Pharmaceutical Research, Vol. 20, No. 4, April 2003 (© 2003)

Research Paper

Validation of the Chloramine-T
Induced Oxidation of Human Serum
Albumin as a Model for Oxidative
Damage in Vivo
Makoto Anraku,1 Ulrich Kragh-Hansen,2
Keiichi Kawai,3 Toru Maruyama,1Yasuomi Yamasaki,4 Yoshinobu Takakura,4 andMasaki Otagiri1,5Received September 11, 2002; accepted January 2, 2003.
Purpose. The validity of using chloramine-T as a model compound
for mimicing oxidative stress was examined using human serum albumin (HSA) as a model. Important sites of oxidation were studied
by mild treatment with chloramine-T and by mutating 34Cys for aserine (C34S).
Methods. High-performance liquid chromatography (HPLC) combined with fluorescence detection to confirm the validity of chloramine-T as an oxidizing agent was used. Oxidized amino acid residues
were detected by reaction with 5,5⬘-dithiobis(2-nitro benzoic acid),
digestion with cyanogen bromide, followed by capillary electrophoresis. Protein conformation was examined by spectroscopic techniques.
Results. From the HPLC analysis of human serum, the validity ofusing chloramine-T as an oxidizing agent was confirmed. At low chloramine-T concentrations (CT0.1-HSA, CT1-HSA), 34Cys and Met
residues were oxidized, at medium concentrations (CT10-HSA), thetryptophan residue also appeared to be oxidized, and at the highest
concentration (CT50-HSA), the net charge of Site II of HSA wasfound to be more negative. The two highest levels of oxidation of
HSA (CT10-HSA, CT50-HSA) resulted in conformational changes
with an increased exposure of hydrophobic regions, decreased highaffinity bindings of warfarin and ketoprofen and a reduced esteraselike activity. The latter protein also has a shorter plasma half-life andan increased liver clearance.
Conclusions. We succeeded in imitating oxidative damage to HSA
using chloramine-T and the findings show that Site II is more affected
than Site I and 34Cys, when HSA is exposed to oxidative stress.
KEY WORDS: human serum albumin; cysteine mutation; chloramine-T; Site II; oxidative damage; pharmacokinetics.

INTRODUCTION
Covalent modification of proteins by oxidative systems
has been implicated in various physiological and pathologicalconditions (1). To protect against this toxicity, organisms havedeveloped a battery of antioxidant defenses. Thus, bloodplasma, which is known to be exposed to continuous oxidative
1

Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1
Oe-honmachi, Kumamoto 862-0973, Japan.
2
Department of Medical Biochemistry, University of Aarhus, DK8000 Aarhus C, Denmark.
3
School of Health Sciences, Faculty of Medicine, Kanazawa University, 5-11-80 Kodatsuno, Ishikawa 920, Japan.
4
Department of Drug Delivery Research, Graduate School of
Pharmaceutical Sciences, Kyoto University, Sakyo-ku 606-8501,
Kyoto, Japan.
5
To whom correspondence should be addressed. (e-mail:otagirim@gpo.kumamoto-u.ac.jp)0724-8741/03/0400-0684/0 © 2003 Plenum Publishing Corporationstress, contains albumin as a major and predominant circulating antioxidant (2). Human serum albumin (HSA) is a single,
nonglycosylated polypeptide of 585 amino acids which is organized in the form of a heart-shaped protein having about
67% ␣-helix but no ␤-sheet structure (3). All but one (34Cys)of the thirty-five cysteine (Cys) residues are involved in theformation of stabilizing disulfide bonds. Human serum albumin is also a comprehensive depot and transport protein, andplays an important role with respect to endogenous and exogenous compounds. Therefore, it can be anticipated that, forexample, the distribution and half life of ligands might begreatly effected, if the functional properties of HSA were to
be modified by disease or aging associated oxidation.
In a previous study, we oxidized HSA using several different procedures and found that several types of amino acidresidues are important for its antioxidant activity (4). However, our experimental system was limited to in vitro conditions. Thus, it was difficult to judge whether our experimental
conditions actually reflect physiological conditions. In a recent study, Era et al. (5) reported on the use of a convenienthigh-performance liquid chromatographic (HPLC) system forthe clear separation of reduced and oxidized albumin, using
an ES-502N column, and extensively studied the conversionof reduced; HSA to an oxidized form in the elderly and invarious pathophysiological states. In general, HSA is a mixture of mercaptalbumin (HMA, reduced form) and nonmercapt albumin (HNA, oxidized form) (6,7). HMA contains one
free sulfhydryl group in 34Cys and HNA is comprised of atleast three types of molecules, namely a mixed disulfide withcysteine or glutathione (HNA(Cys), HNA(Glut)), and products oxidized HNA (HNA(Oxi)) to a greater extent thanmixed disulfide. The chloramine-T method of Shechter et al.
(8) generates the formation of HO⭈ and chloro radicals andmay mimic oxidation reactions that occur under physiologicalconditions. Preliminary results showed an increase in HNA,
when reacting serum was reacted with chloramine-T. Similar
results have been found in aging as well as in certain diseased
states. Furthermore, by varying the experimental conditions,different types of HSA, in different oxidation states could be
prepared easily and reproducibly. Therefore, we chose to further investigate this procedure for oxidizing HSA.
The authors also prepared a recombinant mutant of HSA
in which 34Cys was replaced by a serine, because 34Cys represents a potentially important residue of HSA for oxidative
stress. The impact of the mutation and four levels of chloramine-T induced oxidations on the structural properties of albumin was investigated using a variety of spectroscopic techniques. The influence of oxidation on various functional properties, namely its ligand-binding and esterase-like ability,
were also studied. Finally, the effect of the mutation and oxidations on the plasma half-life and organ clearances of HSA
was evaluated in mice.
ABBREVIATIONS: HSA, human serum albumin; rHSA, recombinant HSA; HMA, mercaptalbumin; HNA, nonmercaptalbumin;
CT0.1-HSA, CT1-HSA, CT10-HSA and CT50-HSA is HSA treatedwith 0.1, 1, 10 and 50 mM of chloramine-T, respectively; DTNB,
5,5⬘-dithiobis (2-nitro benzoic acid); bis-ANS, 1,1-bis-4-anilinonaphthalene-5,5-sulfonic acid; CNBr, cyanogen bromide; CD, circular dichroism; Cys, cysteine; Met, methionine; Trp, tryptophan; Tyr,tyrosine.

