Characterization of and Mechanism for Copper-Induced Thioureation

Aug 20, 2008 - To whom correspondence should be addressed. Graduate Institute of Medical Sciences, Taipei Medical University, 250 Wu-Hsing Street, ...
0 downloads 0 Views 209KB Size
Download PDF
1822

Bioconjugate Chem. 2008, 19, 1822–1830

Characterization of and Mechanism for Copper-Induced Thioureation of Serum Albumin Yu-Wei Wu† and Yu-Hui Tsai*,†,‡ Graduate Institute of Pharmacy and Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan. Received November 10, 2007; Revised Manuscript Received July 30, 2008

Thioureas (Tus) are widely used in chemical and pharmaceutical industries. This study demonstrated that copper induced the disulfide-linkage between Tus, such as R-naphthylthiourea (ANTU) and fluorescein-5-isothiocyanate cadaverine (FTC), with albumin (Alb), a major carrier protein in plasma with multiple functions. This reaction was absolutely copper-dependent, whereas cobalt, nickel, calcium, magnesium, zinc, iron, and manganese ions could not induce the same reaction. The reaction was substrate dose-dependent, and occurred optimally at pH 6.5. The resulting conjugated product was heat-labile, but stable in pH 6.0-8.0 buffer at 25 °C. The linkage could be reduced by Cu(I) (in acidic pH) and thiol-reducing agents. The mechanism of albumin thioureation was concluded: (i) the binding of Cu(II) with albumin is not necessary for the reaction, while the formation of TusCu(II) complex is essential; (ii) thioureation resulted from the attack of Tus-Cu(II) at Alb-Cys34-SH to form the Alb-Cys34-S-S-Tus complex accompanied by the release of Cu(I); (iii) the released Cu(I) would back inhibit the reaction because of its competition with Cu(II) for Tus binding. These phenomenons may have important implications for the pharmacokinetics of thiourea-based drugs in plasma.

INTRODUCTION 1

Thiourea (Tu ) and its derivatives are widely used in chemical, agricultural, metallurgical, and pharmaceutical industries (1). Exposure of human and animals to Tu-containing compounds has been considered as an environmental risk factor that may cause various illnesses such as thyroid hyperplasia, pulmonary edema, pleural effusion, tumors, and so forth (1-3). Physiologically, Tu is absorbed from the gastrointestinal tract in humans and animals and is mostly excreted unchanged in urine (1, 4). The accumulation of radiolabeled Tu in rats was found mainly in thyroid tissue and to a lesser degree in the kidney, blood cells, lung, and liver (4, 5). The metabolism of Tu in the thyroid gland and liver is quite different. In the thyroid gland, Tu is oxidized by thyroid peroxidase to form dithioformamidine (Tu-S-S-Tu), which is later decomposed to cyanamide, sulfur, and Tu. Both Tu and cyanamide are inhibitors of thyroid peroxidase and finally lead the dysfunction of the thyroid gland (6). In the liver, Tu is metabolized to formamidine sulfenate and formamidine sulfinate by microsomal flavincontaining monooxygenase (FMO)-catalyzed S-oxygenation (7). In addition, radiolabeled Tu was found covalently and nonenzymatically binding to thyroid and lung proteins (8, 9). The binding of Tu to thyroid proteins resulted from thiol-disulfide exchange between the thiol of Tu with the intramolecular * To whom correspondence should be addressed. Graduate Institute of Medical Sciences, Taipei Medical University, 250 Wu-Hsing Street, Taipei, Taiwan 110. Tel: 886-2-2736-1661ext. 3417 and 3411. Fax: 886-2-2377-8620. E-mail: [email protected]. † Graduate Institute of Pharmacy. ‡ Graduate Institute of Medical Sciences. 1 Abbreviations: Alb, albumin; HSA, human serum albumin; Tus, thioureas; Tu, thiourea; FTC, fluorescein-5-isothiocyanate cadaverine; FCC, fluorescein-5-carboxamide cadaverine; DC, dansylcadaverine; ANTU, R-naphthylthiourea; EDTA, ethylenediaminetetraacetic acid; DTT, dithiothreitol; SDS, sodium dodecyl sulfate; NCP, neocuproine; NAC, N-acetyl-L-cysteine; GSSG, oxidized glutathione; MD-Charcoal, magnetic dextran-coated charcoal; CFA, charcoal-based fluorescence assay.

disulfide bridge of proteins to form the protein-S-S-Tu complex. The complex was finally hydrolyzed to protein-SSH and urea (8). Tu is known to interact with sulfhydryl-containing compounds such as cysteine or glutathione in vitro (10, 11). The oxidation of cysteine catalyzed by Tu in the presence of hydrogen peroxide in acid solution was illustrated in reactions A and B. The formation of the Cys-S-S-Tu complex was found in the reaction of cysteine with equimolar or excess amount of dithioformamidine (Tu-S-S-Tu) (reaction C). Cys-S-S-Tu was also produced by reacting cysteine with Tu (reaction D). Finally, the Cys-SS-Tu formed in reactions C and D was spontaneously hydrolyzed (reaction E). 2Tu + H2O2 f Tu-S-S-Tu + 2H2O 2Cys-SH + Tu-S-S-Tu f Cys-S-S-Cys + 2Tu Cys-SH + Tu-S-S-Tu f Cys-S-S-Tu + Tu (Tu-S-S-Tu g Cys-SH) Cys-S-S-Cys + Tu f Cys-S-S-Tu + Cys-SH Cys-S-S-Tu + H2O f Cys-SOH + Tu

(A) (B) (C) (D) (E)

The protective role of thioureas (Tus) was found in the clearance of free radicals (12-14). In addition, Tus were known to interact with miscellaneous metal ions including copper (15-17). It was speculated that the binding of copper with Tu may protect cells from copper-induced oxidative damage (18). In pharmaceutical applications, Tu-containing compounds are designed as anti-HIV drugs (19), high-density lipoprotein (HDL)-elevating agents (20), appetite suppressants (21), and so forth. Some of these compounds were found to hydrophobically interact with albumin (22, 23). Albumin is the most abundant protein in blood plasma with miscellaneous functions. Human serum albumin (HSA) itself is a 67 KD single chain protein with 35 cysteine residues; however, it has only one free thiol group of cysteine residues at position 34 (Cys34); the other 34 cysteine residues form 17 disulfide bridges for its tertiary structure. HSA with the free thiol in Cys34, known as mercaptoalbumin, is capable of

10.1021/bc7004158 CCC: $40.75  2008 American Chemical Society Published on Web 08/20/2008

