282.0 Glycated HSA-Cys

h 1306.34 66572.3 132.0 HSA-Hcy

i 1308.26 66670.3 230.0 A disulfide bond

cleavage of

HSA-Hcy ﬁ-SO

+HO

S-

j 1309.51 66734.0 293.7 Glycated HSA-Hcy

Characterization of oxidized human serum albumin A. Kawakami et al.

3348 FEBS Journal 273 (2006) 3346–3357 ª2006 The Authors Journal compilation ª2006 FEBS

molecule [M + nH]

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⁄z851–858, 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⁄z856.1109 (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

plasma HSA [nonoxidized HSA, HSA(red)% ¼

73.0%] and highly oxidized HSA [HSA-Cys,

HSA(red)% ¼8%]. As shown in Fig. 3A, both nonox-

idized HSA and highly oxidized 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

ALVLIAFQYLQQ

CPFEGHFEDVK

Hcy

ALVLIAFQYLQQ

CPFEGHFEDVK

Cys

856 . 4452

856 . 1109 856 . 7793

857 . 1134

856 . 4443 856 . 7782

856 . 1099

856 . 1083 856 . 4427

857 . 1117

851 . 7714

852 . 1052

851 . 4365

852 . 4404

852 . 1066

851 . 7724

851 . 4382

Intensity

854852 856 858 m/

Fig. 2. ESI-FTMS spectrum, identification of binding site of adduct

to albumin. Mass range displayed from m⁄z850.5–858.5. (A) 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.

N H N H N H N H N H

01248

(h)

(kDa)

Non-oxidized HSA

Highly oxidized HSA

Undigested HSA (%)

100

120

02468

Non-oxidized HSA

Highly oxidized HSA

Trypsin treated time (h)

Fig. 3. Susceptibility of reduced HSA and S-cysteinylated HSA to

tryptic proteolysis. (A) SDS ⁄PAGE of HSA after tryptic digestion.

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.

A. Kawakami et al. Characterization of oxidized human serum albumin

FEBS Journal 273 (2006) 3346–3357 ª2006 The Authors Journal compilation ª2006 FEBS 3349

digested 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 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 bind-

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)% ¼73.0] with mildly oxidized HSA

[HSA(red)% ¼55.4], 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

) 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)% ¼73.0%] quenched up to 68.7% of the

hydroxyl radical signal, HSA-Cys [HSA(red)% ¼8%]

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)% ¼54.4%],

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 H

. 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

Table 2. Binding of L-Trp, cefazolin and verapamil to purified HSA

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 (%)

L-Trp Cefazolin Verapamil

Purified non-oxidized HSA 78.5 50.4 17.9 63.3

Mildly oxidized HSA 54.4 65.9 62.3 51.1

Characterization of oxidized human serum albumin A. Kawakami et al.

3350 FEBS Journal 273 (2006) 3346–3357 ª2006 The Authors Journal compilation ª2006 FEBS

Báo cáo khoa học: Identiﬁcation and characterization of oxidized human serum albumin A slight structural change impairs its ligand-binding and antioxidant functions

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