684

Human Serum Albumin as a Model for Oxidative Damage

685

Based on these data, the validity of chloramine-T treatment as a physiological model and the importance of Site I
and Site II in HSA with respect to oxidative damage, arediscussed.

by a combination of precipitation with 60% (w/v) (NH4)2SO4,
followed by purification on a Blue Sepharose CL-6B column(Amersham Pharmacia Co., Uppsala, Sweden). Isolated protein was defatted using the charcoal procedure described byChen (9), deionized, freeze-dried and then stored at −20°C
until used. The resulting albumins (treated with dithiothreitol) appeared as a single band on SDS/PAGE and all therecombinant proteins migrated at the same position as native
HSA. Density analysis of protein bands stained with Coomassie Brilliant Blue showed that the purity of the recombinant albumins was in excess of 97%.

MATERIALS AND METHODS
Materials
HSA was donated by the Chemo-Sera-Therapeutic Research Institute (Kumamoto, Japan) and was defatted by using the charcoal procedure described by Chen (9), deionized,
freeze-dried, and then stored at −20°C until used. HSA andthe oxidized HSAs prepared in this study gave only one bandon SDS-PAGE (data not shown), and the molecular mass of
all the albumins were assumed to be 67 kDa. Sera from five
healthy young male subjects, who had no renal or hepatic
dysfunctions, were obtained from Faculty of Pharmaceutical
Sciences, Kumamoto University. Their ages ranged from 21
to 26 years (23.2 ± 1.92. mean ± S.D.), as previously described(10). Blood sera were obtained by centrifugation and storedat −80°C until used for analysis. Chloramine-T, 5,5⬘dithiobis(2-nitro benzoic acid) (DTNB) and p-nitrophenyl acetate were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). The fluorescence probe 1,1-bis-4-anilino-naphthalene5,5-sulfonic acid (bis-ANS) and fluoresceinamine (isomer II)
were obtained from Sigma (St Louis, MO, USA). Potassium
warfarin (Eisai Co., Tokyo, Japan) and ketoprofen (Sanwakagaku Co., Tokyo, Japan) were obtained as pure substancesfrom the manufacturers. 111InCl3 (74 Mbq/mL in 0.02 N HCl)
was a gift from Nihon Medi-Physics (Takarazuka, Japan). Allother chemicals were of analytical grade, and all solutions
were prepared in deionized and distilled water. Phosphatebuffer, 67 mM and pH 7.4, was used as a standard buffer, andwas prepared from sodium phosphate dibasic and sodium
phosphate monobasic salts.
Synthesis and Purification of rHSA Forms
The recombinant DNA techniques used to produce wildtype rHSA and the single-residue mutant C34S were essentially those described by Watanabe et al. (11). A chimeric
plasmid (pJDB-ADH-L10-HSA-A) having a cDNA for themature form of HSA along with an L10 leader sequence wasa gift from Tonen Co. (Tokyo, Japan). The mutagenic primer
used (underlined letters indicate mismatches) was 5⬘CTTCAGCAGTCTCCATTTGAAG-3⬘. The L10-HSA coding region was amplified by PCR with a forward and a reverse
primer carrying a 5⬘-terminal EcoRI site and cloned into the
EcoRI-digested pKF19k vector (Takara Shuzo Co., Kyoto,
Japan). Mutagenesis was performed with a site-directed mutagenesis kit (oligonucleotide-directed dual amber method)
obtained from Takara Shuzo Co. The mutation was confirmed by DNA sequencing of the entire HSA coding region
with the dideoxy chain termination method on a PerkinElmer
ABI Prism 310 Genetic Analyzer. For constructing the HSA
expression vector pHIL-D2-HSA, an L10-HSA coding region
without or with the desired mutation site was incorporated
into the methanol-inducible pHIL-D2 vector (Invitrogen Co.,San Diego, CA, USA). The resulting vector was introduced
into the yeast species P. pastoris (strain GS115) to express
rHSA. Secreted rHSA was isolated from the growth medium