Mechanism of Albumin Thioureation

participating in numerous redox reactions. Albumin is known to bind small molecules of many types. Metal ions such as copper, nickel, cobalt, zinc, and so forth are carried by albumin. Endogenous and exogenous ligands, metabolites, and drugs are generally bound to Sudlow’s site-I or site-II of albumin, with some exceptions (24-26). In addition, covalent interactions with albumin including glycosylation (27), acylation (28), thiolation (29, 30), S-nitrosation (31, 32), and N-homocysteinylation (33) are also found. Thiolation of HSA (HSA-S-R) occurs at Cys34 with low molecular weight thiol-containing substances (e.g., cysteine, homocysteine, glutathione, and drugs such as cisplatin, D-penicillamine, and N-acetyl-cysteine) via disulfide bridge formation (24-26). Thiolation of albumin has not been previously reported as a copper-dependent reaction; however, a fast S-nitrosation of albumin was mediated by copper (32). In our previous study, a charcoal-based fluorescence assay (CFA) was developed to rapidly analyze the incorporation of small fluorescent molecules into large protein molecules (34). By using the method, a copper-dependent FITC-binding protein was found in human plasma. This protein was subsequently purified and identified as albumin (35). The mechanism of copper-induced albumin thioureation was proposed, evaluated, and established in this study. Furthermore, the importance of this finding is discussed.

EXPERIMENTAL PROCEDURES Materials. Essentially fatty acid free, globulin free albumin with 99% in purity from human (HSA; A3782), bovine (BSA; A0281), and porcine (PSA; A1173) sera were purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human serum albumin (rHSA), containing 584 amino acid residues with a single deletion at Asp1 residue that reduces its binding affinity for copper and nickel, was from United States Biological (Swampscott, MA, USA). Fluorescein-5-isothiocyanate cadaverine (FTC) and fluorescein-5-carboxamide cadaverine (FCC) were from AnaSpec, Inc. (San Jose, CA, USA). BioMag magnetic dextran-coated charcoal (MD-Charcoal) concentrate (40 mg/mL, approximately 1.5 µm particle size) was supplied by Bangs Laboratories, Inc. (Fishers, IN, USA). Other chemicals used were of analytical grade from either Sigma-Aldrich, or Merck (Darmstadt, Germany), unless otherwise stated. NHS-activated Sepharose 4 Fast Flow media were from Amersham Biosciences AB (Uppsala, Sweden). The ultrafiltration unit was from Millipore (Bedford, MA, USA). V96 microwell plate (#249945) was from Nalge Nunc International (Rochester, NY, USA). Slot blot manifold (Hoefer PR648) was from Hoefer, Inc. (San Francisco, CA, USA). Stocks of 1 mM FTC, FCC, and dansylcadaverine (DC) were dissolved in 20% dimethyl sulfoxide and stored at 4 °C till use. Cuprous chloride (CuCl) of 10 mM was freshly prepared in 50 mM HCl. L-Cystine was prepared as 1 mM stock solution in 50 mM HCl. Solution of 50 mM neocuproine (NCP) was dissolved in 50% methanol. MD-Charcoal concentrate was 5-fold diluted with water and stored at 4 °C for subsequent application. The water used in this study was prepared by the Milli-Q system (Millipore, Bedford, MA, USA). Modification of Albumin. Albumin used in this study was mercaptoalbumin (Alb-Cys34-SH) unless otherwise stated. Mercaptoalbumin preparation was modified from a previous publication (36). Briefly, 1 mM HSA (or BSA, PSA, and rHSA) in sodium phosphate buffer (0.1 M Na2PO4, pH 6.86, 0.3 M NaCl, and 1 mM EDTA) was treated with 5 mM DTT at 25 °C for 45 min. The phosphate buffer of the specimen was then changed to HBS buffer (10 mM Hepes-pH 7.0, 0.15 M NaCl) by dialysis at 4 °C. One mole of albumin was confirmed to contain 1 mol of free thiol by Ellman’s method (37). Alkylation of albumin (Alk-HSA) was performed according to Glowacki et al. with modification (33). Briefly, 1 mM

Bioconjugate Chem., Vol. 19, No. 9, 2008 1823

iodoacetamide was incubated with 0.1 mM human mercaptoalbumin containing 0.1 M Hepes-pH 7.5 and 0.2 mM EDTA at 37 °C for 2 h. The buffer of resulting products was changed to HBS buffer by dialysis at 4 °C. Free thiol content of Alk-HSA was calculated to be 0.13 (M/M). For cysteinylation, human mercaptoalbumin (50 µM) was treated with 1 mM L-cystine in 0.1 M Hepes-pH 7.5 buffer containing 0.1 mM EDTA at 37 °C for 21 h. The mixture was subject to HBS buffer exchange by using a 30 K ultrafiltration unit (Amicon Ultra-15). This protein product (50 µM) was further treated with 1 mM IAN in 0.1 M Hepes-pH 7.5 buffer containing 0.1 mM EDTA at 37 °C for 3 h. The buffer of the protein was changed to HBS buffer as described above. This protein product was termed Cys-HSA. To determine the extent of albumin cysteinylation, Cys-HSA (165 µM) was treated with 5 mM DTT at room temperature for 45 min. The buffer of the reduced protein was changed to HBS buffer again as described above. The product from this step was termed r-Cys-HSA (thiolreduced Cys-HSA). Free thiol content for Cys-HSA and r-CysHSA was calculated to be 0.08 and 0.50 (M/M), respectively. The contents of r-Cys-HSA and Cys-HSA were thus estimated as follows: r-Cys-HSA contained Alb-SH (∼50%) and AlkHSA (∼50%); Cys-HSA contained Alb-SH (∼8%), Alb-S-SCys (∼42%), and Alk-HSA (∼50%). Analysis of Ligand-Binding by CFA. A high-throughput CFA method modified from our previous publication was used to analyze the binding capacity of albumin for fluorescent dyes (see Supporting Information, Figure 1) (34). Typically, 30 µL per well of protein samples (2 µM HSA, unless otherwise stated) dissolved in HBS buffer (10 mM Hepes-pH 7.0, 0.15 M NaCl) were added to microplates at 4 °C. After 30 µL of freshly prepared reagent-A (100 mM Hepes-pH 7.0, 0.2 mM CuCl2, and 6 µM FTC) was added into each well, the reaction was initiated by incubating the microplate in a 37 °C humidified incubator for 10 min and terminated by adding 60 µL of reagent-B (2 mM EDTA, 0.5 M Hepes-pH 8.0; 4 °C). Suspended MD-Charcoal (200 µL per well) was then added and incubated at room temperature for 5 min to remove all of the free fluorescent dyes. After precipitating MD-Charcoal by magnet for 1 min, the fluorescence intensity in each well was measured by a microplate reader (Plate Chameleon, Hidex Oy, Finland) with excitation wavelength setting at 485 nm and emission wavelength at 535 nm (Ex485/Em535); gain 35. Transfer of supernatant to a new plate for fluorescence counting is not necessary because the precipitated MD-Charcoal did not influence the fluorescence in the supernatant. Background level of fluorescence intensities were measured by replacing protein samples with HBS buffer. The binding capacity of albumin for fluorescent dye was expressed as net or relative fluorescence (fluo.) intensity per reaction. All experiments were performed in triplicate for each datum point, and the data were presented as the means ( SD. In addition, supplementation of metal ions or small molecules such as cysteine, NCP, and so forth in the reaction mixture did not influence fluorescence counting by using our assay system (see Supporting Information Figures 2 and 3). Beads Preparation. Tu- and Cys-beads were prepared by coupling each ligand with NHS-activated Sepharose 4 matrix. In detail, Tu (0.1 M) or cysteine (0.1 M) dissolved in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4, pH 7.4) was mixed with equal volume of the drained NHS-activated Sepharose 4 Fast Flow media at 25 °C for 2 h with constant shaking. The resulting matrix was washed with basic buffer (0.5 M Tris, pH 9.0, and 0.5 M NaCl) and then with acidic buffer (0.1 M acetate and 0.5 M NaCl, pH 4.0) for 3 cycles. Finally, these beads were washed with HBS buffer

