Effect of oxidative stress on the structure and function of human serum albumin

Article abstract of DOI:10.1023/A:1011029226072

Purpose. Human serum albumin (HSA) was mildly oxidized by a metal-catalyzed oxidation system (MCO-HSA), chloramine-T (CT-HSA) or H₂O₂ (H₂O₂-HSA), and the effects of these treatments on the structural, drug-bindi

Full text of DOI:10.1023/A:1011029226072

Pharmaceutical Research, Vol. 18, No. 5, 2001
Research Paper
multifunctional transport protein. Thus, several lines of evi-
dence exist which indicate that albumin also has significant
antioxidant activity (1–6) and that the protein, in fact, may
represent the major and predominant circulating antioxidant
in plasma, which is known to be exposed to continuous oxi-
dative stress (3). A direct protective effect of albumin is in-
dicated from many epidemiological studies (3). The results of
in vitro experiments also lend support to this hypothesis be-
cause they show that albumin protects human low density
lipoproteins against copper-mediated oxidation and blood
against hemolysis by free radicals (3), protects primary cul-
tures of murine m␾ and renal tubular epithelial cells against
reactive oxygen species (4) as well as, in the form of serum,
protects porcine thoracic aorta endothelial cells and certain
Effect of Oxidative Stress on the
Structure and Function of Human
Serum Albumin
1
1
Makoto Anraku, Keishi Yamasaki,
1
2
Toru Maruyama, Ulrich Kragh-Hansen, and
1
,3
Masaki Otagiri
Received December 15, 2000; accepted January 31, 2001
Purpose. Human serum albumin (HSA) was mildly oxidized by a types of porcine lung fibroblasts against affect by free radicals
metal-catalyzed oxidation system (MCO-HSA), chloramine-T (CT-
(
6). The antioxidant properties of albumin can also help pro-
HSA) or H O (H O -HSA), and the effects of these treatments on
2
2
2
2
tecting the eyes against contact lens disinfectant solutions
which often contain 3% H O (7).
the structural, drug-binding and esterase-like properties were studied.
Methods. Protein conformation was examined by calorimetric, chro-
matographic, electrophoretic and spectroscopic techniques. Drug
binding was studied by ultrafiltration method, and esterase-like ac-
tivity was determined using p-nitrophenyl acetate as a substrate.
2
2
HSA is a single, nonglycosylated polypeptide which is
organized in the form of a heart-shaped protein having about
67% ␣-helix but no ␤-sheet (8,9). The protein has three ho-
Results. Far-UV and near-UV CD spectra indicated that significant mologous domains (I–III), each of which is comprised of two
structural changes had occured as the result of treatment with MCO- subdomains, (A and B), that possess common structural ele-
HSA and CT-HSA but not with H O -HSA. However, SDS-PAGE ments. All but one of the thirty-five cysteine (Cys) residues
2
2
analysis does not provide precise information on gross conforma- are involved in the formation of stabilizing disulfide bonds.
tional changes such as fragmentation, cross-linking and SDS-resistant
Several studies, on the antioxidant activity of HSA in differ-
polymerisation. The results of differential scanning calorimetry, the
fluorescence of the hydrophobic probe 1,1-bis-4-anilino-naphthalene-
ent clinical situations, have focused on the importance of this
single, free sulfhydryl group of Cys (10–12). In the present
work, we report on an evaluation of the importance of other
amino acid residues as well by mild oxidation of HSA by a
metal-catalyzed oxidation system (MCO-HSA) (13), chlora-
34
5
,5-sulfonic acid and the elution time from a hydrophobic HPLC
column indicated that MCO-HSA and CT-HSA in particular, have a
more open structure and a higher degree of exposure of hydrophobic
areas than unoxidized HSA. In all cases, high-affinity binding of war-
farin remained unchanged for all the oxidized HSAs. However, high- mine-T (CT-HSA) (14) or H O (H O -HSA) (15). The im-
2 2 2 2
affinity binding of ketoprofen to CT-HSA and, especially, MCO- pact of the oxidative modifications on albumin structure was
HSA was diminished. In addition, the esterase-like activity of these studied by circular dichroism (CD), differential scanning calo-
proteins were all decreased to the same low level.