HPLC System
An ion exchange HPLC system was used in this study.
The system was used to examine the redox state of albumin inserum and consisted of a Model MIX 100 double-plunger
pump and a Model FP-2025 fluorescence detector (excitation
wavelength, 280 nm; emission wavelength, 340 nm), in conjunction with a Model PU-2080 system controller, all obtained from Jasco Co., Tokyo, Japan. A Shodex Asahipak
ES-502N (Showa Denko Co., Tokyo, Japan) ion-exchange
column was used. The column temperature was 37°C. Measurements were carried out by solvent gradient elution withincreasing ethanol concentrations from 0 to 5% in 0.05 M
sodium acetate-0.40 M sodium sulfate (pH 4.85) and a flowrate of 1.0 mL/min. Samples were injected with fixed volumesof 5 ␮l of serum. To determine the value for each fraction ofalbumin, (i.e., f(HMA) ⳱ [HMA:(HMA + HNA-1 + HNA2)], f(HNA-1) ⳱ [HNA-1:(HMA + HNA-1 + HNA-2)], andf(HNA-2) ⳱ [HNA-2:(HMA + HNA-1 + HNA-2)]) the
HPLC profiles obtained were subjected to numerical curve
fitting; each peakshape was approximated by a Gaussianfunction. The Student’s t-test was used to evaluate the significance of the differences. Values are expressed as mean ± SD
(12).
Oxidation of HSA and Serum by Chloramine-T
To prepare CT-HSA, HSA (15 ␮M) was incubated for 1
h in phosphate buffer (pH 8.0) at 37°C in an oxygen-saturated
solution containing 0.1 mM (CT0.1-HSA), 1 mM (CT1-HSA),
10 mM (CT10-HSA) or 50 mM (CT50-HSA) chloramine-T.
After incubation, the oxidation reactions were stopped bycooling followed by extensive dialysis of solutions against water. The control involved incubating albumin dissolved inbuffer alone, and in all cases the proteins were freeze-dried
after dialysis and stored at −20°C until used.
Amino Acid Residues Oxidized
Reactivity of 34Cys Residue with DTNBAlbumin solutions 1.0 × 10−4 M, 2 mL 0.067 M phosphatebuffer) were preincubated at 37°C. Absorbance increases at
412 nm were monitored against time after the addition of
DTNB (final concentration 2.0 × 10−4 M) (13).
Oxidation of Met Residues
Oxidized HSA, mutated rHSA, and control albumins, allat a concentration of 10 ␮M, were reduced with dithiothreitol(100 mM) in the presence of EDTA (10 mM) in denaturingAnraku et al.

686
buffer (6 M guanidine hydrochloride in 0.25 M Tris, pH 8.0).
The samples were incubated for 16 h at 37°C. After this treatment, the albumins were pyridylethylated and then digested
with CNBr (CNBr: Met molar ratio of 200:1) in the dark for
24 h at 37°C. The digestions were stopped by the addition ofacetone. After evaporation of the acetone and washing withethanol, the proteins were dissolved in 0.1% trifluoroacetic
acid. Protein concentrations were determined by a Bradford
assay (14), and the same amounts of proteins were used in the
SDS-PAGE analysis. It is evident that the cleavage of oxidized HSAs was suppressed by the oxidation of Met residues.
Changes in Protein Net Charge
Changes in the net charge of albumin were evaluated bya modification of the capillary electrophoresis method described by Pande et al. (15). One mL of an albumin sample (2␮M) was run in 100 mM borate buffer (pH 9.0 and 20°C), andthe migration time was determined by means of a CE990/99010 type capillary electrophoresis from Jasco Co. (Tokyo, Japan).
Carbonyl Group Determination
Protein-bound carbonyl groups were quantitated using
the method of Climent et al. (1). In summary, the groups were
derivatized with fluoresceinamine and their number calculated from the absorbance of the complexes at 490 nm (Jasco
Ubest-35 UV/VIS spectrophotometer).

