Identification and characterization of oxidized human serum albumin
A slight structural change impairs its ligand-binding and antioxidant functions Asami Kawakami1,*, Kazuyuki Kubota2,*, Naoyuki Yamada2, Uno Tagami2, Kenji Takehana1, Ichiro Sonaka1, Eiichiro Suzuki2 and Kazuo Hirayama2
1 Pharmaceutical Research Laboratories, Ajinomoto Co. Inc., Kawasaki, Japan 2 Institute of Life Science, Ajinomoto Co. Inc., Kawasaki, Japan
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)
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.
doi:10.1111/j.1742-4658.2006.05341.x
[1]. HSA (66 kDa)
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 sulfonic acid (-SO3H), or S-nitroso acid (-SO2H), [5,6]. As the major oxidized form is (-SNO) thiol reversibly oxidized HSA, the proportion of reduced HSA [HSA(red)%] changes according to surrounding conditions:
Human serum albumin (HSA) is the most abundant protein in plasma ((cid:1)40 mgÆmL)1 or 0.6 mm), and total plasma protein (75– accounts for 50–60% of 80 mgÆmL)1) 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
Abbreviations HNA, nonmercarptoalbumin; HMA, mercarptoalbumin; HSA, human serum albumin; LC-ESI-TOFMS, liquid chromatography-electron spray ionization-time of flight.
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Intensity
fHSA(red)% ¼ ½reduced HSA=ðreduced HSA
c
A
þ reversibly oxidized HSAÞ(cid:2) (cid:3) 100g
d e
a b
d’
B
f g
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.
h
C
i
j
m/z
1290
1295
1300
1305
1310
1315
1320
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].
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.
evaluated several
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 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.
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 (cid:1)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 than is 78.5. Peak d was 119.8 Da heavier pool 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.
observed
The positive ionized albumin was observed with ions distributed from [M + 66H]66+ to [M + 36H]36+. Figure 1A shows the ESI mass spectrum of albumin purified by affinity chromatography. Here, the eluted fraction by affinity chromatography was defined as purified albumin. In the mass range (m ⁄ z 1290–1320), all observed peaks were [M + 51H]51+ ions. These peaks were named from lower mass in alphabetical order: m ⁄ z 1300.09 (peak a), 1301.55 (peak b), 1303.75 (peak c), 1306.10 (peak d) and 1306.94 (peak e), all respectively. Because peaks were [M + 51H]51+ ions, the deconvoluted molecular
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
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Table 1. Estimation of the various HSA structure based on LC- ESI-TOFMS information. All observed masses between m ⁄ z 1290 and 1320 were [M + 51H]51+ ions in LC-ESI-TOFMS spectrum.
the
Observed mass [M + 51H]51+
Peak
Molecular weight
Difference in mass from peak c
Estimated structure of HSA
a
1300.09
66253.6
)186.7
b
1301.55
66328.1
)112.2
c d e f
1303.75 1306.10 1306.95 1308.02
66440.3 66560.1 66603.2 66658.1
0 119.8 162.9 217.8
HSA(red)% value was originally 78.5. After removing excess Cys ⁄ cystine with a low molecular weight cut-off ultrafilter membrane, sample was applied to LC-ESI-TOFMS in order to determine the structure and purity of the HSA-Cys. In this mass spectrum, although the molecular-related ion of reduced HSA was hardly observed, peak d¢ showed the most signifi- cant intensity in the range of m ⁄ z 1290–1320. The m ⁄ z- value of peak d¢ (Fig. 1B) was identical to peak d (Fig. 1A). The HSA(red)% of HSA-Cys was only 5%, therefore HSA-Cys accounted for 95% with the excep- tion of the other peaks in the cysteinylated HSA sam- ple solution. The difference of relative molecular mass of the peak d and the peak g (162.2 Da) and that of peak c and peak e (162.9 Da) was consistent within experimental error. Therefore, peak g was probably glycated HSA-Cys.
g h i
1309.28 1306.34 1308.26
66722.3 66572.3 66670.3
282.0 132.0 230.0
j
1309.51
66734.0
293.7
Deficient form (N-terminal Asp-Ala) Deficient form (C-terminal Leu) Reduced HSA HSA-Cys Glycated HSA A disulfide bond cleavage of HSA-Cys fi -SO3H + HO3S- Glycated HSA-Cys HSA-Hcy A disulfide bond cleavage of HSA-Hcy fi -SO3H + HO3S- Glycated HSA-Hcy
Although the structure of peak f was unknown, it could be due to a partially cleaved and irreversibly oxidized S–S bond resulting in sulfenic acid (-SO3H). This is supported by the mass difference between peak d¢ and peak f (98.0 Da) corresponding to the mass of six oxygen atoms.