Conclusions. Mild oxidation of HSA has no detectable effect on the
rimetry (DSC), the use of a hydrophobic HPLC column and
a variety fluorescence methods.
binding of drugs to site I in subdomain IIA. In contrast, both the
HSA is also able to serve as a depot and transport pro-
ligand binding property of site II and the esterase-like activity of
tein in the circulation, because it can bind reversibly a large
oxidized HSAs are decreased, most probably due to conformational
number of endogenous and exogenous compounds. Two ma-
changes in subdomain IIIA.
jor drug binding regions, Site I and Site II (16), are located in
subdomain IIA and IIIA, respectively (8). In order to test
whether the molecular changes associated with the oxidations
KEY WORDS: human serum albumin; oxidative stress; structural
changes; warfarin binding; ketoprofen binding; esterase-like activity.
have an effect on the drug binding properties of albumin, the
INTRODUCTION
high-affinity binding of warfarin (Site I-ligand) and ketopro-
fen (Site II-ligand) was examined for different protein prepa-
rations by ultrafiltration. Since ketoprofen binding, in con-
trast to warfarin binding, was affected in most cases, we also
examined the potential effects of oxidation on the esterase-
Human serum albumin (HSA) is the most abundant pro-
tein in mammalian plasma and is generally considered to be a
1
Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe- like activity of albumin, an activity which is associated with
honmachi, Kumamoto 862-0973, Japan.
Site II (17).
2
3
Guest professor at Kumamoto University. Permanent address: De-
partment of Medical Biochemistry, University of Aarhus, DK-8000
Aarhus C, Denmark.
To whom correspondence should be addressed. (e-mail: otagirim@
gpo.kumamoto-u.ac.jp)
MATERIALS AND METHODS
Materials
ABBREVIATIONS: HSA, human serum albumin; MCO-HSA,
HSA was donated by the Chemo-Sera-Therapeutic Re-
search Institute (Kumamoto, Japan). It was defatted by treat-
ments with an equaous suspension of activated charcoal at
metal-catalyzed oxidized HSA; H O -HSA, HSA oxidized by hydro-
2
2
gen peroxide; CT-HSA, HSA oxidized by chloramine-T; DTNB, 5,5Ј-
Dithiobis (2-nitro benzoic acid); Bis-ANS, 1,1-bis-4-anilino-
naphthalene-5,5-sulfonic acid; CNBr, cyanogen bromide; CD, circu-
0
°C, after which it was acidified with H SO to pH 3, deion-
2 4
lar dichroism; DSC, differential scanning calorimetry; Cys, cysteine; ized, freeze-dried and stored at −20°C until used (18). HSA
Met, methionine; Trp, tryptophan; Tyr, tyrosine. and oxidized HSAs gave only one band on SDS-PAGE (data
0
724-8741/01/0500-0632$19.50/0 © 2001 Plenum Publishing Corporation
632

Effect of Oxidative Stress on Structure and Function of Human Serum Albumin
633
not shown), and the molecular mass of all the albumins were chloride in 0.25 M Tris, pH 8.0). The samples were incubated
assumed to be 67 kDa. Chloramine-T and 5,5Ј-Dithiobis (2- for 16 h at 37°C. After this treatment, the albumins were
nitro benzoic acid) (DTNB) was purchased from Nacalai pyridylethylated and then digested with CNBr (CNBr : Me-
Tesque, Inc. (Kyoto, Japan), and the fluorescence probe 1,1- thionine molar ratio of 200 : 1) in the dark for 24 h at 37°C.
bis-4-anilino-naphthalene-5,5-sulfonic acid (Bis-ANS) and The digestions were stopped by the addition of acetone. After
fluoresceinamine (isomer II) were obtained from Sigma (St evaporation of acetone and washing with ethanol, the pro-
Louis, MO, USA). Potassium warfarin (Eisai Co., Tokyo, Ja- teins were dissolved in 0.1% trifluoroacetic acid. The protein
pan) and ketoprofen (Sanwakagaku Co., Tokyo, Japan) were concentrations were determined by a Bradford assay (21),
obtained as pure substances from the manufacturers. All and the same amounts of proteins were used in the SDS-
other chemicals were of analytical grade, and all solutions PAGE analysis (22). It is evident that the cleavage of oxidized
were prepared in deionized and distilled water. Phosphate HSAs were suppressed by the oxidation of Met residues.
buffer, 67 mM and pH 7.4, was used as a standard buffer, and
was prepared from sodium phosphate dibasic and sodium
phosphate monobasic salts.