Functional Properties of Native, Oxidized, andMutant HSAs
Ligand Binding Experiments
To study the binding of ligands to the albumins, warfarin
or ketoprofen was added to a solution of albumin (10 ␮M)in phosphate buffer to give a final drug concentration of 5
␮M. The unbound ligand fractions were separated using an
Amicon MPS-1 micropartition system with YMT ultrafiltration membranes by centrifugation (2000 g, 25°C, 40 min).
Adsorption of warfarin or ketoprofen to the filtration membranes and apparatus was found to be negligible. The concentration of unbound ligand was determined by HPLC. The
HPLC system consisted of a Hitachi 655A-11 pump and a
Hitachi F1000 variable fluorescence monitor or a Hitachi
655A variable wavelength UV monitor. LiChrosorb RP-18(Cica Merck, Tokyo, Japan) was used as the stationary phase.
The mobile phase consisted of 200 mM sodium acetate buffer(pH 4.5)/acetonitrile (40:60, v/v) for warfarin and of 200 mM
sodium acetate buffer (pH 4.5)/acetonitrile (60:40, v/v) for
ketoprofen. The flow rates in both cases were 1 mL/min. Warfarin was quantitated fluorometrically by using 320 nm and
400 nm for excitation and emission, respectively, and ketoprofen was detected at 270 nm by means of UV monitoring.
The unbound fraction (%) was calculated as follows: Unbound fraction (%) ⳱ [ligand concentration in filtered fraction/total ligand concentration (before ultrafiltration)] × 100.
Determination of Esterase-Like Activity

Structural Properties of Native, Oxidized, andMutant HSAs
Circular Dichroism (CD)Measurements were done using a Jasco J-720 type spectropolarimeter (Jasco Co., Tokyo, Japan) at 25°C. Far-UV
and near-UV spectra were recorded at protein concentrationsof 20 ␮M in phosphate buffer using 1 mm and 1 cm quartz
cells, respectively.

The reaction of p-nitrophenyl acetate with the albumins
was followed spectrophotometrically at 400 nm (Jasco Ubest35 UV/VIS spectrophotometer) by monitoring the rate of appearance of p-nitrophenol. The reaction mixtures contained 5
␮M p-nitrophenyl acetate and 20 ␮M protein in phosphatebuffer. Reactions were followed at 25°C. Under these conditions, a pseudo-first-order rate constant analysis could be applied (17), and the apparent hydrolysis rate constants (kobs)
were calculated.

Effective Hydrophobicity of Native, Oxidized, andMutant HSAs

In Vivo Experiments

The effective hydrophobicity of the albumins (1 ␮M), as
dissolved in phosphate buffer, was probed with bis-ANS (10␮M) at 25°C. The compound was excited at 394 nm (16), andfluorescence spectra were recorded on a Jasco FP-770 fluorometer (Tokyo, Japan) using 1 cm quartz cells, thermostateddevices and 5 nm excitation and emission band widths.

Animals

Effect on Aromatic Amino Acid Residues
Steady-state fluorescence measurements were made using a Jasco FP-770 fluorometer with 1 cm quartz cells andthermostated devices. All studies were performed using aprotein concentration of 2 ␮M at 25°C using 5 nm excitationand emission band widths. A fluorescence excitation wavelength of 295 nm (tryptophan (Trp) residue) or 280 nm (tyrosine (Tyr) residues) was employed.
Absorbance spectra (200–400 nm) of the albumins (20␮M) were recorded at 25°C with 1 cm quartz cells by using the
Jasco UV/VIS spectrophotometer.

Male ddY mice (6-weeks old, 25–35 g) were purchasedfrom the Shizuoka Agricultural Cooperative Association forLaboratory Animals (Shizuoka, Japan). The animals weremaintained under conventional housing conditions. This
study was carried out in accordance with the Principles ofLaboratory Animal Care as adopted and promulgated by theUnited States National Institutes of Health.
Biodistribution Experiment
HSAs were radiolabeled with 111In using DTPA anhydride as described previously (18). Each radiolabeled derivative was purified by gel-filtration chromatography using aSephadex G-25 column eluted with 0.1 M acetate buffer (pH6.0). The absorbency of the eluants was measured at 280 nm,
and the protein-containing fractions were pooled. The resulting solution was concentrated and washed with 0.9% NaCl by
ultrafiltration. The specific activity of each derivative was ap-