1824 Bioconjugate Chem., Vol. 19, No. 9, 2008

Wu and Tsai

Figure 1. Chemical structures of Tu derivatives and analogues. (a) Tu; (b) ANTU; (c) FTC; (d) urea; (e) DC; (f) FCC.

and stored at 4 °C. The Cys-bead was treated with 10 mM DTT at 25 °C for 30 min before use. Alkylation of the Cys-bead (Alk-Cys-bead) was performed by incubating the bead with an equal volume of 10 mM iodoacetamide at 37 °C for an hour with shaking. The bead was then washed with HBS buffer and stored at 4 °C. Copper pretreated Tu- or Cys-beads were produced by mixing the Tu- or Cys-beads with an equal volume of 5 mM CuCl2 at 25 °C for 15 min with constant shaking. After supplementation with 20 mM EDTA, the beads were then washed with HBS buffer and then stored at 4 °C till use. Formation and Purification of the HSA-FTC Complex. The HSA-FTC complex was produced by reacting 2 µM human mercaptoalbumin with an equal volume of reagent-A (100 mM Hepes-pH 7.0, 0.2 mM CuCl2, and 6 µM FTC) at 37 °C for an hour. After supplementation with 2 mM EDTA, the protein fraction was concentrated, and the buffer was changed to HBS buffer by ultrafiltration using a 10 kDa cutoff filter device. The resulting product (0.72 mg/mL) was stored at 4 °C till use within 24 h.

RESULTS Copper-Induced Specific Binding of Tus to HSA. The analysis of albumin-bound fluorescent dyes was performed by CFA. As shown in Figure 2, no dyes bound to HSA unless copper was added. By comparing the structural analogues (ANTU versus DC, and FTC versus FCC), HSA-bound fluorescent dyes, either ANTU or FTC, have similar Tu functional groups (Figure 1). Thus, the Tu group is speculated to interact with albumin upon copper induction. FTC was selected for subsequent HSA binding analyses. The coupling reaction showed a positive dose-dependence on HSA, FTC, and copper contents (Figure 3). Disulfide Bridge Formation between Tus and Cysteine. To identify the type of linkage between HSA and FTC, the purified HSA-FTC complexes were treated with/without NaCl, urea, DTT, or SDS (Figure 4a). While urea- or SDS-treatment damaged up to 40% of complexes, DTT-treatment almost eliminated the fluorescence signal of protein fractions (>90%). This indicates that the linkage between HSA and FTC is a disulfide bond. In addition, a high concentration of salt (1 M NaCl) did not significantly influence the stability of HSA-FTC complexes. To confirm that thioureation occurred at the Cys34 residue of albumin, Cys34-modified albumins, Cys-HSA, r-Cys-HSA, or Alk-HSA, were employed in the reaction (Figure 5). The binding capacity of FTC with Cys-HSA, r-Cys-HSA, or Alk-HSA corresponded to their free thiol content, as compared to that with HSA. In addition, the interaction of cysteine with FTC

Figure 2. Analysis of copper-induced binding using the CFA method. Aliquots of 60 µL of reaction mixture containing (a) 7.52 µM HSA, 30 µM ANTU (or dansyl-cad), 50 mM Hepes-pH 7.0, with/without 0.1 mM CuCl2, or (b) 1 µM HSA, 3 µM FTC (or FCC), 50 mM HepespH 7.0, with/without 0.1 mM CuCl2, were incubated at 37 °C for 30 or 10 min, respectively. After the addition of equal volume reagent-B (2 mM EDTA, 0.5 M Hepes-pH 8.0), unbound fluorescent dyes were removed by MD-Charcoal (0.2 mL/well). MD-Charcoal was precipitated, and the fluorescence intensities were measured at Ex340/Em460 for ANTU, Ex340/Em353 for dansyl-cad, and Ex485/Em535 for FTC and FCC. Gain value was set at 35. Data presented are the means ( SD (n ) 3).

was also further confirmed by using the Cys-bead and AlkCys-bead in copper-induced reaction (Figure 4b). Similar to albumin, the Cys-bead interacts with FTC through the disulfide bond, as compared to that with Alk-Cys-bead (as control). Furthermore, the Tu-bead was also used to confirm the participation of the Tu group of FTC in disulfide bridge formation, and the Cys-bead was used as the control (Figure 4c). In the presence of copper, HSA was also found to form a disulfide bond with the Tu-bead. The same phenomenon was also observed for Cys-beads. In the absence of copper, however, only the Cys-bead that was pretreated with copper could bind HSA. The mechanism of copper-induced cysteinylation appears different from that of copper-dependent thioureation of HSA. The Influence of Ligand-Binding for Albumin Thioureation. Various reagents were evaluated for their effects on the formation of the HSA-FTC complex by the CFA method (Table 1). It was found that free thiol-containing molecules, such as NAC and cysteine, greatly reduced the binding of HSA with FTC, while cystine and GSSG had no significant effect. Because cysteine was also observed to interact with FTC under copper treatment (Figure 4b), the inhibitory effects of free thiolcontaining molecules are speculated to compete with HSA for interacting with the Tu-Cu(II) complex. As expected, Tu and ANTU competed with FTC for HSA-binding as compared to the Tu analogue, urea. The competition of Tu was not as effective as ANTU. In addition, dansylglutamine and dansylglycine, the site I and II binding probes of albumin (25), respectively, did not affect the interaction of FTC with HSA. The Effect of pH on Thioureation. Copper-induced HSAFTC complex formation was greatly influenced by the pH of the reaction solution (Figure 6a). The optimal pH for the reaction was pH 6.5. Above pH 6.5, the reaction was gradually reduced with increasing pH and is completely abolished at pH above 8.0. The relative initial reaction rate constants of reactions at various pH values were also calculated (Figure 6b), and the kinetic data did support the observations shown in Figure 6a. In the absence of albumin, the interaction between FTC and Cu(II) was also influenced by pH (Figure 6c). Upon Cu(II) supplementation, the fluorescence intensity of FTC is severely