34
Reactivity of Cys Residue with DTNB
−
4
A HSA solution (1.0 × 10 M, 2ml 0.067M phosphate
buffer) was preincubated at 37°C. The absorbance increase at
Oxidation of HSA
4
12 nm was monitored against time after the addition of
For preparing MCO-HSA, HSA (300 ␮M) was incubated
in phosphate buffer at 37°C in an oxygen-saturated solution
−4
DTNB (final concentration 2.0 × 10 M) (23).
containing sodium ascorbate (100 mM) and FeCl (10 ␮M)
2
3
+
Structural Properties of Native and Oxidized HSAs
(
13). Ascorbate was added for reducing the Fe formed back
2
+
to Fe . Aliquots were withdrawn after different time inter-
vals, and the oxidative process was terminated by cooling and Circular Dichroism (CD)
removing the oxidants by extensive dialysis against water.
H O -HSA (300 ␮M) and CT-HSA (300 ␮M) were prepared
The measurements were done using a Jasco J-720 type
2
2
in phosphate buffer by aerobic incubation at 37°C with hy- spectropolarimeter (Jasco Co., Tokyo, Japan) at 25°C. Far-
drogen peroxide (200 mM) (15) and chloramine-T (10 mM) UV and near-UV spectra were recorded at protein concen-
(
14), respectively. Aliquots were withdrawn at different time trations of 20 ␮M in phosphate buffer.
intervals, and the oxidative processes were stopped by cooling
and the addition of acetone. After the reactions were stopped
the aliquots were dialyzed extensively against water.
Differential Scanning Calorimetry (DSC)
Prior to dialysis, all solutions contained 0.05% NaN . For
For a thermodynamic evaluation of the structural prop-
3
all three types of modification, controls were made by incu- erties of native and oxidized HSA, DSC was carried out on a
bating albumin dissolved in buffer alone, and in all cases the MicroCal MC-2 ultrasensitive DSC (micro-Cal Inc.,
proteins were freeze-dried after dialysis and stored at −20°C Northampton, MA, USA) at heating rates of 1 K/min, using
until used.
sample concentrations of 100 ␮M. The obtained DSC data
were applied to nonlinear fitting algorithms, in order to cal-
culate the thermodynamic parameters, thermal denaturation
temperature (Tm), calorimetric enthalpy (⌬H) and the van’t
Hoff enthalpy (⌬Hv), from the temperature dependence of
excess molar heat capacity, Cp, by employing Using Origin™
scientific plotting software.
Amino Acid Residues Oxidized
Carbonyl Group Determination
Protein-bound carbonyl groups were quantitated using
the method of Climent et al. (19). In summary, the groups
were derivatized with fluoresceinamine and their number cal-
culated from the absorbancy of the complexes at 490 nm
Effective Hydrophobicity of Native and Oxidized HSAs
(
Jasco Ubest-35 UV/VIS spectrophotometer). However, in
The effective hydrophobicity of oxidized and control
HSAs (1 ␮M), as dissolved in phosphate buffer, was probed
with Bis-ANS (10 ␮M) at 25°C. The compound was excited at
our hands the reproducibility of the results was improved by
increasing the amount of reactants by 40-fold.
3
94 nm (24), and fluorescence spectra were recorded on a
Changes in Protein Net Charge
Jasco FP-770 fluorometer (Tokyo, Japan) using 1 cm quartz
cells, thermostated devices and 5 nm excitation and emission
band widths.
Effective hydrophobicity was also evaluated by HPLC
chromatography by using a TSK-Gel Phenyl-5PW column
Changes in the net charge of albumin were evaluated by
a modification of the capillary electrophoresis method de-
scribed by Pande et al. (20). One mL of a HSA sample (2 ␮M)
was run in 100 mM borate buffer (pH 8.5 and 20°C), and the
migration time was determined by using a CE990/990-10 type
capillary electrophoresis from Jasco Co. (Tokyo, Japan).