Human Serum Albumin as a Model for Oxidative Damage

687

proximately 37 MBq/mg protein. The mice received a 0.1 mg/
kg dose of a 111In-HSA conjugate in saline by tail vein injection and were housed in metabolic cages for urine collection.
At given time points, blood was collected from the vena cavawith the animal under ether anesthesia, and plasma was obtained by centrifugation. The heart, lung, liver, spleen, andkidney were excised, rinsed with saline, weighed, and examined for radioactivity. The amount of radioactivity in urine
was determined by collecting urine both excreted and remaining in the bladder. 111In radioactivity was counted in a welltype NaI scintillation counter (ARC-500; Aloka, Tokyo, Japan).
Pharmacokinetic Analysis
Tissue distribution patterns of the 111In-HSA derivatives
were evaluated using organ uptake clearance according to aprevious reported method (19). In the early period after injection, the efflux of 111In radioactivity from organs is assumed to be negligible, because the degradation products of
111
In-labeled ligands using DTPA anhydride cannot easily
pass through biological membranes (19). This assumption was
supported by the fact that no 111In was detectable in the urine.
Using this assumption, organ uptake clearance was calculatedby dividing the amount of radioactivity in an organ at 120 min
by the area under the plasma concentration-time curve(AUC) at the same time point. AUC was calculated by fitting
an equation to the plasma concentrations of the derivatives
using the nonlinear least-squares program MULTI (20). Tissue distribution patterns were evaluated using tissue uptake
clearances according to the integration plot analysis. Tissue
accumulation at time t was proportional to the AUC0-t. By
dividing the tissue accumulation at time t (␹t) and the AUC0-t
by the plasma concentration (Ct), CL tissue was obtained
from the slope of the plot of ␹t/Ct versus AUC0-t/Ct. A previous report (21) has shown, that 111In is not suitable for
evaluating the dynamic phase of a protein for which the invivo half life is long. Therefore, we estimated the plasma
half-life and liver clearance within the 120 min, period.
Statistics
Where possible, statistical analyses were performed using the Student’s t test.
RESULTS
HPLC Studies on Serum and Oxidized Serum From
Healthy Young Subjects
Figure 1 shows a representative HPLC profile of serum
and oxidized serum from a healthy young subject, obtained byelution from an ES-502N column with an increasing ethanol
concentration from 0 to 5% in the acetate-sulfate buffer (pH4.85). Peaks 1, 2, and 3 in Fig. 1 correspond well to HMA,
HNA-1, and HNA-2 with the retention times of 15.8, 25.8,
and 31.2 min, respectively (Fig. 1). Increasing the chloramine-T concentration from 0 mM to 0.1 or 1 mM resulted in
an increase in, especially, HNA-2 (Table I). When the chloramine-T concentration was increase to 10 or 50 mM (TableI), or the exposure time was increased from 3 min to 1 h (Fig.
1), the three protein peaks disappeared. The reason is that,
under these conditions, HSA was modified to such an extent

Fig. 1. HPLC profile of control serum and sera exposed to chloramine-T (1 mM) for 3 min or 1 h. The serum was from a healthy young
male subject (23 years of age) and was eluted from an ES-502N
column with an ethanol gradiant of 0 to 5% in acetate-sulfate buffer(pH 4.85). The ethanol concentration gradient was as follows; 0–5min, 0%; 5–30 min, linear increase from 0 to 5%; 30–35 min, linear
decrease from 5 to 0%; 35–40 min, 0%. Peaks 1, 2, and 3 correspondto HMA, HNA-1 and HNA-2, respectively, and their relative values
are included in Table I.

that it remained bound to column and could only be eluted at
a high ethanol concentrations (not shown).
Extent of Oxidation of Amino Acid Residues
The effect of oxidation on the reactivity of 34Cys was
examined using DTNB. The reactivity constants for pure
HSA and rHSA were found to be the same (i.e., 0.07 min−1).
The constant was decreased only slightly for CT0.1-HSA (i.e.,
0.05 min−1 P < 0.05), and considerably decreased in the case of
CT1-HSA and CT10-HSA (i.e., 0.02 min−1; P < 0.001), and noreactivity at all could be detected for CT50-HSA. As expected, no reactivity was found for the C34S mutant. These
results indicate an increased oxidation of the cysteine residue
with increasing concentrations of chloramine-T.
To examine the potential effect of the oxidations on thesix Met residues of albumin, the proteins were reduced andtreated with CNBr and then examined by SDS-PAGE. As
seen in Fig. 2, the C34S mutant (lane 2) and CT0.1-HSA (lane3) showed the same fragmentation pattern as normal HSA(lane 1) and wild-type rHSA (not shown). However, in thecase of CT1-HSA (lane 4), a slight decrease in the amount of
low-molecular weight fragments and a corresponding increasein high-molecular weight fragments and intact protein wereobserved. This tendency is more pronounced at higher con-

Anraku et al.

688
Table I. f(HMA), f(HNA-1) and f(HNA-2) Values (%) for HSA and Oxidized HSA in Serum from
Normal Subjectsa

After 3 minf(HMA)
f(HNA-1)
f(HNA-2)
After 1 hf(HMA)
f(HNA-1)
f(HNA-2)

Serum

Serum +CT (0.1 mM)

Serum +CT (1 mM)

Serum +CT (10 mM)

Serum +CT (50 mM)

74.9 ± 0.99
22.1 ± 1.51
2.97 ± 1.03

72.4 ± 2.50
22.5 ± 1.38
2.62 ± 0.78

65.2 ± 3.38*
22.4 ± 3.95
12.4 ± 1.28*

N.D.b
N.D.
N.D.

