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 Pharmaceutical Research Laboratories, Ajinomoto Co Inc., Kawasaki, Japan 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 However, little is known regarding the pathophysiological significance of oxidation 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-ESITOFMS 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 nonoxidized (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 Human serum albumin (HSA) is the most abundant protein in plasma ($40 mgỈmL)1 or 0.6 mm), and accounts for 50–60% of total plasma protein (75– 80 mgỈmL)1) [1] HSA (66 kDa) is a single-chain polypeptide of 585 residues, which has heterogeneity as a result of post-translational nonenzymatic modifications 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 nonmercaptoalbumin) Reduced HSA contains 17 disulfide bonds and one free thiol group at Cys34 [2] Oxidized HSA is a generic name for those proteins that have various modifications at Cys34 HSA is a mixture of reversibly and irreversibly oxidized HSA Reversibly oxidized HSA has mixed disulfide bonds with a thiol compound such as cysteine, homocysteine [3,4] or glutathione In irreversibly oxidized HSA, Cys34 is more highly oxidized to sulfenic acid (-SOH), sulfinic acid (-SO2H), sulfonic acid (-SO3H), or S-nitroso thiol (-SNO) [5,6] As the major oxidized form is reversibly oxidized HSA, the proportion of reduced HSA [HSA(red)%] changes according to surrounding conditions: Abbreviations HNA, nonmercarptoalbumin; HMA, mercarptoalbumin; HSA, human serum albumin; LC-ESI-TOFMS, liquid chromatography-electron spray ionization-time of flight 3346 FEBS Journal 273 (2006) 3346–3357 ª 2006 The Authors Journal compilation ª 2006 FEBS A Kawakami et al fHSA(red)% ẳ ẵreduced HSA=reduced HSA ỵ reversibly oxidized HSAị 100g HSA(red)% tends to be lower in patients with various diseases or conditions such as hepatic disease [7], diabetes [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 pathophysiological significance HSA has various functions, such as: (a) maintenance of colloid osmotic pressure; (b) binding and transport of a wide variety of metabolites including steroids, fatty acids, bilirubin, tryptophan and hemin; (c) supplying an amino acid source during times of malnutrition; and (iv) acting as an antioxidant by radical scavenging [13–19] The goal of this study is to clarify the structure– function relationship between reduced and oxidized HSA in healthy human plasma, and to assess the pathophysiological significance of change in HSA(red)% In this report, we determined an exact adduct bound to Cys34 residue of oxidized HSA using mass spectrometry in order to prepare both reduced and oxidized HSA as standard samples for functional studies Using these, we evaluated several functional differences between purified HSA samples with various states of oxidation Results Analysis of the structure of oxidized albumin purified from human plasma using LC-ESI-TOFMS Structural heterogeneity exists in plasma HSA, which is a mixture of reduced, oxidized and glycated albumin We analyzed the purified HSA from healthy human plasma by LC-ESI-TOFMS in order to determine structure based on mass information The positive ionized albumin was observed with ions distributed from [M + 66H]66+ to [M + 36H]36+ Figure 1A shows the ESI mass spectrum of albumin purified by affinity chromatography Here, the eluted fraction by affinity chromatography was defined as purified albumin In the mass range (m ⁄ z 1290–1320), all observed peaks were [M + 51H]51+ ions These peaks were named from lower mass in alphabetical order: m ⁄ z 1300.09 (peak a), 1301.55 (peak b), 1303.75 (peak c), 1306.10 (peak d) and 1306.94 (peak e), respectively Because all observed peaks were [M + 51H]51+ ions, the deconvoluted molecular Characterization of oxidized human serum albumin Intensity c A a b de B d’ f g C h i j 1290 1295 1300 1305 1310 1315 m/z 1320 Fig [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 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) calculated from the known primary amino acid sequence of HSA after subtracting 34 Da due to 17 pairs of disulfide bonds Therefore, we regarded peak c as reduced HSA, and the others were due to post-translational modification resulting in mass differences compared with peak c When plasma was incubated at 37 °C to promote aerobic oxidation, we observed gradual increase in the intensity of peak d, while the intensity of peak c was reciprocally decreased Thus, we regarded peak d as oxidized HSA From peak heights, we estimated that