The
cysteine,
results of our ESI-TOFMS measurements showed that oxidized HSA in healthy human plasma showed mainly 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
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.
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-Hcy; HSA(red)% ¼ 5% and HSA(red)% ¼ 9%] and the purified nonoxidized HSA [HSA(red)% ¼ 78.5] from healthy plasma with Lys-C. The digested peptides were analyzed using FTMS. FTMS has high performance in high mass resolution and accuracy. If the precise mass is known, the exact composition formula of a low molecular weight com- pound can be determined.
The peptide containing Cys34 generated by Lys-C enzyme reaction is from Ala21 to Lys40 (ALVLIA- FAQYLQQCPFEDHVK). When the peptide was cysteinylated through a S–S bond binding at Cys34, the multiply-protonated the mono-isotopic mass of
The difference of relative molecular masses of peaks h and j (161.7 Da) and those of peaks c and e (162.9 Da) was again within experimental error, sug- gesting that peak j was glycated HSA-Hcy. Peak i cor- responded to peak f, possibly due to sulfenic acid formation, as described above. If the thiol group at Cys34 of reduced HSA was sulfenized (-SO3H), the difference in relative molecular mass against reduced HSA will be 49.0 Da due to the addition of three oxy- gen atoms. However, peaks with a relative molecular mass difference of 49 Da were hardly observed on the baseline level. Calculated molecular weight and the predicted structure of HSA corresponding to each peak observed in the LC-ESI-TOFMS measurements were listed in Table 1.
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Intensity
A
(h)
1
4
8
ALVLIAFQYLQQ34CPFEGHFEDVK
856 . 4452 856 . 1109 856 . 7793
A
2 0 N H N H N H N H N H
Hcy
857 . 1134
851 . 7724
ALVLIAFQYLQQ34CPFEGHFEDVK
64 (kDa)
852 . 1066
851 . 4382
B
Cys
852 . 4404
856 . 4443
856 . 7782
856 . 1099
B
120
851 . 7714
852 . 1052
Non-oxidized HSA Non-oxidized HSA
851 . 4365
C
)
100
856 . 1083
%
Highly oxidized HSA Highly oxidized HSA
856 . 4427 857 . 1117
(
80
852
854
856
858 m/z
60
40
i
A S H d e t s e g d n U
20
(C) Spectrum of
Fig. 2. ESI-FTMS spectrum, identification of binding site of adduct to albumin. Mass range displayed from m ⁄ z 850.5–858.5. (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. the Lys-C digested peptides from purified HSA.
0
0
2
4
6
8
Trypsin treated time (h)
molecule [M + nH]n+ was theoretically calculated to be 2552.2682 (1+), 1286.6380 (2+) and 851.4279 (3+), respectively, from peptide sequence information. For the homocysteinylated peptide, these masses were calculated to be 2566.2838 (1+), 1283.6458 (2+) and 856.0998 (3+).
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.
856.1109
and 851.4382
(Fig. 2A)
and
highly
Figure 2A,B shows FTMS spectra in the mass range m ⁄ z 851–858, which includes the peptides following Lys-C digestion of the HSA-Hcy and HSA-Cys stand- ards, respectively. Both of the multiply-charged ions were 3+. The mono-isotopic ions were observed at m ⁄ z (Fig. 2B), respectively. These values were within experimental the theoretical mono-isotopic values (m ⁄ z error of 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
whether this affects its susceptibility to proteolysis. We compared the proteolytic sensitivity of purified healthy plasma HSA [nonoxidized HSA, HSA(red)% ¼ oxidized HSA [HSA-Cys, 73.0%] HSA(red)% ¼ 8%]. 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
After we had determined the modification on Cys34 examined residue of oxidized albumin, we next
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found to be slightly increased. These results suggest that reduced HSA and oxidized HSA have different ligand-binding properties.