N.D.
N.D.
N.D.

72.8 ± 2.11
23.9 ± 2.39
3.21 ± 1.13

61.2 ± 3.63*
23.7 ± 4.23
14.6 ± 1.23*

N.D.
N.D.
N.D.

N.D.
N.D.
N.D.

N.D.
N.D.
N.D.

a

Average values of five experiments (± S.D.).
N.D. denotes for not detectable.
* P < 0.001 as compared with Serum.
bcentrations of chloramine-T (lanes 5 and 6). These results
show a concentration-dependent oxidation of methionine
residues.
The net charge on the albumins was investigated by determining their migration times by capillary electrophoresis.
The migration times for the C34S mutant (18.59 ± 0.11 min),
CT0.1-HSA (18.66 ± 0.08 min), CT1-HSA (18.46 ± 0.04 min)and CT10-HSA (18.45 ± 0.04 min) were the same or similar tothat of HSA and rHSA (18.59 ± 0.11 min and 18.58 ± 0.19
min). By contrast, the migration time for CT50-HSA was increased significantly (20.03 ± 0.42 min, P < 0.005). Thus, thenet negative charge on albumin is increased, only at the highest chloramine-T concentrations used.
The oxidation of proteins frequently results in the formation of carbonylated amino acid residues. Therefore, theformation of such groups was measured as a function of chloramine-T concentration. The results (mol carbonyl groups/
mol protein) showed concentration-dependent increases in
the level of these groups: 0.098 ± 0.045 for CT0.1-HSA, 0.158 ±0.030 for CT1-HSA, 0.331 ± 0.013 for CT10-HSA and 0.769 ±
0.038 for CT50-HSA. In comparison, the level in control HSA
was only 0.045 ± 0.017.
Structural Properties of Native, Mutant, andOxidized HSAs
The structural properties of the albumins were examinedusing several different spectroscopic methods. Figures 3A and3B show the far-UV and near-UV CD spectra, respectively.
As can be seen in Fig. 3A, the characteristics of the CD spectra of the C34S mutant, rHSA, CT0.1-HSA and CT1-HSA
were similar to that of native HSA. In the case of CT10-HSA
and CT50-HSA, the ␪obs-values are slightly higher in the range
ca. 207–240 nm indicating some loss in ␣-helix structure.
However, in all cases, the spectra were very different fromthat obtained for albumin, when dissolved in phosphatebuffer, pH 7.4, containing 6 M guanidine hydrochloride. Fig.
3B shows that the tertiary structures of CT10-HSA and CT50HSA, but not that of the other samples, had been modified.
However, again the structural changes are not so pronounced
as those observed for albumin that had been denatured withguanidine hydrochloride.
The effect of oxidation on the exposure of hydrophobic
areas was examined using the fluorescence probe bis-ANS.
The results (Fig. 4A) indicate that neither the mutation of34
Cys nor oxidation at the low chloramine-T concentrations
had any measurable effect on the accessible albumin hydrophobicity. However, treatment with higher concentrations ofchloramine-T (CT10-HSA and CT50-HSA) resulted in an increase in accessible hydrophobic regions.
The effect of mutating34Cys and of the oxidations on the
intrinsic fluorescence of albumin is shown in Fig. 4B. This
fluorescence, which mainly is due to the excitation of 214Trp,
is only affected to a small extent by the mutation and treatment with 0.1 mM or 1 mM chloramine-T. In contrast, 10 mM

Fig. 2. SDS-PAGE electrophoresis of the reduced forms of control albumin (HSA ⳱ rHSA), the C34S mutant and chloramine-T oxidized
HSAs treated with CNBr. Left lane, molecular weights of protein markers (M.W.). The gel was stained Coomassie Brillant Blue.

Human Serum Albumin as a Model for Oxidative Damage

689

Fig. 3. Far-UV (A) and near-UV CD spectra (B) of native, mutated or oxidized HSAs. (1) native HSA, (2) wild-type rHSA, (3) the C34S
mutant, (4) CT0.1-HSA, (5) CT1-HSA, (6) CT10-HSA, (7) CT50-HSA, (8) native HSA dissolved in phosphate buffer, pH 7.4, containing 6 M
guanidine hydrochloride. Spectra are the averages of three determinations.

and especially 50 mM chloramine-T had a pronounced effecton the fluorescence spectrum. The fluorescence at ␭max was
decreased to 76.8% and 23.8% of the normal level, respectively, and the ␭max was blue shifted in both cases from 339
nm to 333 nm. These findings suggest that in CT10-HSA and
CT50-HSA minor conformational changes occurred in the vicinity of the Trp residue. However, the possibility that the Trp
residue itself could have been oxidized cannot be excluded.
The type of changes in light absorption spectra (data notshown) suggest that this seems to be the case for CT10-HSA
and CT50-HSA (4).
The nature of the Tyr residues in the different albumins
was examined by fluorescence measurements using an excitation wavelength of 280 nm and emission wavelengths from
290 nm to 370 nm (data not shown). In this case no effects as
the result of the mutation or oxidations seem to have takenplace.