Mechanism of Albumin Thioureation

Bioconjugate Chem., Vol. 19, No. 9, 2008 1825

Figure 3. Dose dependence of HSA, FTC, and CuCl2 for albumin thioureation. Reaction mixtures containing (a) HSA (0-1.5 µM), 3 µM FTC, 0.1 mM CuCl2, and 50 mM Hepes-pH 7.0; (b) FTC (0-5.0 µM), 1 µM HSA, 0.1 mM CuCl2, and 50 mM Hepes-pH 7.0; or (c) CuCl2 (0-48 µM), 1 µM HSA, 3 µM FTC, and 50 mM Hepes-pH 7.0 were incubated at 37 °C for 10 min and analyzed by the CFA method as described in Experimental Procedures. Data presented are the means ( SD (n ) 3).

Figure 4. Disulfide bridge formation between (a) HSA and FTC; (b) cysteine and FTC; and (c) HSA and Tu. In (a), 28 µg of HSA-FTC complex in HBS buffer was treated with 1 M NaCl, 3 M urea, 5 mM DTT, or 0.1% SDS, separately, at 37 °C for 15 min. The mixtures were then ultrafiltrated using Microcon YM-30 tubes. Protein fractions were collected in microplates and adjusted to pH 8.0 for fluorescence counting. In (b), Cys-beads or Alk-Cys-beads (drained volume ) 30 µL/reaction) after reaction with 3 µM FTC (in 50 mM Hepes-pH 7.0) in the presence or absence of CuCl2 (0.1 mM) at 37 °C for 30 min were washed with 0.5 M NaCl (in 10 mM Hepes-pH 7.0) and then treated with 5 mM DTT at 25 °C for 15 min. The supernatants supplemented with 50 mM Hepes-pH 8.0 were measured for fluorescent intensities to assess the bound FTC molecules. In (c), reaction mixtures containing HSA (0.5 mg) and Tu- or Cys-beads (0.1 mL drain matrix) with/without 2 mM CuCl2 were incubated at 25 °C for 30 min. Copper-pretreated Tu- or Cys-beads were also tested for HSA binding in the absence of copper. The copper-containing reaction was terminated by adding 2 mM EDTA. After washing with 0.5 M NaCl (in 10 mM Hepes-pH 7.0), the bead-bound proteins were eluted with DTT followed by slot blotting the eluted proteins onto nitrocellulose membranes. The existence of protein on the membrane was visualized by a MemCode reversible protein stain kit (Pierce Biotechnology, Rockford, IL, USA). Table 1. Influence of Thiol-Containing Agents, Tu Analogues, Ligands, and Cu(I) on HSA-FTC Complex Formationa reagents

relative fluorescence intensity (%)

control thiol-containing molecules: DTT, 10 µM NAC, 10 µM Cys-SH 10 µM cystine, 10 µM GSSG, 10 µM Tu and Tu analogues: Tu, 10 µM Tu, 100 µM ANTU, 10 µM Urea, 100 µM ligands: dansylglutamine, 10 µM dansylglycine, 10 µM Cu(I): CuCl, 10 µM CuCl, 100 µM

100.0 ( 3.4 7.4 ( 0.9 5.6 ( 0.3 5.6 ( 0.5 98.2 ( 2.6 96.2 ( 1.9 79.1 ( 0.9 33.5 ( 2.0 31.3 ( 0.9 99.0 ( 1.4 98.7 ( 1.5 99.8 ( 3.2

Figure 5. Evaluation of FTC binding capacity by various albumins. Various albumins (1 µM) were incubated with 3 µM FTC, 0.1 mM CuCl2, and 50 mM Hepes-pH 7.0 at 37 °C for 10 min. The copperinduced coupling reactions were assessed by the CFA method as described in Experimental Procedures. The fluorescence intensity of each was calculated relative to that of HSA. Data presented are the means ( SD (n ) 3). The calculated free thiol content for mercaptoalbumin (HSA, rHSA, BSA, or PSA), Cys-HSA, r-Cys-HSA (thiolreduced Cys-HSA), and Alk-HSA were 1.00, 0.08, 0.50, and 0.13 (M/ M), respectively.

a Various reagents were included in the reaction of 1 µM HSA, 3 µM FTC, 0.1 mM CuCl2, and 50 mM Hepes-pH 7.0. The complex formation of HSA-FTC was analyzed by the CFA method, and shown as relative fluorescence intensities comparing to control (no reagents added). Data represents the means ( SD (n ) 3).

quenched (∆Q). However, the fluorescence of the FTC-Cu(II) mixture is greatly enhanced (∆H1) with increasing pH above 7.5, as compared to that observed in the absence of copper (∆H2). The increased fluorescence intensity (∆H1) at high pH was speculated to be the result of the dissociation of FTC from Cu(II) ions.

Participation of Metal Ions in Thioureation. Various metal ions (0.1 mM in reaction), including CuCl, CuCl2, CoCl2, NiCl2, CaCl2, MgCl2, ZnCl2, MnCl2, FeCl2, and FeCl3, were tested for Alb-FTC complex formation by using the CFA method. As shown in Figure 7, only Cu(II) induced the thioureation reaction among various metal ions; the small amplitude of the

83.0 ( 1.5 34.9 ( 0.6

1826 Bioconjugate Chem., Vol. 19, No. 9, 2008

Wu and Tsai

Figure 6. pH effect on Alb-FTC complex formation. (a) Reaction mixtures as described in Figure 5 were incubated at various pH values at 37 °C for 10 min to evaluate the influence of pH on the formation of the Alb-FTC complex. (b) The kinetics of the reaction at various pH values. Reactions at various pH values (pH 6.0-8.0) were performed at 37 °C for designated time intervals (0-60 s). The fluorescence intensity of the reaction at pH 7.0 for 1 min was set as 1.0, and the relative initial rate was calculated. The slopes of the trend lines of the reaction data points at various pH values correspond to the relative rate constants for the reactions. (c) The effect of pH on the interaction between Cu(II) and FTC. The fluorescence intensities of 3 µM FTC with/without 0.1 mM CuCl2, in 50 mM Hepes at various pH values, were measured to assess the interaction between FTC and Cu(II). Data presented are the means ( SD (n ) 3). ∆Q: quenched fluorescence intensity of FTC upon the addition of Cu(II). ∆H1: increased fluorescence intensity of the FTC-CuCl2 mixture with increasing pH. ∆H2: increased fluorescence intensity of FTC with increasing pH.