(
7.5 mm × 75 mm), obtained from Tosoh (Tokyo, Japan). The
HPLC system consisted of a Hitachi 655A-11 pump and a
Hitachi 655A variable wavelength UV monitor set at 280 nm.
The proteins were eluted using the following ratios of solvent
A (1.5 M (NH ) SO ) and solvent B (phosphate buffer): from
Oxidation of Methionine Residues
4
2
4
Oxidized HSA (10 ␮M) and control HSA (10 ␮M) were 0 to 5 min the gradient was changed linearly from 50:50 to
reduced with dithiothreitol (100 mM) in the presence of 13:87, from 5 to 25 min to 0:100 and then maintained at that
EDTA (10 mM) in denaturing buffer (6 M guanidine hydro- value for an additional 15 min.

634
Anraku et al.
Effect on Aromatic Amino Acid Residues
literature (13–15). However, information concerning incuba-
tion times, does not appear to be available.
Steady-state fluorescence measurements were made us-
ing a Jasco FP-770 fluorometer with 1 cm quartz cells and
thermostated devices. All studies were performed at 25°C
using 5 nm excitation and emission band widths. A fluores-
cence excitation wavelength of 295 nm (tryptophan (Trp)
residue) or 280 nm (tyrosine (Tyr) residues) was employed.
Absorbance spectra were recorded at 25°C with 1 cm
quartz cells by using the Jasco UV/VIS spectrophotometer.
The oxidation of proteins usually results in the formation
of carbonylated amino acid residues. Therefore, the forma-
tion of such groups was measured as a function of time. As
seen in Fig. 1, a clearly detectable increase in the amount of
albumin-bound carbonyl groups was found for MCO-HSA
and CT-HSA. The formation of these groups reached a pla-
teau after 144 h and 10 h of incubation, respectively, and
longer times of incubation resulted in the developement of
turbidity, indicating the denaturation of the protein. In con-
trast, treatment with H O resulted in the formation of rela-
Functional Properties of Native and Oxidized HSAs
2
2
tively few carbonyl groups, and a prolonged incubation failed
to increase the amount of these groups. Incubation times of
Ligand Binding Experiments
1
44 h, 10 h, and 12 h for MCO-HSA, CT-HSA, and H O -
To study the binding of ligands to HSA and oxidized
HSAs, 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 the Amicon MPS-1 micropartition system
with YMT ultrafiltration membranes by centrifugation (2000
g, 25°C, 40 min). The adsorption of warfarin or ketoprofen to
the filtration membranes and apparatus was found to be neg-
2 2
HSA, respectively, appear to be a reasonable compromise
between the efficiency of oxidation and non-denaturing con-
ditions for the protein.
Extent of Oxidation of Amino Acid Residues
As a first step, we determined the amount of carbonyl
ligible. The concentration of unbound ligand was determined groups in the three types of oxidized HSAs after fixed incu-
by HPLC. The HPLC system consisted of a Hitachi 655A-11 bation times. The results (mol carbonyl groups/mol protein)
pump and a Hitachi F1000 variable fluorescence monitor or a showed pronounced increases in the level of these groups in
Hitachi 655A variable wavelength UV monitor. LiChrosorb MCO-HSA (0.144 ± 0.007 (S.D.), n ס 3) and CT-HSA (0.145
RP-18 (Cica Merck, Tokyo, Japan) was used as the stationary ± 0.004), but only a moderate increase in the case of H O -
2
2
phase. The mobile phase consisted of 200 mM sodium acetate HSA (0.056 ± 0.003) as compared with control HSA (0.037 ±
buffer (pH 4.5)/acetonitrile (40:60, v/v) for warfarin and of 0.002).
200 mM sodium acetate buffer (pH 4.5)/acetonitrile (60:40,
The net charge of the albumins was investigated by de-
v/v) for ketoprofen. The flow rates in both cases were 1 mL/ termining their migration times in capillary electrophoresis
min. Warfarin was quantitated fluorometrically by using 320 (Fig. 2). The migration time for H O -HSA (7.01 ± 0.09 min
2
2
nm and 400 nm for excitation and emission, respectively, and (S.D.), n ס 3) was only slightly increased as compared with
ketoprofen was detected at 270 nm by means of UV moni- that of normal HSA (6.81 ± 0.21 min). However, the times for
toring. The unbound fraction (%) was calculated as follows: MCO-HSA (9.48 ± 0.25 min) and CT-HSA (9.80 ± 0.13 min)
Unbound fraction (%) ס [ligand concentration in filtered were increased considerably more, indicating additional nega-
fraction/total ligand concentration (before ultrafiltration)] ×
100.