specialized cavities in subdomain IIA and IIIA, respectively(3). The potential effects of mutation and oxidation at thesesites were examined using warfarin and ketoprofen as representative drugs. As seen in Table II, the high-affinity bindingof warfarin which takes place at site I (23), was not affected by
the mutation or mild oxidation by chloramine-T. However,binding was slightly decreased by a more severe oxidation of
HSA (CT10-HSA and CT50-HSA). Essentially, the same effects, but to a more dramatic extent, were observed for thehigh-affinity binding of ketoprofen (Table II) which takes
place at Site II (24).
Among its many functional properties HSA also possesses an esterase-like activity that is largely due to the close
proximity of 410Arg and 411Tyr in Site II (17). Figure 5 showsthat this activity is significantly reduced in the case of CT10HSA and, especially, CT50-HSA, whereas the remaining albumins have comparable activities.

Functional Properties of Native, Mutant andOxidized HSAs

Pharmacokinetic Analysis of Native, Mutant, andOxidized HSAs

The unique drug binding properties of HSA can, to agreat extent, be explained by the presence of two major binding sites; Site I and Site II (22), which are located within

In an attempt to evaluate whether the C34S mutation or
the chloramine-T induced oxidations had any effect on the
biological fate of HSA, plasma half-lifes and uptake clearance

Fig. 4. Effect of different protein modifications on the fluorescence of albumin-bound bis-ANS (A) and on the intrinsic fluorescence ofalbumin as excited at 295 nm (B). (1) native HSA, (2) wild-type rHSA, (3) the C34S mutant, (4) CT0.1-HSA, (5) CT1-HSA, (6) CT10-HSA,(7) CT50-HSA. Spectra are the averages of three determinations.

Anraku et al.

690
Table II. Binding of Warfarin and Ketoprofen to Native, Mutatedand Oxidized HSAs at pH 7.4 and 25°Ca

ProteinFree fraction (%)(warfarin)

Free fraction (%)(ketoprofen)

Native HSA
Wild-type rHSA
C34S
CT0.1-HSA
CT1-HSA
CT10-HSA
CT50-HSA

24.71 ± 2.07
25.48 ± 1.14
25.01 ± 1.33
26.54 ± 1.93
27.41 ± 0.93
31.26 ± 1.54***35.60 ± 1.01**

3.78 ± 0.52
3.92 ± 0.67
3.99 ± 1.17
3.83 ± 2.11
3.89 ± 2.34
33.9 ± 2.44**
46.5 ± 3.58*

aAverage values of three experiments (± S.D.). * P < 0.001, ** P <0.01 and *** P < 0.05 all as compared with native HSA.

of the HSAs were determined in mice (Table III). Except for
CT50-HSA, the albumins had the same half-life (i.e., about 20
min). To better understand the reason for the decreased
plasma half-life of CT50-HSA (8.69 min) organ uptakes wasstudied. In Table III, the decreased plasma half-life of CT50HSA can be explained by a very pronounced increment inliver clearance. By contrast, neither the mutation nor the
milder oxidation of albumin had any effect on liver clearance.
The uptake by heart, lung, spleen, and kidney of CT50-HSA,
and the other modified albumins, were comparable to thoseof native albumin (results not shown).
DISCUSSION
The Validity of Chloramine-T as an Oxidant
Albumin, which is thought to have antioxidant properties, is the most abundant protein in extracellular fluids. Oxidation of protein sulfhydryl groups to mixed disulfides andtheir conversion to sulfhydryls by reduction might represent
an effective antioxidant system in extracellular fluids. In thissense, albumin, which possess a single sulfhydryl residue(34Cys), is responsible for the largest fraction of reactive sulfhydryl groups in extracellular fluids and the mercaptnonmercapt conversion (intermolecular sulfhydryl disulfide

Table III. Half-Lifes and Liver Clearances of 111In-HSAs in Micea

ProteinHalf-life(min)

Liver uptake clearance(␮L/h)

Native HSA
Wild-type rHSA
C34S
CT0.1-HSA
CT1-HSA
CT10-HSA
CT50-HSA

20.7 ± 1.26
20.1 ± 2.85
19.2 ± 1.39
20.8 ± 1.40
20.5 ± 2.40
20.3 ± 2.93
8.69 ± 1.39*

13.37 ± 2.57
13.47 ± 2.71
14.00 ± 1.37
12.15 ± 1.98
13.17 ± 1.86
12.93 ± 2.12
146.0 ± 8.91*

aAverage values of three experiments (± S.D.). * P < 0.001 as compared with native HSA.