HSA(red)% of healthy human plasma pool is 78.5 Peak d was 119.8 Da heavier than reduced HSA (peak c), corresponding to being the Cys-adduct of HSA via an S–S bond We speculated peak a to be the N-terminal Asp-Ala truncated form We also suggested peak b to be the C-terminal Leu truncated form and peak e to be glycated HSA Subsequently, we prepared highly oxidized HSA with Cys (HSA-Cys) as a standard Figure 1B shows the ESI mass spectrum of HSA-Cys Under the reaction conditions, excess Cys ⁄ cystine solution was added to purified HSA of healthy human plasma pool, whose FEBS Journal 273 (2006) 3346–3357 ª 2006 The Authors Journal compilation ª 2006 FEBS 3347 Characterization of oxidized human serum albumin A Kawakami et al HSA(red)% value was originally 78.5 After removing excess Cys ⁄ cystine with a low molecular weight cut-off ultrafilter membrane, the sample was applied to LC-ESI-TOFMS in order to determine the structure and purity of the HSA-Cys In this mass spectrum, although the molecular-related ion of reduced HSA was hardly observed, peak d¢ showed the most significant intensity in the range of m ⁄ z 1290–1320 The m ⁄ zvalue 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 exception of the other peaks in the cysteinylated HSA sample solution The difference of relative molecular mass of the peak d and the peak g (162.2 Da) and that of peak c and peak e (162.9 Da) was consistent within experimental error Therefore, peak g was probably glycated HSA-Cys Although the structure of peak f was unknown, it could be due to a partially cleaved and irreversibly oxidized S–S bond resulting in sulfenic acid (-SO3H) This is supported by the mass difference between peak d¢ and peak f (98.0 Da) corresponding to the mass of six oxygen atoms Figure 1C shows the ESI mass spectrum of the prepared 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 sample 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 incorporated by an S–S bond The HSA(red)% was only 9%, therefore HSA-Hcy accounted for 91% with the exception of the other peaks in the homocysteinylated HSA sample solution The difference of relative molecular masses of peaks h and j (161.7 Da) and those of peaks c and e (162.9 Da) was again within experimental error, suggesting that peak j was glycated HSA-Hcy Peak i corresponded 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 oxygen 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 3348 Table Estimation of the various HSA structure based on LCESI-TOFMS information All observed masses between m ⁄ z 1290 and 1320 were [M + 51H]51+ ions in LC-ESI-TOFMS spectrum Observed Difference in Estimated mass Molecular mass from structure Peak [M + 51H]51+ weight peak c 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 66658.1 119.8 162.9 217.8 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 + HO3SGlycated HSA-Cys HSA-Hcy A disulfide bond cleavage of HSA-Hcy fi -SO3H + HO3SGlycated HSA-Hcy The results of our ESI-TOFMS measurements showed that oxidized HSA in healthy human plasma showed mainly cysteine, and not homocysteine, adducts The comparative FTMS measurement of peptide mixture derived from highly oxidized HSA standard (HSA-Cys) and purified HSA of healthy human plasma From the result of ESI-TOFMS, we deduced that the main form of oxidized albumin in healthy human plasma is a cysteine adduct on reduced HSA In order to prove exactly where cysteine bonds to reduced HSA, we digested the standard highly oxidized HSA [HSA-Cys; HSA(red)% ¼ 5% and HSA-Hcy; 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 compound can be determined The peptide containing Cys34 generated by Lys-C enzyme reaction is from Ala21 to Lys40 (ALVLIAFAQYLQQCPFEDHVK) When the peptide was cysteinylated through a S–S bond binding at Cys34, the mono-isotopic mass of the multiply-protonated FEBS Journal 273 (2006) 3346–3357 ª 2006 The Authors Journal compilation ª 2006 FEBS A Kawakami et al Characterization of oxidized human serum albumin Intensity A ALVLIAFQYLQQ34CPFEGHFEDVK 856 4452 856 1109 856 7793 857 1134 Hcy 851 7724 851 4382 852 1066 A N H N H N H N (h) H N H 64 (kDa) ALVLIAFQYLQQ34CPFEGHFEDVK B Cys 852 4404 856 1099 856 4443 856 7782 B 120 C 856 1083 852 854 856 856 4427 857 1117 858 m/z Fig ESI-FTMS spectrum, identification of binding site of adduct to albumin Mass range displayed from m ⁄ z 850.5–858.5 (A) Spectrum of the Lys-C digested peptide containing Cys34 from HSA-Hcy conjugate (B) Spectrum of the Lys-C digested peptide contains Cys34 from HSA-Cys conjugate (C) Spectrum of the Lys-C digested peptides from purified HSA Non-oxidized