Figure 7. Necessity of metal ions for thioureation. Various metal ions (0.1 mM) incubated with 1 µM HSA, 3 µM FTC, and 50 mM HepespH 7.0 at 37 °C for 10 min were analyzed by the CFA method as described in Experimental Procedures. Data presented are the means ( SD (n ) 3).

Cu(I)-induced reaction may result from the oxidation of Cu(I) to Cu(II) during material preparation. The necessity of Cu(II) for thioureation was demonstrated by the dose-dependent reaction (Figure 3c), and the reduction of Cu(II) to Cu(I) during thioureation processes was speculated. NCP, a specific Cu(I) chelator, was used to confirm the formation of Cu(I). In the presence of NCP, the formation of HSA-FTC is greatly enhanced and reaches its plateau within 10 min as compared to that observed in the absence of NCP (Figure 8a). Similarly, the cross-linking of FTC with cysteine (which was evaluated by Cys-beads) was also elevated by NCP treatment (Figure 8b). In addition, the supplement of Cu(I) did decrease the Cu(II)induced formation of HSA-FTC complexes (Table 1). These results implied that Cu(I) was a byproduct associated with the Cu(II)-induced thioureation of Cys34 of albumin and that the released Cu(I) would back inhibit thioureation. Stability of Thioureated Products. The purified HSA-FTC complex was used for stability evaluation (Figure 9). This product appears stable at 25 °C, pH 7.0, within an hour but is unstable with increasing temperature (Figure 9a). As to the pH effect, the HSA-FTC complex was stable at pH 6-8 at 25 °C but degraded significantly in acidic (pH < 6.0) or basic (pH > 8.0) conditions (Figure 9b). In acidic conditions, Cu(I) was capable of reducing the Alb-FTC complex (Figure 9c). The presence of thiol-containing agents, including DTT, NAC,

Figure 8. Effect of NCP on thioureation of HSA. (a) Reaction mixtures as described in Figure 5 were incubated at 37 °C in the presence or absence of NCP for indicated time-intervals. The relative fluorescent intensities were calculated relative to that of the 10-min reaction without NCP treatment. (b) Drained Cys-beads (30 µL) supplemented with 30 µL of HBS buffer and 30 µL of reagent-A (100 mM Hepes-pH 7.0, 0.2 mM CuCl2, and 6 µM FTC), with/without 0.1 mM NCP, were incubated at 37 °C for 15 min. After the addition of 2 mM EDTA, the beads were washed with HBS buffer. Bead-bound FTC was finally eluted with 5 mM DTT (dissolved in 0.1 M Hepes-pH 8.0) and subjected to fluorescence measurement. Data presented are the means ( SD (n ) 3).

cysteine, and Tu, ANTU caused the release of FTC from the HSA-FTC complex (Figure 9d). However, cystine and GSSG did not reduce the linkage of Alb-FTC.

DISCUSSION This study demonstrated the essential role of copper in the cross-linking of albumin with Tus, such as ANTU or FTC (Figure 2). To facilitate the assessment of the albumin-Tus interaction, FTC was chosen for subsequent analyses in this study. It was found that the level of HSA-FTC complex formation depends on the amount of HSA, FTC, and copper included in the reaction (Figure 3). As few as 6 µM Cu(II) could induce a detectable HSA-FTC complex reaction in vitro (Figure 3c). The copper concentration in blood plasma was about 15 µM (38); thus, the thioureation of albumin may actually occur in physiological conditions. Our data show that the linkage between HSA and FTC is a disulfide bridge (Figure 4a). Because there is only one reactive thiol group in the Cys34 residue of albumin (26), FTC was

Mechanism of Albumin Thioureation

Bioconjugate Chem., Vol. 19, No. 9, 2008 1827

Figure 9. Stability of the thioureated complex. The influence of (a) temperature, (b) pH, (c) Cu(I), or (d) thiol-containing molecules on the stability of Alb-FTC was evaluated by using a modified CFA method (see Supporting Information, Figure 1). Aliquots of purified HSA-FTC (3 µg/well) were (a) incubated at the indicated temperature in 50 mM Hepes-pH 7.0 for designated time intervals; (b) incubated in various pH (in 50 mM sodium acetate-pH 4.0-5.0; 50 mM Hepes-pH 6.0-9.0) at 25 °C for an hour; (c) mixed with 0.1 mM Cu(I) in 50 mM Hepes-pH 5.5-7.0, at 25 °C for an hour; (d) mixed with 0.1 mM thiol-containing agents in 50 mM Hepes-pH 7.0 at 25 °C for 3 h. Reagent-B (60 µL/well) and MD-Charcoal (200 µL/well) were then added to adjust pH and remove the released FTC, respectively. After magnetic precipitation with MD-Charcoal, the fluorescent intensities were measured. Data presented are the means ( SD (n ) 3).

speculated to cross-link with cysteine at this site. This notion was further confirmed by using Cys34-modified albumin (CysHSA, r-Cys-HSA, and Alk-HSA) and the fact that FTC did not interact with cysteinylated or alkylated albumin (Figure 5). Furthermore, copper-induced cross-linking was also demonstrated for the interaction of FTC with cysteine (Figure 4b) and that of Tu with HSA (Figure 4c). The difference between albumin thioureation and cysteinylation could be distinguished by comparing the interaction of HSA with Tu-beads and HSA with Cys-beads. As shown in Figure 4c, both Tu- and Cysbeads were found to conjugate with HSA in the presence of copper. In the absence of copper, however, only Cys-beads that were pretreated with copper could react with HSA. As reported by other investigators (29, 32, 39), the binding of HSA with Cys-beads in this study is probably caused by the coppercatalyzed autoxidation of cysteine to cystine, followed by the interaction of cystine with HSA through thiol-disulfide exchange. However, copper-pretreated Tu-beads did not induce the same reaction as did Cys-beads. We speculate that the pretreatment with copper did not form dithioformamidine (Tu-S-S-Tu) in Tu-beads; instead, the Tu-Cu(II) complex was formed as described by Doona and Stanbury (15). The thioureation reaction is highly sensitive to pH changes (Figure 6a and b). The decreased thioureation reaction at low pH may be due to the instability of the resulting product (Alb-FTC) in acidic conditions (Figure 9b). However, in basic conditions, the thioureation reaction is greatly reduced although the thioureated product is stable (at pH 8.0, Figure 9b). Such decreased thioureation reaction at high pH is likely due to the insolubility of copper in basic buffers, thus leading to the decomposition of the FTC-Cu(II) complex (Figure 6c). This