Determination of Esterase-like Activity
The reaction of p-nitrophenyl acetate with HSA and the
oxidized HSAs was followed spectrophotometrically at 400
nm (Jasco Ubest-35 UV/VIS spectrophotometer) by monitor-
ing the rate of appearance of p-nitrophenol. The reaction
mixtures contained 5 ␮M p-nitrophenyl acetate and 20 ␮M
protein in phosphate buffer. Reactions were followed at 25°C.
Under these conditions, pseudo-first-order rate constant
analysis could be applied (17), and the apparent hydrolysis
rate constants (kobs) were calculated.
Statistics
Where necessary, statistical analyses were performed by
the Student t test.
RESULTS
The Oxidation Processes as a Function of Time
Most of the information concerning parameters such as
molar ratios between the reactants, the temperature of incu- tion time. ᭿: MCO-HSA, ᭹: CT-HSA, ᭡: H O -HSA. The bars rep-
Fig. 1. Carbonyl content of oxidized HSAs as a function of incuba-
2
2
bation and how best to stop the reactions is available in the resent standard deviations (n ס 3).

Effect of Oxidative Stress on Structure and Function of Human Serum Albumin
635
3
4
not shown). These results indicate the possibility that Cys is
oxidized to some extent in the case of CT-HSA and H O -
2
2
HSA.
Structural Properties of Native and Oxidized HSAs
The structural properties of the oxidized HSAs were ex-
amined by a variety of methods. Thus, Fig. 4A and 4B show
the far-UV and near-UV CD spectra, respectively. As can be
seen in Fig. 4A, the characteristics of the CD spectra of the
three oxidized proteins were similar to that of native HSA. In
the case of MCO-HSA and CT-HSA, the ␪obs-values are
slightly higher in the range ca. 207–240 nm. However, in all
cases, the spectra were very different from that obtained for
albumin, when dissolved in phosphate buffer, pH 7.4, con-
taining 6 M guanidine hydrochloride. These findings indicate
that the oxidative processes have, at the most, only a slight
affect on the secondary structures of albumin. Figure 4B
shows that the tertiary structures of MCO-HSA and CT-
Fig. 2. Electrophoretograms of native and oxidized HSAs. (1) Native HSA, but not that of H O -HSA, have been modified. How-
2
2
HSA, (2) H O -HSA incubated for 12 h, (3) CT-HSA, (4) MCO- ever, again the structural changes are not so pronounced as
2
2
HSA incubated for 144 h, (4) MCO-HSA incubated for 10 h. The
absorbance spectra are the averages of three determinations.
those observed for albumin which had been denatured with
guanidine hydrochloride.
The effect of the oxidations on the thermal denaturation
of albumin was examined by means of DSC measurements.
As seen in Table I, the denaturation temperature (Tm) was
not affected by oxidation. In contrast, both the denaturation
enthalpy (⌬H) and the van’t Hoff enthalpy (⌬Hv) were di-
tive net charges and, therefore, a greater extent of modifica-
tion of these albumins.
To examine the potential effect of the oxidations on the
six methionine residues of albumin, the proteins were re-
duced and treated with CNBr and then examined by SDS-
PAGE. As seen in Fig. 3, MCO-HSA (lane 2) showed the
same fragmentation pattern as normal HSA (lane 1). In the
case of H O -HSA (lane 3), however, a lower amount of
minished as follows: native HSA > H O -HSA > CT-HSA >
2
2
MCO-HSA. Therefore in these results, broading of these
peaks was made in that order (data not shown). The progres-
sive decrease in the ⌬H-values suggests that the oxidized
forms are progressively more easily denatured which again
implies that the protein structures are more open (i.e., more
hydrophobic regions are exposed to the solvent) at 25°C. The
parallel diminution in the ⌬Hv-values indicates that, although
the thermal denaturation of the proteins takes place at the
same temperature, the denaturation process involves an in-
creasing number of intermediary steps.
