| Summary: | 博士 === 臺北醫學大學 === 藥學系(博士班) === 96 === In this study, a protein purified by fluorescein isothiocyanate (FITC)-affinity chromatography from human plasma was identified as albumin by MALDI-TOF-MS. Albumin (Alb) is the major carrier protein in the circulation and exhibits multiple functions. Albumin was found to conjugate with FITC-labeled molecules through a copper-dependent reaction. The formation of this complex was confirmed by methods including a newly developed “charcoal-based fluorescence assay” (CFA), gel-filtration, affinity chromatography, and ultrafiltration. The binding was identified as disulfide bridge formation. The thiol group found in FITC molecule is a thiourea (Tu) group ((R1R2N)(R3R4N)C=S). Thioureas (Tus), which are widely used in chemical and pharmaceutical industries, however, have not been reported to covalently conjugate with albumin. In this study, we showed that copper induces the cross-linking of albumin with Tus such as α-naphthylthiourea (ANTU) and fluorescein-5-isothiocyanate cadaverine (FTC) through disulfide bridge formation. 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 inhibited by thiol-containing molecules. Albumin without copper-binding site exhibited the same reaction, indicating that the binding of copper with albumin is not necessary for the thioureation reaction. Albumin that was cysteinylated or alkylated in the Cys34 residue can not be thioureated. The optimal reaction occurred at pH 6.5, and the complex formation was reduced with the increasing pH. The resulting conjugated product was heat-labile, and stable at pH between 6.0 ~ 8.0. The linkage could be reduced by Cu(I) (in acidic pH) and thiol-containing agents such as cysteine (but not cystine). The mechanism of albumin thioureation was concluded: (i) the formation of Tus-Cu(II) complex is essential for the reaction; (ii) the thioureation is resulted from the attack of Tus-Cu(II) at Alb-Cys34-SH to form Alb-Cys34-S-S-Tus complex accompanied with the release of Cu(I); (iii) the released Cu(I) would back inhibit the reaction due to its competition with Cu(II) for Tus binding. This is the first to demonstrate that copper induces a covalent binding of Tus with albumin. These phenomenons may have important implications for the pharmacokinetics of Tu-based drugs in plasma. Since albumin is the major thiol-containing protein in plasma, Tu-based drugs may require special evaluation for patients with copper metabolism disorders such as Wilson’s disease.
|
|---|