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 antioxidant property of albumin is impaired in oxidized HSA
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- including l-trp, genous small molecular compounds, 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.
HSA is the major antioxidant in blood due to its free thiol at Cys34. In this study, we investigated the poten- tial effect of oxidation on the antioxidant capacity of HSA by comparing the radical scavenging activities of HSA in various states of oxidation. The hydroxyl radical scavenging activity of nonoxidized HSA and highly oxidized HSA-Cys (10 mgÆmL)1) was studied using ESR. The typical 1 : 2 : 2 : 1 four-peak ESR spectrum of the hydroxyl radical was observed and is shown in Fig. 4A. Addition of HSA caused a decrease in the ESR signal intensities. While nonoxidized HSA [HSA(red)% ¼ 73.0%] quenched up to 68.7% of the hydroxyl radical signal, HSA-Cys [HSA(red)% ¼ 8%] reduced it by only 54.4% compared with the control. This suggests that the radical scavenging activity of reduced HSA is greater than that of HSA-Cys. When we used mildly oxidized HSA [HSA(red)% ¼ 54.4%], the signal decreased by 62.3%. As shown in Fig. 4B, the HSA(red)% and hydroxyl radical scavenging activ- ities for each sample show a high positive correlation. To eliminate the possibility that varying iron binding affinity of HSA decreased radical generation, we gener- ated the hydroxyl radical by a different reaction, UV photolysis of H2O2. The same results were obtained, showing that oxidized HSA had a decreased radical scavenging activity (Fig. 4C). Therefore, we concluded that oxidation of HSA reduced its radical scavenging activity.
Discussion
The binding affinity of each compound to purified HSA was evaluated by ultrafiltration. The results are expressed as unbound fraction (%) in Table 2. All the values tended to be relatively high in our experiments compared with their binding capacities to human plasma in the literature for unknown reasons. When we compared the unbound fractions of nonoxodized HSA [HSA(red)% ¼ 73.0] with mildly oxidized HSA [HSA(red)% ¼ 55.4], l-Trp bound less strongly to mildly oxidized HSA. The same result was obtained when cefazolin was used as a ligand. While l-Trp and cefazolin showed decreased affinity to mildly oxidized HSA, verapamil binding to mildly oxidized HSA was
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,
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%.
Unbound fraction (%)
L-Trp Cefazolin
Verapamil
HSA sample
HSA (red)%
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.
Purified non-oxidized HSA 78.5 54.4 Mildly oxidized HSA
50.4 65.9
17.9 62.3
63.3 51.1
To distinguish the different functional properties of oxidized HSA, it was first necessary to prepare clearly
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A
NaCl/Pi Nonoxidized HSA
330.5
332.5
334.5
336.5
338.5
340.5
Field (mT)
B
80
70
) l o r t n o c f o %
60
50
i
40
30
i
20
0
20
40
60
80
100
( y t i v i t c a g n g n e v a c s l a c d a r
HSA(red)%
C
70
60
) l o r t n o c f o %
50
40
defined and highly purified HSA samples. Initially, we attempted to analyze the structure of native oxidized HSA from healthy human plasma. Oxidized HSA has generally been regarded to have various modifications on free thiol group at Cys34. There are a number of previous reports in which diverse attempts to identify the actual structures of oxidized albumin in detail have been made. Sugio et al. [21] determined the X-ray structures of HSA, derived from human pooled plasma or from a Pichia pastoris expression system, at a reso- lution of 2.5 A˚ . However, they were not able to investigate in detail the final differences in the electron density map around the sulfhydryl side chain of Cys34. Therefore, X-ray analysis has not been able to precisely observe the structure of oxidized HSA. Bar-Or et al. [22] showed profiles of both typical commercial albu- min preparations and normal healthy volunteer human serum albumin by LC-ESI-TOFMS measurement. Sengupta et al. [23] also showed that thiols (Cys and Hcy) disulfide-bonded to albumin-Cys34 could be removed by treatment with dithiothreitol to form albu- min-Cys34-SH by ESI-TOFMS. In these studies, the structure of oxidized albumin was determined by MS. However, it is impossible to determine the exact bind- ing site for thiols by measurement of the mass of the whole albumin molecule. Additionally, Kleinova et al. [24] showed that the structure of pharmaceutical-grade HSA were mainly oxidized HSA. However, as intact albumin from human plasma was not used in their study, the true structure of physiologically oxidized HSA was never proven. Moreover, reduced HSA was indirectly measured after alkylation with 4-vinylpyri- dine and subsequent tryptic digestion. Therefore, for definitive analysis of albumin oxidation it is necessary to show that the oxidized albumin has no thiol group and has conjugated to cysteine, homocysteine or gluta- thione through a disulfide bond at Cys34.