observation implicates the importance of Tus-Cu(II) complex formation in the process of albumin thioureation. Other metal ions tested in this study, including ferric ion, did not induce the same reaction as copper did, indicating the specific nature of copper in the reaction (Figure 7). It is difficult to clarify the role of copper in Alb-S-S-Tus complex formation since both albumin and Tus were known to interact with copper (15, 16, 26). The interaction site of copper with HSA was at its N-terminal copper-binding site (N-Asp-Ala-His-Lys-) and the Cys34 residue (26, 40). However, the truncated rHSA, which has a single deletion at Asp1 and exhibits a reduced copper-binding affinity, did not reduce the binding of FTC, as compared to intact HSA (Figure 5). It appears that the binding of copper at the N-terminal copper-binding site appears unnecessary for the thioureation of HSA. Similarly, PSA, which exhibits low-affinity for copper (there is a single residue change in PSA, His3f Tyr3, as compared to BSA at the N-terminal Asp-Thr-His-Lys- sequence), interacted more efficiently with FTC than did BSA. We thus exclude the N-terminal copperbinding site of albumin from the reaction. The possible interaction of Cu(II) with Cys34 of albumin was considered. In copper-induced S-nitrosation of albumin (32), it was suggested that the interaction of Cu(II) with Cys34 of albumin leads to the formation of a copper-thiol complex, which could further react with NO to form BSA-SNO. According to Zhang et al., Cu(II) bound with high affinity to Cys34 of albumin at higher pH (approximately 9) (41). If the formation of HSA-Cys34-Cu(II) is essential for thioureation, more HSA-FTC would be formed at high pH. However, our data show that the incorporation of FTC to HSA was greatly reduced with increasing pH, and the conjugation was abolished com-

1828 Bioconjugate Chem., Vol. 19, No. 9, 2008

pletely above pH 8.5 (Figure 6a). Although the interaction of Cu(II) with Cys34 may occur, it was not essential for the incorporation of FTC into albumin. Consequently, a mechanism similar to S-nitrosation is not suitable for albumin thioureation. Because Tus were known to form complexes with copper (15-17), we suggest that the formation of the Tus-Cu(II) complex is essential for the reaction. In copper-induced reactions, Tus including FTC, ANTU, and Tu-beads, which were in their copper-bound form would further interact with albumin (Figures 2 and 4c). There are two possible reaction mechanisms for the thioureation of cysteine or Cys34 of albumin: (a) CysSH or Alb-Cys34-SH attacked by Tus-Cu(II) as shown in reactions F and G, respectively (the release of Cu(I) will be discussed later); (b) Cys-S-S-Cys or dimerized albumin (AlbCys34-S-S-Cys34-Alb) reacting with Tus by thiol-disulfide exchange as described in reaction D (see Introduction). Cys-SH + Tus-Cu(II) f Cys-S-S-Tus + Cu(I) + 2H+ (F) Alb-Cys34-SH + Tus-Cu(II) f Alb-Cys34-S-S-Tus + Cu(I) + 2H+ (G) The scheme b described above was probably excluded from the copper-induced fast coupling reaction because (i) copper may not participate in the formation of dimerized albumin. In fact, the treatment of albumin with Cu(II) did not lead to the oxidation of Cys34 and the dimerization of BSA (32). (ii) CysHSA (Alb-Cys34-S-S-Cys) could not complex with FTC in a 10-min reaction without copper (Figure 5). Therefore, it is not the major mechanism in the copper-induced reaction, although thiol-disulfide exchange described in reaction D may occur. According to Stubauer et al., Cu(II)-induced albumin Snitrosation accompanied the release of Cu(I) as judged by using NCP, a Cu(I) specific chelator (32). Similarly, by using NCP, the formation of HSA-S-S-FTC and Cys-S-S-FTC is predominantly increased (Figure 8). Thus, we speculate that the release of Cu(I) is involved in reactions F-G. There are two possible mechanisms for the Cu(I)-inhibited formation of Alb-Tus at neutral pH: (i) the Cu(I) decomposes the disulfide bridge of Alb-S-S-Tus; and (ii) the competition of Cu(I) with Cu(II) for Tus. The former is suggested by the report of Kato et al. that oxidized D-penicillamine (PSSP) and glutathione (GSSG) were reduced by Cu(I) with the release of Cu(II) (42). Stubauer et al. also described that the decomposition of BSA-SNO by Cu(I) reduced the S-nitrosation reaction (32). At neutral pH in this study, Cu(I) did decrease the formation of Alb-FTC (Table 1). However, once Alb-FTC formed, Cu(I) did not significantly decompose the complex at pH 7.0 (Figure 9c), indicating that the first scheme is not likely to occur at neutral pH. Alternatively, the second scheme that Cu(I) competes with Cu(II) for Tu is, thus, a more feasible model at neutral pH because Tu was also known to form a complex with Cu(I). It was also reported that the formation of a redox-inactive Tu-Cu(I) complex prevents copper-mediated protein oxidation (18). Therefore, we speculate that the Cu(II) bound Tus is active for the progression of thioureation while Cu(I)-bound Tus is inactive. The stability of thioureated albumin, Alb-FTC, was also assessed. As shown in Figure 9a and b, Alb-FTC was unstable at high temperature (37 °C and 45 °C), under acidic (pH < 6.0) or basic conditions (pH > 8.0). Although the breakdown of the disulfide bond is considered, other possibilities may not be excluded: (i) Cys-S-S-Tu was hydrolyzed to Cys-SOH and Tu in reaction E (10, 11); (ii) thioureated protein (protein-SS-Tu) in thyroid tissue was hydrolyzed to thiosulfenic acid (protein-S-SH) and urea (8). The latter implied that the decomposition of cross-linked product may be influenced by surrounding amino acids near the binding site. The hydrolytic products may vary among various Tu derivatives. Further experiments are required to establish the hydrolytic mechanism.