HSA Undigested HSA (%) 851 7714 852 1052 851 4365 100 80 60 40 20 n+ molecule [M + nH] was theoretically calculated to be 2552.2682 (1+), 1286.6380 (2+) and 851.4279 (3+), respectively, from peptide sequence information For the homocysteinylated peptide, these masses were calculated to be 2566.2838 (1+), 1283.6458 (2+) and 856.0998 (3+) Figure 2A,B shows FTMS spectra in the mass range m ⁄ z 851–858, which includes the peptides following Lys-C digestion of the HSA-Hcy and HSA-Cys standards, respectively Both of the multiply-charged ions were 3+ The mono-isotopic ions were observed at m ⁄ z 856.1109 (Fig 2A) and 851.4382 (Fig 2B), respectively These values were within experimental error of the theoretical mono-isotopic values (m ⁄ z 856.0998, 851.4279) Accordingly, both HSA-Cys and HSA-Hcy standards would be derived from a Cys and a Hcy being incorporated into reduced HSA at Cys34 via an S–S bond, respectively Figure 2C shows the FTMS mass spectrum (m ⁄ z 851–858) of purified nonoxidized HSA using affinity chromatography from healthy human plasma A 3+charged mono-isotopic ion was observed at m ⁄ z 851.4365 This is consistent with that of the digested peptide from HSA-Cys standard Therefore, HSA-Cys with cysteine incorporated at Cys34 via a disulfide bond is the main form of purified HSA in healthy human plasma S-Cysteinylation affects susceptibility of albumin to trypsin digestion After we had determined the modification on Cys34 residue of oxidized albumin, we next examined Highly oxidized HSA Trypsin treated time (h) Fig 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 procedures 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 undigested HSA and the open arrow indicates the major tryptic fragment 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 whether this affects its susceptibility to proteolysis We compared the proteolytic sensitivity of purified healthy plasma HSA [nonoxidized HSA, HSA(red)% ¼ 73.0%] and highly oxidized HSA [HSA-Cys, HSA(red)% ¼ 8%] As shown in Fig 3A, both nonoxidized HSA and highly oxidized HSA (HSA-Cys) degraded in a time-dependent manner, but showed different susceptibility to digestion by trypsin, with highly oxidized HSA being degraded far faster than nonoxidized 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 proceeded, 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 FEBS Journal 273 (2006) 3346–3357 ª 2006 The Authors Journal compilation ª 2006 FEBS 3349 Characterization of oxidized human serum albumin A Kawakami et al digested peptide by Edman degradation The N-terminal 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 ligand binding HSA binds many endogenous and exogenous small molecular compounds, including l-trp, fatty acids, bilirubin, and drugs; HSA also plays an important role in delivering these compounds to target tissues Several specific binding sites of these ligands on HSA have been identified, and the two major binding sites are designated as sites I and II [20] The binding 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 properties 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 ligands All these ligands are known for their high binding efficiencies to HSA The binding affinity of each compound to purified HSA was evaluated by ultrafiltration The results are expressed as unbound fraction (%) in Table 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 Table 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 (%) HSA sample HSA (red)% L-Trp Cefazolin Verapamil Purified non-oxidized HSA Mildly oxidized HSA 78.5 54.4 50.4 65.9 17.9 62.3 63.3 51.1 3350 found to be slightly increased These results suggest that reduced HSA and oxidized HSA have different ligand-binding properties HSA is the major antioxidant in blood due to its free thiol at Cys34 In this study, we investigated the potential 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 mgỈmL)1) was studied using ESR The typical : : : 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 activities for each sample show a high positive correlation To eliminate the possibility that varying iron binding affinity of HSA decreased radical generation, we generated the hydroxyl radical by a different reaction, UV photolysis of H2O2 The same results were obtained, showing that oxidized HSA had a decreased radical scavenging activity (Fig 4C) Therefore, we concluded that oxidation of HSA reduced its radical scavenging activity Discussion Oxidation of HSA has been reported in numerous diseases 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 conformation and its functional properties