30
i
20
10
( y t i v i t c a g n g n e v a c s l
0
i
a c d a r
Non- oxidized HSA
Highly oxidized HSA
In this study, to solve the structure of physiological oxidized HSA, we purified HSA from healthy human plasma. We employed ESI-TOF-MS analysis of HSA as a whole molecule and FTMS analysis of proteolytic digests. By combining both results, we succeeded in revealing that the majority of oxidized HSA in healthy human plasma has only a single modification at Cys34, which is due to a disulfide bond to cysteine.
Fig. 4. Scavenging effects of purified HSA with various HSA(red)% on hydroxyl radical. (A) ESR spectra of the spin adducts of DMPO- OH. Grey spectrum corresponds to the radical generated without HSA and dotted spectrum corresponds to the one generated when nonoxidized HSA was added to the reaction. (B) Radical scavenging activities are plotted against HSA(red)% of the samples. (C) Radical scavenging activities of nonoxidized and highly oxidized HSA against the hydroxyl radical generated by UV photolysis. Values are the mean ± SD (n ¼ 3, P < 0.005).
After the determination of the molecular structure of oxidized HSA, we subsequently prepared standard forms of nonoxidized HSA and oxidized HSA, using purified HSA from healthy human plasma. The identi- ties of prepared HSA samples were confirmed by LC-ESI-TOFMS (Fig. 1) and FTMS analysis (Fig. 2). Therefore, well characterized and highly purified oxid- ized HSA samples were used for functional analysis.
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[25]
the
Fig. 5. Molecular dynamics simulation of reduced HSA and HSA- Cys. Blue ribbon represents reduced form of HSA and green ribbon represents S-cysteinylated oxidized form. Five regions of HSA-Cys, highlighted by orange, were conformationally changed from reduced HSA. The Cys34 residue is shown in pink.
ship between reduced and oxidized HSA, we examined two functional properties, ligand-binding properties and antioxidant activities, using both purified nonoxi- dized and oxidized HSA samples.
the H-bond between Cys34 and Tyr84,
In order to examine if the localized modification at Cys34 of oxidized HSA causes an overall conforma- tional changes in albumin, we investigated the differ- ence in the susceptibility of the two types of HSA to tryptic proteolysis. As shown in Fig. 3, highly S- cysteinylated oxidized HSA showed increased suscepti- bility to tryptic proteolysis. Consistent results were reported by Glowacki et al. in their study that compared the proteolytic susceptibility of HSA-Cys with dithiothreitol-treated, highly reduced HSA. These findings suggest that HSA undergoes a conformational change upon S-cysteinylation, thereby making the clea- vage sites more accessible. We identified the cleavage site of tryptic digested HSA by Edman sequencing and revealed that region encompassing Glu82 in domain I becomes more susceptible to proteolysis. To gain further insight into the molecular basis for the effects of oxidation on conformational change, molecu- lar modeling of HSA-Cys was performed using the crystal structure of human serum albumin (RSC PDB ID: 1BM0). The expected modeled structures of HSA- Cys were subjected to molecular dynamics simulations using insightii (Accelrys Inc., San Diego, CA, USA). The conditions of calculation were as follows: force field ¼ discover3, run time ¼ 1000 fs, temperature ¼ 298 K. These calculations showed that the conforma- tional changes induced by S-cysteinylation of HSA were not large scale, but were localized to five regions of the molecule (indicated as orange ribbons in Fig. 5). One of the five conformationally altered regions, a loop between Thr79 and Leu85, which is located near to the Cys34 residue in the three-dimensional structure, may be the reason for increased susceptibility to pro- teolysis, as it contains the specific site for tryptic diges- tion, Glu82. Stewart et al. [2] recently analyzed X-ray structures of recombinant HSA and showed that the sulfur of Cys34 is tethered to the hydroxyl oxygen of Tyr84 by a hydrogen bond. They speculated that the formation of disulfide at Cys34 would lead to the loss of thereby resulting in a conformational change. In fact, using 1H NMR study, they demonstrated that S-cysteinyla- tion altered the conformation and dynamics of the entire domain I, and also the domain I ⁄ II interface. All these results suggest that oxidation, a single modifi- cation at Cys34, could result in a number of regional conformational changes of HSA resulting in increased susceptibility to proteolysis.