2
2
low-molecular weight fragments was found. The most pro-
nounced effect was observed for CT-HSA (lane 4). In this
case, mainly high-molecular weight fragments and intact pro-
tein were detected.
3
4
The possible oxidation of only one Cys residue, Cys
3
4
was determined by the reactivity of Cys with DTNB on the
native and oxidized HSA. CT-HSA and H O -HSA showed
2
4
2
3
The effect of oxidation on the exposure of hydrophobic
areas was also examined by using the fluorescence probe bis-
ANS and the phenyl-substituted HPLC column. The results
obtained with bis-ANS (Fig. 5) indicate that the formation of
CT-HSA and of MCO-HSA in particular, invove the forma-
tion of increased accessible hydrophobic regions, whereas
only small changes take place in the case of H O -HSA. The
an average reduction in the reactivity of Cys of about 3%
compared with that of native albumin, whereas only neg-
ligeble changes were found in the case of MCO-HSA (data
2
2
results obtained with the hydrophobic column are consistent
with these findings, since the elution time of control HSA was
6
.35 ± 0.10 min (S.D., n ס 3), whereas those of H O -HSA,
2 2
CT-HSA and MCO-HSA were increased to 7.72 ± 0.24 min,
9.36 ± 0.19 min and 10.02 ± 0.11 min, respectively.
The effect of the oxidations on the single Trp residue in
albumin was monitored by fluorescence measurements (Fig.
A). Treatment with H O resulted in a small increment in
6
2
2
the fluorescence maximum (by 3.4%) but it had no effect on
␭max which was the same as that observed for native HSA
(
338 nm). In contrast, MCO-HSA and especially CT-HSA
had much lower fluorescence intensities at ␭max, 62.1% and
5.8% of the normal, respectively, and ␭max itself was, in both
cases, blue shifted to 333 nm. These findings suggest that in
gel was stained by using Coomassie Brillant Blue and then computer MCO-HSA and CT-HSA minor conformational changes had
scanned. taken place in the vicinity of the Trp residue (25). However,
Fig. 3. Pattern of SDS-PAGE electrophoresis of the reduced forms
of native and oxidized HSAs treated with CNBr. The lanes represent:
control; native HSA, MCO; MCO-HSA, H O ; H O -HSA, CT; CT-
4
2
2
2
2
HSA. Left lane, molecular weights of protein markers (M.W.). The

636
Anraku et al.
Fig. 4. Far-UV (A) and near-UV CD spectra (B) of native and oxidized HSAs. (1) native HSA, (2) H O -HSA, (3)
2
2
CT-HSA, (4) MCO-HSA, (5) native HSA dissolved in phosphate buffer, pH 7.4, containing 6 M guanidine hydro-
chloride. In all cases, the protein concentration was 20 ␮M. The spectra are the averages of three determinations.
4
10
411
the possibility also exists that the Trp residue itself could have largely due to the close proximity of Arg-and Tyr in Site
been oxidized. According to the light absorption spectra II (17). Since ligand binding to this site, in contrast to Site I,
shown in Fig. 6B, this seems indeed to be the case for both was effected in MCO-HSA and CT-HSA we also examined
MCO-HSA and CT-HSA.
the enzymic properties of the albumin preparations using p-
The status of the Tyr residues in the different albumins nitrophenyl acetate as a substrate. From Table III, it can be
was examined by fluorescence measurements using an exci- seen that the activity of H O -HSA is the same as that of
2
2
tation wavelength of 280 nm and emission wavelengths from native HSA (p > 0.1). In contrast, the activities of MCO-HSA
90 nm to 370 nm (data not shown). In this case no effects as and CT-HSA were much reduced and to the same level (p >
2
the result of the oxidations appear to have taken place.
0.1).
