新穎的人類血清白蛋白硫尿化之研究
Study on a Novel Thioureation of Human Serum Albumin
中文摘要
在本研究中，我們以FITC 親和性層析法從人類血漿中純化出單一蛋白質。經
「基質輔助雷射脫附游離飛行時間質譜儀」(MALDI-TOF-MS) 的鑑定，確定此蛋
白質為人類血清白蛋白 (HSA)。白蛋白是血液循環系統中最主要的攜帶性蛋白，
具有多重功能。白蛋白與 FITC 標記的分子之結合是一種依銅型的反應。在此研
究中，我們發展出一套「活性炭螢光分析法」(CFA) 來分析白蛋白與 FITC 標記分
子的結合，並佐以膠質過濾法、親和性層析法，以及超濾技術來確認此反應複合
物的存在。白蛋白與FITC 之間的結合是經由雙硫鍵的形成，而 FITC 分子上的
硫是屬於硫尿基 ((R1R2N)(R3R4N)C=S)。硫尿及其衍生物廣泛的被運用在化學 及製藥工業上，然而，過去並沒有報告指出這些物質會與白蛋白形成共價鍵結。
在本研究中，我們證實了銅離子會誘發硫尿物質（例如：α-naphthylthiourea (ANTU)、fluorescein-5-isothiocyanate cadaverine (FTC)）與白蛋白形成雙硫鍵鍵結 這種反應是絕對依銅型的反應，其他金屬離子，例如鈷、鎳、鈣、鎂、鋅、鐵、錳，
都不會誘發此反應。該反應與反應物的劑量成線性關係，且受到含硫氫基（- SH）物質的抑制。缺乏銅離子結合區的白蛋白也可以進行硫尿化的反應，據此
推測白蛋白與銅離子的結合對反應並不重要。白蛋白的Cys34 氨基酸經過半胱氨
酸化（cysteinylation）或烷基化（alkylation）後，會失去反應能力。白蛋白硫尿 化的最佳反應 pH 值為 6.5，且隨著 pH 值增高而遞減。硫尿化的白蛋白具有幾項 特性：(a) 熱不穩定性，在溫度高於 25℃時，雙硫鍵會逐漸斷裂；(b) 在 pH 6.0
~ 8.0 之間穩定；(c)在酸性情況下其鍵結可被一價銅離子還原；(d) 其鍵結可被 帶有硫氫基（-SH）或硫尿基的分子還原。綜合實驗結果，我們推論白蛋白的硫 尿化機轉為：(1) 二價銅離子與硫尿物質形成「硫尿-銅(II)」複合物；(2) 「硫尿-銅 (II)」複合物攻擊白蛋白 Cys34 氨基酸的硫氫基，形成「白蛋白-Cys34-S-S-硫尿」
複合物，並釋放一價銅離子；(3) 反應中釋出的一價銅離子會與二價銅離子共同 競爭硫尿的結合，從而抑制了二價銅離子所誘發的硫尿化反應。本論文是第一篇 提出銅離子會誘發白蛋白硫尿化反應並推論其機轉的研究。這些研究結果對於以 硫尿結構所設計的藥物，在藥物動力學分析上將有重要意義。由於白蛋白為血漿 中最主要的蛋白質，因此對於銅離子代謝性疾病，例如威爾森氏症的病人，硫 尿藥物的使用應該受到進一步的評估。
英文摘要
So far, there are still many traditional Chinese medical theories and practices not supported with good enough scientific evidences. We are interested in the mechanism involved in purgation that traditional Chinese doctors used when their patients
suffered with internal heat, stagnation, and fullness syndromes. Three Chen-Chi-Tang
decoctions (CCTD), including Ta-Chen-Chi-Tang (TCCT), Xiao-Chen-Chi-Tang (XCCT), Tiao-Wei-Chen-Chi-Tang (TWCCT) are three famous purgative formulas In this study, we first conducted a systemic review for evaluating the clinical efficacy of these purgative decoctions on gastrointestinal problems and obesity and
hyperlipidemia. We also designed many in vitro and in vivo studies in order to delineate the efficacy and underlying mechanisms involved in purgation. From the systemic review, there is moderate evidence to support the efficacy of CCTD (especially TCCT) in the management of gastro-intestinal problems, but most of the studies do not support anti-obesity effect of purgative decoctions, and clinical
evidence of purgatives on hyperlipidemia is still controversial. Through in vitro and in vivo experiments, we have shown that TCCT is a potent anti-inflammatory agent, and XCCT is a potent anti-oxidantant. Inflammation and oxidative stress are two
pathophysiological processes involved in many diseases suffered by modern man today. Future clinical application of CCTDs may based on these findings in order to improve the efficacy of these traditional Chinese medicine in the present world. 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.