In this study, in order to clarify the pathological consequences of oxidation, we identified an exact adduct and position of the modification of oxidized HSA from human plasma and characterized its specific functional properties by comparing among the purified HSA samples which had distinct HSA(red)% values To distinguish the different functional properties of oxidized HSA, it was first necessary to prepare clearly FEBS Journal 273 (2006) 3346–3357 ª 2006 The Authors Journal compilation ª 2006 FEBS A Kawakami et al Characterization of oxidized human serum albumin A NaCl/Pi Nonoxidized HSA 330.5 332.5 334.5 336.5 338.5 340.5 Field (mT) radical scavenging activity (% of control) B 80 70 60 50 40 30 20 20 40 60 80 100 HSA(red)% radical scavenging activity (% of control) C 70 60 50 40 30 20 10 Nonoxidized HSA Highly oxidized HSA Fig Scavenging effects of purified HSA with various HSA(red)% on hydroxyl radical (A) ESR spectra of the spin adducts of DMPOOH 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) 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 albumin 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 albumin-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 binding 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-vinylpyridine 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 glutathione through a disulfide bond at Cys34 In this study, to solve the structure of physiological oxidized HSA, we purified HSA from healthy human plasma We employed ESI-TOF-MS analysis of HSA as a whole molecule and FTMS analysis of proteolytic digests By combining both results, we succeeded in revealing that the majority of oxidized HSA in healthy human plasma has only a single modification at Cys34, which is due to a disulfide bond to cysteine After the determination of the molecular structure of oxidized HSA, we subsequently prepared standard forms of nonoxidized HSA and oxidized HSA, using purified HSA from healthy human plasma The identities of prepared HSA samples were confirmed by LC-ESI-TOFMS (Fig 1) and FTMS analysis (Fig 2) Therefore, well characterized and highly purified oxidized HSA samples were used for functional analysis FEBS Journal 273 (2006) 3346–3357 ª 2006 The Authors Journal compilation ª 2006 FEBS 3351 Characterization of oxidized human serum albumin A Kawakami et al In order to examine if the localized modification at Cys34 of oxidized HSA causes an overall conformational changes in albumin, we investigated the difference in the susceptibility of the two types of HSA to tryptic proteolysis As shown in Fig 3, highly Scysteinylated oxidized HSA showed increased susceptibility to tryptic proteolysis Consistent results were reported by Glowacki et al [25] in their study that compared the proteolytic susceptibility of HSA-Cys with dithiothreitol-treated, highly reduced HSA These findings suggest that HSA undergoes a conformational change upon S-cysteinylation, thereby making the cleavage sites more accessible We identified the cleavage site of tryptic digested HSA by Edman sequencing and revealed that the region encompassing Glu82 in domain I becomes more susceptible to proteolysis To gain further insight into the molecular basis for the effects of oxidation on conformational change, molecular modeling of HSA-Cys was performed using the crystal structure of human serum albumin (RSC PDB ID: 1BM0) The expected modeled structures of HSACys 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 conformational 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 proteolysis, as it contains the specific site for tryptic digestion, Glu82 Stewart et al [2] recently analyzed X-ray structures of recombinant HSA and showed that the sulfur of Cys34 is tethered to the hydroxyl oxygen of Tyr84 by a hydrogen bond They speculated that the formation of disulfide at Cys34 would lead to the loss of the H-bond between Cys34 and Tyr84, thereby resulting in a conformational change In fact, using H NMR study, they demonstrated that S-cysteinylation 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 modification at Cys34, could result in a number of regional conformational changes of HSA resulting in increased susceptibility to proteolysis The structure of the protein should be highly associated 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 relation3352 Fig Molecular dynamics simulation of reduced HSA and HSACys 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 nonoxidized and oxidized HSA samples Ligand-binding properties are the most significant functions of HSA [26] HSA binds and transports