Wu and Tsai

Figure 10. Schematic mechanism of copper-induced thioureation. The complex of Tus-Cu(II) is actively catalyzing the thioureation between Tus and cysteine or Cys34 residue of mercaptoalbumin (Alb-Cys34SH) at weak acidic or physiological pH. The resulting product is thioureated cysteine (Cys-S-S-Tus) or albumin (Alb-Cys34-S-S-Tus) with the formation of a disulfide bridge and the release of Cu(I). The released Cu(I) is speculated to compete with Cu(II) for Tus, thus inhibiting the progress of thioureation. The complex, Alb-Cys34-S-STus, was unstable at high temperature (>25 °C) and either acidic (pH 8.0). Both Cu(I) (at acidic pH only) and thiolreducing agents degraded the Alb-Cys34-S-S-Tus complex.

In addition, Cu(I) was found to reduce the linkage between albumin and FTC in acidic conditions (Figure 9c). The diminished reduction effect of Cu(I) with increasing pH may be due to the decreased solubility of Cu(I) in basic conditions (considering that CuCl must be prepared in 50 mM HCl). Finally, because of the formation of disulfide linkage between thiourea and albumin, the produced thioureated albumin could also be reduced or modified by thiol-containing compounds presented in blood plasma, such as cysteine (but not cystine) as demonstrated in Figure 9d. The mechanism of copper-induced thioureation is summarized and illustrated in Figure 10. Tus were considered as hydroxyl radical scavengers and may help to suppress metal-induced protein damage (12-14). However, high levels of Tus are intrinsically toxic physiologically (1-3). It is noteworthy that the combination of copper with Tus (e.g., phenylthiourea, ANTU, and pyrrolidine dithiocarbamate) was much more toxic than copper or Tus alone for cultured cells (43, 44). The treatment of these cells with copperbinding protein such as albumin would decrease the cytotoxic effect (44). In our opinion, the decreased cytotoxicity in these cases may be due to the dysfunction of some important cellular proteins or enzymes that were thioureated by Tus-Cu(II) and finally resulted in cell death. Although the diminished cytotoxicity by albumin-treatment may be due to the competition of albumin with Tus for copper, the possible contribution of sacrificial albumin for Tu-binding (that forms Alb-Tus to prevent thioureation of important cellular proteins) should not be ignored. We therefore speculate that albumin may play a role in the clearance of Tus in the presence of copper. This may be important for reducing the toxicity of these compounds in circulation. In today’s pharmaceutical industry, many drugs are developed in the form of Tus (19-21). Since albumin is the major thiol-containing protein in plasma, the pharmacokinetics of these Tu-based drugs may require special evaluation for patients with copper metabolism disorders such as Wilson’s disease (45).

ACKNOWLEDGMENT The study was supported by NSC 94-2314-B-038-034 (ROC) and NSC 96-2314-B-038-015 (ROC).

Mechanism of Albumin Thioureation

Supporting Information Available: Schemes of the CFA method and the influence of metal ions and small molecules on fluorescence intensity by using the CFA method. This material is available free of charge via the Internet at http://pubs.acs.org.

LITERATURE CITED (1) IPCS (2003) Thiourea, in CICADs 49 (2003 IPCS, Ed.) WHO, Geneva, Switzerland. (2) Scott, A. M., Powell, G. M., Upshall, D. G., and Curtis, C. G. (1990) Pulmonary toxicity of thioureas in the rat. EnViron. Health Perspect. 85, 43–50. (3) Onderwater, R. C., Commandeur, J. N., and Vermeulen, N. P. (2004) Comparative cytotoxicity of N-substituted N′-(4-imidazole-ethyl)thiourea in precision-cut rat liver slices. Toxicology 197, 81–91. (4) Schulman, J., Jr., and and Keating, R. P. (1950) Studies on the metabolism of thiourea. I. Distribution and excretion in the rat of thiourea labeled with radioactive sulfur. J. Biol. Chem. 183, 215–221. (5) Hollinger, M. A., Giri, S. N., Alley, M., Budd, E. R., and Hwang, F. (1974) Tissue distribution and binding of radioactivity from 14C-thiourea in the rat. Drug Metab. Dispos. 2, 521–525. (6) Davidson, B., Soodak, M., Strout, H. V., Neary, J. T., Nakamura, C., and Maloof, F. (1979) Thiourea and cyanamide as inhibitors of thyroid peroxidase: the role of iodide. Endocrinology 104, 919–924. (7) Ziegler, D. M. (1978) Intermediate metabolites of thiocarbamides, thioureylenes and thioamides: mechanism of formation and reactivity. Biochem. Soc. Trans. 6, 94–96. (8) Maloof, F., and Soodak, M. (1961) Cleavage of disulfide bonds in thyroid tissue by thiourea. J. Biol. Chem. 236, 1689–1692. (9) Hollinger, M. A., and Giri, S. N. (1990) Non-enzymatic covalent binding of radioactivity from [14C]thiourea to rat lung protein. Toxicol. Lett. 52, 1–5. (10) Pirie, N. W. (1933) The oxidation of sulphydryl compounds by hydrogen peroxide: Catalysis of oxidation of cysteine by thiocarbamides and thiolglyoxalines. Biochem. J. 27, 1181–1188. (11) Toennies, G. (1937) Relations of thiourea, cysteine, and the corresponding disulfides. J. Biol. Chem. 120, 297–313. (12) Ziegelstein, R. C., Zweier, J. L., Mellits, E. D., Younes, A., Lakatta, E. G., Stern, M. D., and Silverman, H. S. (1992) Dimethylthiourea, an oxygen radical scavenger, protects isolated cardiac myocytes from hypoxic injury by inhibition of Na(+)Ca2+ exchange and not by its antioxidant effects. Circ. Res. 70, 804–811. (13) Kelner, M. J., Bagnell, R., and Welch, K. J. (1990) Thioureas react with superoxide radicals to yield a sulfhydryl compound. Explanation for protective effect against paraquat. J. Biol. Chem. 265, 1306–1311. (14) Wasil, M., Halliwell, B., Grootveld, M., Moorhouse, C. P., Hutchison, D. C., and Baum, H. (1987) The specificity of thiourea, dimethylthiourea and dimethyl sulphoxide as scavengers of hydroxyl radicals. Their protection of alpha 1-antiproteinase against inactivation by hypochlorous acid. Biochem. J. 243, 867– 870. (15) Doona, C. J., and Stanbury, D. M. (1996) Equilibrium and redox kinetics of copper(II)-thiourea complexes. Inorg. Chem. 35, 3210–3216. (16) Foye, W. O., and Chao, C. C. (1984) Antiradiation compounds XIX: metal-binding abilities of thioureas. J. Pharm. Sci. 73, 1284–1286. (17) Piro, O. E., Piatti, R. C., Bolzan, A. E., Salvarezza, R. C., and Arvia, A. J. (2000) X-ray diffraction study of copper(I) thiourea complexes formed in sulfate-containing acid solutions. Acta Crystallogr., Sect. B 56, 993–997. (18) Zhu, B. Z., Antholine, W. E., and Frei, B. (2002) Thiourea protects against copper-induced oxidative damage by formation of a redox-inactive thiourea-copper complex. Free Radical Biol. Med. 32, 1333–1338.