exchange reaction) of serum albumin might be a component
in such a system. In this study, the authors attempted to construction in vivo oxidation model of albumin by treatment
with CT using on HPLC system. As shown in Fig. 1, in thecase of serum treated with CT (1 mM) for 3 minutes, thef(HMA) value of 65.2% was significantly lower than that of
74.9% for healthy young male subjects. A significant decreasein the f(HMA) value as a result of aging and some diseases
has previously been reported (10,12). The HPLC profile observed in Fig. 1 was similar to that of serum treated with CT(0.1 mM) for 1 h (Table. I). This finding leads to the suggestion that the oxidation of HSA is dependent on the concentration of oxidant as well as incubation time used for theoxidation and that HSA treated with CT for extended periods
is easily oxidized even when low concentration of oxidant areused. Therefore, the authors used this oxidant and carried out
a detail examination of the importance of 34Cys and otherresidues relative to antioxidant properties of HSA.
Amino Acid Residues Oxidized
The type and number of oxidized amino acid residues of
HSA increase with the chloramine-T concentration. The active oxidants in this system are HO⭈ and chloro radicals (4). In
CT0.1-HSA and CT1-HSA, only 34Cys and Met residues wereaffected. Increasing the chloramine-T concentration to 10
mM oxidized Met residues were affected to a more pronounced extent and apparently affected 214Trp as well. Finally, in CT50-HSA no reactivity of 34Cys was detected, Met
residues and 214Trp (or its surroundings) were intensively
modified and the net charge of HSA became more negative.
The latter observation is explained by the oxidation of arginine, lysine, or proline residues to ␭-glutamyl semialdehyde(1).
Structural Properties

Fig. 5. Effect of mutation and oxidation on the rate constants (Kobs)
for the hydrolysis of p-nitrophenyl acetate by albumin at pH 7.4 and
25°C. The data are average values of three experiments (± S.D.). * P< 0.001 and ** P < 0.01 both as compared with control.

The mild oxidation of HSA (CT0.1-HSA and CT1-HSA)
has no measurable effect on its secondary and tertiary structures. However, in CT10-HSA and, especially, CT50-HSA
slight decreases in ␣-helical contents were observed accompanied by tertiary conformational changes. These were detectable by near-UV CD and resulted in an increased exposure of hydrophobic portions of the protein. Large changes inintrinsic fluorescence and light absorption spectra were also
observed. Although these changes are consistent with oxidation of 214Trp, they could also, at least partly, reflect conformational changes in the protein in the vicinity of that residue.

Human Serum Albumin as a Model for Oxidative Damage691

In contrast, no conformational changes of rHSA were observed upon mutating Cys in position 34 to a serine.

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Functional Properties
The high-affinity binding of warfarin and ketoprofen wasstudied to examine whether the exceptional drug binding
properties of HSA had been affected by the mutation and theoxidations. In both examples, only CT10-HSA and CT50-HSA
showed reduced drug binding properties. The decreased binding of warfarin to CT10-HSA can be explained by conformational changes taking place in subdomain IIA and the oxidation of one or more of the Met residues at positions 298, 329
or 446. In CT50-HSA these explanations could be supplemented with oxidation of relevant lysine or arginine residues.
However, in both cases, the oxidation of 214Trp could contribute to the diminished binding, because a previous study
has shown that mutation of 214Trp to an alanine results indecreased warfarin binding (25). The decreased ketoprofen
binding to CT10-HSA and CT50-HSA is most likely the resultof conformational changes involving site II in subdomain IIIA
(7). The decreased binding to the latter protein can also be
caused by oxidation of 410Arg.
The esterase-like activity of HSA is caused by the proximity of 410Arg and 411Tyr in subdomain IIIA (17). This function is only impaired in the case of CT10-HSA and CT50-HSA.
The finding is probably the result of conformational changes
that affect the orientation of the two residues. In the latter
case, however, 410Arg could have been oxidized as well.
Pharmacokinetics
Neither the mutation of 34Cys for a serine nor the relatively extensive oxidation of HSA (CT10-HSA) had any effecton its plasma half-life or liver clearance in mice (Table III).
However, CT50-HSA was modified to such a degree that its
plasma half-life was decreased to a considerable exist from ca.
20 min to ca. 9 min. This finding can be explained by a ca.
11-fold increase in liver uptake clearance. The increased liverclearance is probably due to an increased endocytosis of theprotein by scavenger receptors of endothelial, Kupffer cells(26). In contrast to liver clearance, the uptake by heart, lung,spleen, and kidney were in all cases unaffected by the protein
modifications. The authors have not estimated the clearanceof other tissues or organs. However, in the future detailed
examinations of this will be carried out using cell lines.
CONCLUSION
As illustrated by the column experiments with serum, theauthors succeeded in constructing an oxidation model using
HSA. Functional studies, performed in vitro, with the different albumin preparations indicate that Site II is more effected
than Site I and 34Cys in HSA, when it is exposed to oxidative
stress.
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