Functional Properties of Native and Oxidized HSAs
DISCUSSION
The unique ligand binding properties of HSA can, to a
great extent, be explained by the presence of two major bind-
ing sites; Site I and Site II (16), which are located within
Structural Aspects
Covalent modification of proteins and other macrostruc-
specialized cavities in subdomain IIA and IIIA, respectively tures by oxidative systems has been implicated in various
8). The potential effect of oxidation on these sites was ex-
(
amined by using warfarin and ketoprofen as representative
ligands. As seen from Table II, the high-affinity binding of
warfarin, which takes place at Site I (2), was not affected by
any of the oxidants. In contrast, high-affinity binding of the
Site II-ligand ketoprofen (26) was considerably diminished in
the case of CT-HSA and even more (p < 0.02) in the case of
MCO-HSA (Table II). The pronounced difference between
the free fractions of warfarin and ketoprofen in the case of
native HSA (Table II) is due to differences in the primary
5
−1
binding constants, which are 3.3 × 10 M for warfarin (2)
6
−1
and 2.5 × 10 M for ketoprofen (27).
HSA also possesses an esterase-like activity which is
Table I. Thermodynamic Parameters for Thermal Denaturation of
a
Native and Oxidized HSAs
Tm
(°C)
⌬H
(× 10 J/mol)
5
Protein
⌬Hv/⌬H
Native HSA
H O -HSA
CT-HSA
59.58 ± 0.05
59.45 ± 0.06
58.25 ± 0.10
58.10 ± 0.13
6.91 ± 0.13
5.13 ± 0.19
4.04 ± 0.21
3.13 ± 0.11
0.69 ± 0.01
0.65 ± 0.02
0.42 ± 0.03
0.38 ± 0.02
Fig. 5. Effect of native and oxidized HSAs on the fluorescence of
2
2
bis-ANS. (1) native HSA, (2) H O -HSA, (3) CT-HSA, (4) MCO-
2
2
MCO-HSA
HSA. The concentration of bis-ANS was 10 ␮M, whereas that of the
albumins was 1 ␮M. The spectra are the averages of three determi-
nations.
a
The data are average values of three experiments (± S.D.).

Effect of Oxidative Stress on Structure and Function of Human Serum Albumin
637
Fig. 6. Intrinsic fluorescence spectra (A) and light absorption spectra (B) of native and oxidized HSAs. The protein
concentration was 2 ␮M (A) or 20 ␮M (B). (1) native HSA, (2) H O -HSA, (3) CT-HSA, (4) MCO-HSA. The spectra
2
2
are the averages of three determinations.
pathological conditions. Albumin seems to be able to provide human high density lipoproteins (Apo AI and Apo AII) (14).
protection against such systems, and recently the antioxidant According to these authors, chloramine-T also oxidizes Cys
activity of HSA has received considerable attention. How- and Met residues. Due to its high sensitivity to free radicals
ever, detailed information about how this activity affects (10–12) the Cys residue of HSA is most probably also oxi-
HSA itself and its other functions is scarce. Therefore, in an dized in the presence of this compound. Therefore, we exam-
attempt to better understand this situation, we studied the ined the effect of chloramine-T on the Met residues of this
effect of three different oxidative systems on the structure, and the two other albumin preparations by CNBr fragmen-
ligand binding properties and esterase-like activity of HSA.
tation and SDS-PAGE (Fig. 3). This test showed that Met
HSA was mildly oxidized by treatment with a metal- residues were oxidized to methionine sulfoxide in CT-HSA
catalyzed oxidation system (MCO-HSA), H O (H O -HSA) and to a lesser extent in H O -HSA but not in MCO-HSA.
2
2
2
2
2
2
or chloramine-T (CT-HSA) because these are widely used as Furthermore, both the intrinsic fluorescence measurements
an in vitro oxidation model. In general, these oxidants are (Fig. 6A) and the light absorption spectra (Fig. 6B) indicated
2
14
known to generate different radicals. For example, metal- extensive modification of Trp in CT-HSA rather than mi-
catalyzed oxidation and chloramine-T generate OH and croenvironmental changes around Trp, because the binding
chloro radicals, and H O involves the formation of active of warfarin to oxidized HSA was nearly the same as that for
2
2
oxygen. This was done in order to study the early alterations native HSA. Thus, the three oxidized albumin forms have
of albumin structure, and, on an average, less than one car- been modified in different manners.