Bioconjugate Chem., Vol. 19, No. 9, 2008 1829 (19) Tsogoeva, S. B., Hateley, M. J., Yalalov, D. A., Meindl, K., Weckbecker, C., and Huthmacher, K. (2005) Thiourea-based nonnucleoside inhibitors of HIV reverse transcriptase as bifunctional organocatalysts in the asymmetric Strecker synthesis. Bioorg. Med. Chem. 13, 5680–5685. (20) Coppola, G. M., Damon, R. E., Eskesen, J. B., France, D. S., and Paterniti, J. R., Jr. (2002) Thiourea-based gemfibrozil analogues as HDL-elevating agents. Bioorg. Med. Chem. Lett. 12, 2439–2442. (21) Bhandari, K., Srivastava, S., and Shankar, G. (2004) Synthesis of tetrahydronaphthyl thioureas as potent appetite suppressants. Bioorg. Med. Chem. 12, 4189–4196. (22) Cui, F., Cui, Y., Luo, H., Yao, X., Fan, J., and Lu, Y. (2006) The interaction between N-n-undecyl-N′-(sodium p-aminobenzenesulfonate) thiourea and serum albumin studied using various spectroscopies and the molecular modeling method. Biopolymers 83, 170–181. (23) Cui, F., Wang, J., Cui, Y., Li, J., Lu, Y., Fan, J., and Yao, X. (2007) Binding of human serum albumin to N-(p-ethoxy-phenyl)N′-(1-naphthyl)thiourea and synchronous fluorescence determination of human serum albumin. Anal. Sci. 23, 719–725. (24) Peters Jr., T. (1996) Ligand Binding by Albumin, in All About Albumin: Biochemistry, Genetics, and Medical Applications pp 76-132, Academic Press, San Diego, CA. (25) Kragh-Hansen, U., Chuang, V. T., and Otagiri, M. (2002) Practical aspects of the ligand-binding and enzymatic properties of human serum albumin. Biol. Pharm. Bull. 25, 695–704. (26) Quinlan, G. J., Martin, G. S., and Evans, T. W. (2005) Albumin: biochemical properties and therapeutic potential. Hepatology 41, 1211–1219. (27) Iberg, N., and Fluckiger, R. (1986) Nonenzymatic glycosylation of albumin in vivo. Identification of multiple glycosylated sites. J. Biol. Chem. 261, 13542–13545. (28) van Breemen, R. B., and Fenselau, C. (1985) Acylation of albumin by 1-O-acyl glucuronides. Drug Metab. Dispos. 13, 318– 320. (29) Sengupta, S., Wehbe, C., Majors, A. K., Ketterer, M. E., DiBello, P. M., and Jacobsen, D. W. (2001) Relative roles of albumin and ceruloplasmin in the formation of homocystine, homocysteine-cysteine-mixed disulfide, and cystine in circulation. J. Biol. Chem. 276, 46896–46904. (30) Sengupta, S., Chen, H., Togawa, T., DiBello, P. M., Majors, A. K., Budy, B., Ketterer, M. E., and Jacobsen, D. W. (2001) Albumin thiolate anion is an intermediate in the formation of albumin-S-S-homocysteine. J. Biol. Chem. 276, 30111–30117. (31) Zhang, H., and Means, G. E. (1996) S-nitrosation of serum albumin: spectrophotometric determination of its nitrosation by simple S-nitrosothiols. Anal. Biochem. 237, 141–144. (32) Stubauer, G., Giuffre, A., and Sarti, P. (1999) Mechanism of S-nitrosothiol formation and degradation mediated by copper ions. J. Biol. Chem. 274, 28128–28133. (33) Glowacki, R., and Jakubowski, H. (2004) Cross-talk between Cys34 and lysine residues in human serum albumin revealed by N-homocysteinylation. J. Biol. Chem. 279, 10864–10871. (34) Wu, Y. W., and Tsai, Y. H. (2006) A rapid transglutaminase assay for high-throughput screening applications. J. Biomol. Screen. 11, 836–843. (35) Wu, Y. W., Chen, S. F., Yang, C. B., and Tsai, Y. H. (2008) Screening, purification, and identification of a copper-dependent FITC-binding protein in human plasma: albumin. J. Chromatogr., B 863, 187–191. (36) Sogami, M., Nagoka, S., Era, S., Honda, M., and Noguchi, K. (1984) Resolution of human mercapt- and nonmercaptalbumin by high-performance liquid chromatography. Int. J. Pept. Protein Res. 24, 96–103. (37) Ellman, G. L. (1959) Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70–77. (38) Samman, S. (2002) Trace elements, in Essentials of Human Nutrition (Mann, J., and Truswell, A. S., Eds.) 2nd ed., pp 164166, Oxford University Press Inc., New York.

1830 Bioconjugate Chem., Vol. 19, No. 9, 2008 (39) Kachur, A. V., Koch, C. J., and Biaglow, J. E. (1999) Mechanism of copper-catalyzed autoxidation of cysteine. Free Radical Res. 31, 23–34. (40) Suzuki, K. T., Karasawa, A., and Yamanaka, K. (1989) Binding of copper to albumin and participation of cysteine in vivo and in vitro. Arch. Biochem. Biophys. 273, 572– 577. (41) Zhang, Y., and Wilcox, D. E. (2002) Thermodynamic and spectroscopic study of Cu(II) and Ni(II) binding to bovine serum albumin. J. Biol. Inorg. Chem. 7, 327–337. (42) Kato, N., Nakamura, M., and Uchiyama, T. (1999) 1H NMR studies of the reactions of copper(I) and copper(II) with

Wu and Tsai D-penicillamine and glutathione. J. Inorg. Biochem. 75, 117– 121. (43) Masuda, A., and Eguchi, G. (1984) Phenylthiourea enhances Cu cytotoxicity in cell cultures: its mode of action. Cell Struct. Funct. 9, 25–35. (44) Chen, S. H., Liu, S. H., Liang, Y. C., Lin, J. K., and LinShiau, S. Y. (2000) Death signaling pathway induced by pyrrolidine dithiocarbamate-Cu(2+) complex in the cultured rat cortical astrocytes. Glia 31, 249–261. (45) Ala, A., Walker, A. P., Ashkan, K., Dooley, J. S., and Schilsky, M. L. (2007) Wilson’s disease. Lancet 369, 397–408. BC7004158