Ligand-binding properties are the most significant functions of HSA [26]. HSA binds and transports numerous endogenous and exogenous compounds, and controls their solubility and toxicity in vivo. Among the several endogenous ligands of albumin, we focused on l-Trp, which circulates in plasma mostly bound to site II on HSA [27]. Because l-Trp is the precursor of sero- tonin, it is hypothesized that increased levels of free l-Trp in plasma enhance serotonin synthesis and release at the brain. Excessive serotonin secretion is observed in many clinical conditions and modulates numerous physiological and psychiatric systems. In this study, highly oxidized HSA showed decreased l-Trp binding. The binding site of l-Trp on HSA is reported to be located at site II [28], which is distant from Cys34 in the three-dimensional structure. However, a spatial correlation between these two regions has been implica- ted in the study by Muscaritoli et al. [29]. In an in vitro study, they reported that the level of free unbound l-Trp increased in the presence of cisplatin, an anti- neoplastic drug that binds to HSA at Cys34. They suggested that cisplatin administration caused l-Trp
The structure of the protein should be highly associ- ated to its specific functional activities. We attempted to investigate whether the conformational change of oxidized HSA affects certain functional properties of albumin. To elucidate the structure–function relation-
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Characterization of oxidized human serum albumin
Cys34. As oxidized HSA has decreased antioxidant activity, decreased HSA(red)% not only reflects the oxidative shift of the redox state of the human body, but also may be a factor influencing the redox state of a number of diseases.
In conclusion, the present study demonstrated that oxidized HSA, primarily cysteinylated via a disulfide bond at Cys34, exhibits various differences in its biolo- gical properties relative to reduced HSA. Although it is still unknown whether highly oxidized HSAs from patients with a number of diseases have a similar structure, we suggest that reduced HSA(red)% may result in impaired function of HSA. We suggest that there may be potential diagnostic and therapeutic benefits of measuring HSA(red)% in a variety of dis- ease conditions.
Experimental procedures
Plasma collection
Preparation of purified HSA with various states of oxidation
Blood (160 mL) was collected using a vacuum tube collec- tion system, using heparin as an anticoagulant, from two healthy volunteers. Plasma fractions of the two volunteers were isolated by centrifugation at 4 (cid:1)C for 20 min (2000 g), and were mixed together. The pooled plasma, which is used as healthy human plasma in this study, was immediately frozen in liquid nitrogen prior to long-term storage at )80 (cid:1)C.
displacement from HSA and enhanced precursor avail- ability for serotonin synthesis and release at the brain, and that might contribute to the pathogenesis of cisp- latin-induced emesis. These findings indicate that the state of Cys34 of HSA affects the l-Trp binding affinity on the other side of the molecule. As shown in Table 2, our study also showed that S-cysteinylation at Cys34 decreased binding affinity towards l-Trp of HSA. This might also be related to increased levels of serotonin and the frequent occurrence of some adverse complica- tions of diseases which have an increased level of oxid- ized HSA. On the other hand, exogenous ligands of albumin such as cefazolin and verapamil also showed different binding affinities between nonoxidized HSA and mildly oxidized HSA. Mera et al. [30] recently demonstrated that purified albumin from hemodialysis patients with decreased HSA(red)% showed reduced drug-binding properties to warfarin and ketoprofen. In our experiment, mildly oxidized HSA displayed bidirec- tional changes in binding properties dependent on dif- ferent types of ligands. These observations indicate that structural changes of HSA caused by oxidative modifi- cation on the thiol moiety of Cys34 affect the drug- binding properties of this protein. These phenomena are important from a therapeutic point of view, as the concentration of unbound free drugs in plasma has an impact on pharmacokinetics, monitoring efficacy and adverse effects. These factors are essential in specifying the patient’s therapeutic regimen. Altered steady-state plasma concentration of drugs is a clinical therapeutic problem for the treatment of various inflammatory dis- eases, especially in elderly patients. It is assumed that reduced HSA(red)%, together with hypoalbuminemia, often found in these patients is responsible for this problem.