bonyl group was detected per albumin molecule (Fig. 1). In
Far-UV CD spectra showed that, in the case of MCO-
addition, as judged by capillary electrophoresis (Fig. 2), only HSA and CT-HSA, the oxidation of amino acid residues re-
minor or small changes in the net charge on albumin was sulted in the same decrease of ␣-helix content (Fig. 4A). The
introduced. According to the literature, proline, lysine, argi- near-UV CD spectra (Fig. 4B) were also modified, especially
nine, and/or histidine residues, but apparently not the Cys that of CT-HSA, and these changes indicate tertiary struc-
residue, have been modified in the case of MCO-HSA tural changes in the environment of the disulfide bonds and
(
(
13,28). Our data (Fig. 6) strongly suggest that the Trp residue aromatic amino acid residues (25). However, fluorescence
2
14
Trp) has been oxidized as well. According to the detailed measurements indicated that Tyr residues were not affected
3
4
study of Finch et al. (15), the SH-group of Cys and Met in the albumins studied in this work (data not shown). This
residues have been oxidized in the case of H O -HSA. The finding is in accord with other reports (13,25). The conforma-
2
2
protocol for preparing CT-HSA was adopted from a study of tional changes registered are most probably of a moderate
Table II. Binding of Warfarin and Ketoprofen to Native and Oxi- Table III. Effect of Oxidation on Hydrolysis Rate Constants (Kobs)
a
a
dized HSAs at pH 7.4 and 25°C
of HSA for p-Nitrophenyl Acetate at pH 7.4 and 25°C
Free fraction (%)
(warfarin)
Free fraction (%)
(ketoprofen)
Kobs
(× 10 sec )
−
2
−1
Protein
Protein
Native HSA
H O -HSA
CT-HSA
26.31 ± 3.21
27.56 ± 1.02
30.56 ± 4.02
27.12 ± 2.45
3.93 ± 0.89
4.56 ± 1.23
38.2 ± 2.96
47.5 ± 2.62
Native HSA
H O -HSA
CT-HSA
1.981 ± 0.21
2.193 ± 0.54
0.456 ± 0.02
0.353 ± 0.08
2
2
2
2
MCO-HSA
MCO-HSA
a
a
The data are average values of three experiments (± S.D.). The
concentration of both ligands was 5 ␮M, whereas that of the albu-
mins was 10 ␮M.
The data are average values of three experiments (± S.D.). The
concentration of both ligands was 5 ␮M, whereas that of the albu-
mins was 10 ␮M.

638
Anraku et al.
nature, because the changes in CD spectra were smaller than (Table III). Since this activity of HSA depends on the close
4
10
411
those observed in the presence of 6 M guanidine hydrochlo- proximity of
Arg and
Tyr (17), the latter finding most
ride (Figs. 4A and 4B), and because the results of SDS-PAGE probably is caused by conformational changes which increase
analysis (data not shown) excluded the possibility of gross the distance between the active groups of these residues. If
4
10
conformational changes such as fragmentation, cross-linking,
Arg had been modified in MCO-HSA, it would be ex-
and SDS-resistant aggregation. In contrast to MCO-HSA and pected to have a negative effect on esterase-like activity. This
CT-HSA, no conformational changes in H O -HSA could be observation seems to be in correlated well with the reduced
2
2
detected by the CD methods used.
ketoprofen binding. Interestingly, the findings obtained here
The conformational changes of MCO-HSA and CT- suggest a decrease in the binding of fatty acids, an endog-
HSA, and, to a lesser extent, of H O -HSA, seem to result in enous substances to oxidized HSA.
2
2
a more open protein molecule with a higher degree of expo-
Thus, the oxidation of albumin probably has individual
sure of hydrophobic areas. Such types of molecular changes effects on the different parts of the protein, and since these
are indicated by the findings that the fluorescence of the hy- parts possess different functional properties, or have them to
drophobic probe bis-ANS is increased considerably in the different degrees, a variety of effects on the function of albu-
case of the two former modifications (Fig. 5), that, in addition, min can be expected. In the case of the present preparations
these preparations have prolonged elution times from a hy- of H O -HSA, MCO-HSA, and CT-HSA the functional im-
2
2
drophobic column, and that the ⌬H-values for their thermal pairments are especially associated with the ligand binding
denaturation were much decreased (Table I).
and the enzymic properties of subdomain IIIA.
Functional Aspects
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3
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214
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