numerous endogenous and exogenous compounds, and controls their solubility and toxicity in vivo Among the several endogenous ligands of albumin, we focused on l-Trp, which circulates in plasma mostly bound to site II on HSA [27] Because l-Trp is the precursor of serotonin, 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 implicated 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 antineoplastic drug that binds to HSA at Cys34 They suggested that cisplatin administration caused l-Trp FEBS Journal 273 (2006) 3346–3357 ª 2006 The Authors Journal compilation ª 2006 FEBS A Kawakami et al displacement from HSA and enhanced precursor availability for serotonin synthesis and release at the brain, and that might contribute to the pathogenesis of cisplatin-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 complications of diseases which have an increased level of oxidized 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 bidirectional changes in binding properties dependent on different types of ligands These observations indicate that structural changes of HSA caused by oxidative modification on the thiol moiety of Cys34 affect the drugbinding 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 diseases, especially in elderly patients It is assumed that reduced HSA(red)%, together with hypoalbuminemia, often found in these patients is responsible for this problem Antioxidant activity is also an important function of albumin The function is believed to be ascribed to its single exposed thiol group at Cys34 [31–33] Because albumin accounts for most of the total plasma thiol content (about 80%), it can act as a major antioxidant in plasma or extracellular fluids where the amounts of antioxidant enzymes are relatively small [34,35] In the former article, Mera et al [30] reported that purified albumin from hemodyalysis patients showed a decreased ability to scavenge chemical synthetic DPPH radicals In this study, we also demonstrated that oxidized HSA has reduced scavenging ability against highly reactive oxygen species, in this case, hydroxyl radicals (Fig 4C) It is found that the degree of hydroxyl radical scavenging activity of HSA is highly correlated with HSA(red)% (Fig 4B) This observation confirms that the antioxidant activity of HSA, at least in part, depends on the state of the thiol at 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 biological 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 disease conditions Experimental procedures Plasma collection Blood (160 mL) was collected using a vacuum tube collection system, using heparin as an anticoagulant, from two healthy volunteers Plasma fractions of the two volunteers were isolated by centrifugation at °C for 20 (2000 g), and were mixed together The pooled plasma, which is used as healthy human plasma in this study, was immediately frozen in liquid nitrogen prior to long-term storage at )80 °C Preparation of purified HSA with various states of oxidation We prepared purified albumin samples with various HSA(red)% from healthy human plasma Non-oxidized HSA Just after plasma collection, the intact purified albumin prepared by affinity chromatography using HiTrapTM Blue HP Column (Amersham Bioscience) was designated as ‘nonoxidized albumin’ HSA(red)% of this sample was high as 78.5% For functional assays (ligand-binding and antioxidant assay), the purified nonoxidized HSA was concentrated by ultrafiltration, followed by incubation at 37 °C for 48 h After the treatments, HSA(red)% of the purified nonoxidized HSA slightly changed to 73.0 Mildly oxidized HSA Mildly oxidized HSA was purified from the plasma which was incubated at 37 °C for 18 h to promote aerobic oxidation The HSA(red)% value of this sample was 54.4% FEBS Journal 273 (2006) 3346–3357 ª 2006 The Authors Journal compilation ª 2006 FEBS 3353 Characterization of oxidized human serum albumin A Kawakami et al Highly oxidized HSA Highly oxidized albumin was prepared by artificial incorporation 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 mgỈmL)1 (0.06 mm) was treated with a 50-fold molar excess of l-cysteine ⁄ cystine by mixing 80 mL of mgỈmL)1 HSA, 72 mL of mm cysteine, and mL of mm cystine All solutions were in 0.1 m calcium carbonate ⁄ hydrogen carbonate buffer (pH 10.0), and the mixture was incubated at 37 °C for 48 h Next, low molecular weight compounds were removed by ultrafiltration through an Ultrafree-3000 Da membrane (Millipore, Billerica, MA, USA) at °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 reaction resulted in high yield and HSA(red)% value of each sample was