Non-oxidized HSA
from hemodyalysis
patients
showed
We prepared purified albumin samples with various HSA(red)% from healthy human plasma.
Mildly oxidized HSA
is found that
Just after plasma collection, the intact purified albumin pre- pared by affinity chromatography using HiTrapTM Blue HP Column (Amersham Bioscience) was designated as ‘nonoxi- dized albumin’. HSA(red)% of this sample was high as 78.5%. For functional assays (ligand-binding and antioxid- ant assay), the purified nonoxidized HSA was concentrated by ultrafiltration, followed by incubation at 37 (cid:1)C for 48 h. After the treatments, HSA(red)% of the purified nonoxi- dized HSA slightly changed to 73.0.
Antioxidant activity is also an important function of albumin. The function is believed to be ascribed to its single exposed thiol group at Cys34 [31–33]. Because albumin accounts for most of the total plasma thiol content (about 80%), it can act as a major antioxidant in plasma or extracellular fluids where the amounts of antioxidant enzymes are relatively small [34,35]. In the former article, Mera et al. [30] reported that purified albumin a decreased ability to scavenge chemical synthetic DPPH radicals. In this study, we also demonstrated that oxidized HSA has reduced scavenging ability against highly reactive oxygen species, in this case, hydroxyl radicals (Fig. 4C). It the degree of hydroxyl radical scavenging activity of HSA is highly correlated with HSA(red)% (Fig. 4B). This observa- tion confirms that the antioxidant activity of HSA, at least in part, depends on the state of the thiol at
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Mildly oxidized HSA was purified from the plasma which was incubated at 37 (cid:1)C for 18 h to promote aerobic oxida- tion. The HSA(red)% value of this sample was 54.4%.
A. Kawakami et al.
Characterization of oxidized human serum albumin
Highly oxidized HSA
Enzymatic digestion for FTMS measurement
following formula: identically charged ion using the HSA(red)% ¼ {(peak height of reduced HSA) ⁄ [(peak height of reduced HSA) + (peak height of oxidized HSA)]} · 100 The average of the eight values was assumed as HSA(red)% of the sample.
carbonate (pH 6.0). This lysylendopeptidase
Digested peptides measurement by FTMS
Lyophilized lysylendopeptidase (20 lg per vial; mass spectr- ometry grade; Wako Pure Chemical Industries, Osaka, Japan) was dissolved in 2 mL of 0.1 m ammonium acetate buffer solution was diluted 2.4 times with 0.1 m ammonium acetate buffer (pH 6.0). Purified fresh nonoxidized HSA and highly oxid- ized HSA-Cys solution buffers were changed to 0.1 m ammonium acetate by membrane filtration using 3000-Da cut-off filters. One hundred microliters of 30 lm purified nonoxidized HSA or HSA-Cys standards were mixed with 100 lL of 0.15 mm lysylendopeptidase, and incubated at 37 (cid:1)C for 8 h. The enzymatic digestion was inactivated with the 200 lL of 0.1% formic acid.
LC-ESI-TOFMS measurement
Highly oxidized albumin was prepared by artificial incor- poration of cysteine or homocysteine into reduced albumin. Cysteinylated HSA (HSA-Cys) and homocysteinylated HSA (HSA-Hcy) were prepared as follows. Purified reduced HSA at a concentration of 4 mgÆmL)1 (0.06 mm) was trea- ted with a 50-fold molar excess of l-cysteine ⁄ cystine by mixing 80 mL of 4 mgÆmL)1 HSA, 72 mL of 3 mm cys- teine, and 8 mL of 3 mm cystine. All solutions were in 0.1 m calcium carbonate ⁄ hydrogen buffer (pH 10.0), and the mixture was incubated at 37 (cid:1)C for 48 h. Next, low molecular weight compounds were removed by ultrafiltration through an Ultrafree-3000 Da membrane (Millipore, Billerica, MA, USA) at 4 (cid:1)C. The HSA(red)% value of this sample was 5–9%. HSA-Hcy was prepared by exactly the same procedure except that dl-homocysteine was used in place of l-cysteine, and homocystine was used in place of cystine. LC-ESI-TOFMS was used to determine the purity of HSA-Cys and HSA-Hcy. The additive reac- tion resulted in high yield and HSA(red)% value of each sample was <10%. We also confirmed the remaining l-cys- teine ⁄ cystine or homocysteine ⁄ homocystine in the standard solutions were minute using LC-MS ⁄ MS measurement.
Each plasma and albumin preparation was analyzed by HPLC (LC-Packings, Amsterdam, Netherlands) coupled to ESI-TOFMS (Bruker Daltonics Inc., Billerica, MA, USA).
Tryptic digestion of HSA
We used a Bruker Daltonics Apex II FT-MS equipped with a Bruker-Magnex actively shielded superconducting magnet operating at 7.0 T and a nano ESI Sources with Microme- talTip stainless emitter (Eisho-metal Co. Ltd, Tokyo Japan). The digested sample was diluted 10-fold with 0.1% formic acid in 50% acetonitrile ⁄ water (v ⁄ v). For syringe infusion experiments, the digested albumin samples were individually 100-fold diluted and infused into the ESI source at a flow rate of 200 nLÆmin)1. MS data were acquired in the positive ionized mode over an m ⁄ z range of 400–2000. Data acquisi- tion was performed using with the xmass software (Bruker Daltonics). We focused on the peptide containing Cys34 in the peptide mixture produced by enzyme digestion.
Plasma or purified HSA samples was diluted to 0.4 mgÆmL)1 (0.06 mm). The diluted sample was filtered with a 0.1 lm cut-off membrane filter microfilter tube. Sub- sequently, aliquots of 100 lL of filtered sample were trans- ferred to a sample vial. Two microliters of each sample was injected into the pre-column (100 mm · i.d. 200 lm, length packed with monolith C18; GL Science Inc., Tokyo, Japan). Albumin was eluted using a 20-min linear gradient method by 75 : 25 water ⁄ acetonitrile containing 0.1% for- mic acid (solution A) to 10 : 90 water ⁄ acetonitrile contain- ing 0.1% formic acid (solution B). All albumin samples were desalted and concentrated by a column switching method.
were mixed volumes equal with
The eluted albumin from the main column was intro- duced into ESI-TOFMS (microTOF(cid:2); Bruker Daltonics Inc.). In all experiments, spectra were acquired over the range m ⁄ z 50–3000. The observed mass spectrums were averaged from 30 scans of the albumin ion. The averaged data were smoothed using a Gauss algorithm and baseline subtracted using the microtof(cid:2) software.
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Purified nonoxodized HSA (300 lg) and highly oxidized HSA (HSA-Cys) (2 mgÆmL)1) were submitted to trypsin digestion by incubating the samples at the final substrate to trypsin (86 units, Wako) ratio of 25 : 1 in NaCl ⁄ Pi at 37 (cid:1)C for 8 h. Aliquots from each digested sample were collected at 0, 0.5, 1, 2, 4, and 8 h after the beginning of proteolysis. Reactants of 2 · SDS ⁄ PAGE sample buffer and denatured for 10 min at 95 (cid:1)C. Twelve micrograms of HSA per lane were subjected to SDS ⁄ PAGE on 12.5% polyacrylamide gels for 30 min at constant voltage (200 V). Protein bands were visualized by Coomasie brilliant blue staining. The gel image was captured using ImageMaster LabScan 5.0 (Amersham Biosciences, GE Healthcare, Piscataway, NJ, USA) and protein bands To determine HSA(red)% of the samples, we focused on the mass range of m ⁄ z 1250–1480 to identify certain signa- ture peaks. Eight pairs (reduced and oxidized) of albumin ions charged from 47+ to 54+ were obtained in this range. HSA(red)% was calculated independently for each
A. Kawakami et al.
Characterization of oxidized human serum albumin
Fenton–reaction
Ligand binding experiments
L-Trp binding property
corresponding to HSA were quantified using imagequant 5.1 software (Molecular Dynamics, Sunnyvale, CA, USA).
to purified added and highly
UV photolysis
A mixture of 5 mm DMPO, 0.025 mm FeSO4, 0.1 mm diethylenetriaminepentaacetic acid (DETAPAC), 0.5 mm H2O2, and purified HSA samples with various states of oxi- dation (10 ngÆmL)1) in NaCl ⁄ Pi (pH 7.3) was transferred to a quartz flat cell and placed in the cavity of the ESR spectro- meter for the measurement. The conditions of ESR measure- ment were as follows: center field 335.5 mT, microwave power 1 mW, modulation frequency 100 Hz, modulation width 0.1 mT, receiver gain 79, sweep width 10 mT, time constant 0.03 s, and sweep time 1 min.
Drug-binding properties
l-Trp was nonoxidized HSA (30 mgÆmL)1) oxidized HSA (HSA-Cys, 30 mgÆmL)1) in NaCl ⁄ Pi (pH 7.3) to give a final concentra- tion of 100 lm. The unbound ligand fractions were separ- ated using ultrafiltration membranes in Centricon YM100 devices (Amicon ⁄ Millipore Inc., Bedford, MA, USA) by centrifugation (3000 g, 5 min, 37 (cid:1)C). Fifty-microliter aliqu- ots of the initial (before ultrafiltration), filtrate, and apical (not ultrafiltered fraction) fractions were mixed with the same amount of 8% (w ⁄ v) trichloroacetic acid solution, respectively. Protein-free supernatants were separated by centrifugation at 3000 g for 5 min. l-Trp concentration of each fraction was determined by LC-MS analysis.
Calculation of the radical scavenging activity (%)
A mixture of 88 mm DMPO and 0.3% H2O2 in NaCl ⁄ Pi (pH 7.3) with or without HSA samples (10 ngÆmL)1) was transferred to a quartz flat cell and irradiated for 8 s under UV lamp at 230–430 nm in the cavity of the ESR spectro- meter. The following measurement was conducted under the conditions as follows: center field 335.5 mT, microwave power 3 mW, modulation frequency 100 Hz, modulation width 0.063 mT, receiver gain 250, sweep width 5 mT, time constant 0.03 s, and sweep time 1 min.
The scavenging effects of HSA on hydroxyl radicals were determined by the following calculation: scavenging activity (%) ¼ [(hPBS ) hHSA) ⁄ Radical hPBS] · 100%
Here, hPBS and hHSA are the ESR signal intensities without and with HSA, respectively. The intensities of the signals were normalized to that of manganese as an internal control. The binding of cefazolin (5 lm) and verapamil (5 lm) to HSA (30 mgÆmL)1) with two oxidative states in NaCl ⁄ Pi (pH 7.3, 37 (cid:1)C) was examined. The ultrafiltration method was the same as in the l-Trp binding experiment. Fifty- microliter aliquots of each fraction was mixed with 300 lL acetonitrile and left at room temperature for 5 min. Pro- tein-free supernatants were obtained by centrifugation at 3000 g for 5 min and the solvents were removed by evapor- ation. Samples were dissolved in the eluent for the follow- ing LC-MS analysis. The concentration of each compound was determined according to the standard curve made by LC-MS analysis.
Acknowledgements
Calculation of the unbound fraction (%)
The unbound fraction (%) of each ligand was calculated as follows:
Unbound fraction (%) ¼ [ligand concentration in filtrate ⁄ the initial ligand concentration (before ultrafiltration)] · 100
ESR spectroscopy
We would like to give our special thanks to Dr Seiichi Era, Department of Physiology and Biophysics, Gifu University Graduate School of Medicine, and Drs Hisataka Moriwaki and Hideki Fukushima, the First Department of Internal Medicine, Gifu University Graduate School of Medicine, for numerous discus- sions and helpful suggestions. We are grateful to Dr Masaichi-Chang-il Lee, Clinical Care Medicine Divi- sion of Pharmacology, Kanagawa Dental College, for valuable advice on ESR analysis. We also thank Dr Itsuya Tanabe and other researchers in Ajinomoto Co., Inc., for technical advice and helpful discussions.
Recovery was validated by the following calculation: Recovery (%) ¼ {[(ligand concentration in filtrate) · fraction · 400)] ⁄ (the 50+ (ligand concentration in apical initial ligand concentration · 450)} · 100
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