[1]张雷,张晓,罗智颖.胆红素氧化酶及其变异体在胆红素血症评价中的应用[J].南方医科大学学报,2005,(11):1337-1340.
 ZHANG Lei,ZHANG Xiao,LUO Zhi-Ying.Value of bilirubin oxidase and its mutants in the diagnosis of hyperbilirubinemia[J].,2005,(11):1337-1340.
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胆红素氧化酶及其变异体在胆红素血症评价中的应用()
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《南方医科大学学报》[ISSN:/CN:]

卷:
期数:
2005年11期
页码:
1337-1340
栏目:
出版日期:
2005-11-01

文章信息/Info

Title:
Value of bilirubin oxidase and its mutants in the diagnosis of hyperbilirubinemia
作者:
张雷1 张晓2 罗智颖3
1. 华南理工大学化工系生物制药研究室, 广东, 广州, 510640;日本金沢大学化学系, 金沢, 920-1192;
2. 河南省南阳市第一人民医院小儿科, 河南, 南阳, 473000;
3. 华南理工大学附属医院, 广东, 广州, 510640
Author(s):
ZHANG Lei1 ZHANG Xiao2 LUO Zhi-Ying3
1. Biopharmaceutical Research Laboratory, Department of Chemical Engineering, South China University of Technology, Guangzhou 510640, China, and Department of Chemistry, Faculty of Science, Kanazawa University, Kanazawa 920-1192, Japan;
2. Department of Pediatrics, Nanyang First People’s Hospital, Nanyang 473000, China;
3. Affiliated Hospital of South China University of Technology, Guangzhou 510640, China
关键词:
胆红素胆红素氧化酶聚合酶链反应高胆红素血症
Keywords:
bilirubinbilirubin oxidasepolymerase chain reactionhyperbilirubinemia
分类号:
R55
摘要:
目的 探讨胆红素氧化酶中配位氨基酸对保持酶活性的重要性和酶促反应的动力学,并评价胆红素氧化酶突变体能否作为一种更好的高胆红素血症的诊断试剂。方法 胆红素氧化酶突变体I402G和C457S通过聚合酶链反应获得,并经氨基酸序列测定加以证实。钌搀杂的突变体C457S通过变异体与钌配合物的直接反应得到。研究了重组胆红素氧化酶及其变异体的光谱学性质,以及重组胆红素氧化酶和变异体I402G的酶促反应的动力学。结果 变异体I402G和C457S被成功的表达和纯化。变异体I402G表现出较低的活性(1.67U/mg),C457S则完全失去了活性(0U/mg),但是钌搀杂的突变体C457S表现出较高的活性(5.50和6.42U/mg)。光谱学实验结果表明I402G突变体在较低的pH时第一类铜呈+1价态,即还原态;突变体C457S中失去第一类铜,使得突变体无法保持完整的活性中心,从而失去了酶活性。酶促反应的动力学实验显示重组胆红素氧化酶和变异体I402G动力学参数kcat分别为235.8min-1和6.9min-1,说明重组胆红素氧化酶具有很高的催化能力。结论 配位的氨基酸对保持重组胆红素氧化酶及其变异体活性中心的完整性和酶活性具有重要的意义。重组胆红素氧化酶远比突变体I402G和C457S的活性高,说明突变体I402G和C457S不适合作为诊断试剂。钌搀杂有助于在突变体C457S中形成新的完整的活性中心,从而产生酶活性。
Abstract:
Objective To elucidate the significance of the coordination amino acid residues in bilirubin oxidase (BO) and their kinetic characteristics, and evaluate whether BO mutants may serve as better diagnostic agent for hyperbilirubinemia. Methods The BO mutants I402G and C457S were obtained by site-directed mutagenesis and confirmed by amino acid sequence analysis. Ru-incorporated C457S mutant was obtained by direct incubation of ruthenium compounds with the mutant. The electron paramagnetic resonance (EPR) spectra of the recombinant BO and the mutants were investigated, and the enzyme kinetics of the recombinant BO and I402G mutant were measured with bilirubin as the substrate at 25 ℃. Results The BO mutants were expressed and purified successfully. The mutant I402G showed low enzyme activity, and had C457S virtually no enzyme activity. Nevertheless Ru-incorporation conferred higher enzyme activity to C457S mutant. The enzyme kinetic investigations revealed that the kinetic parameter kcat of the recombinant BO and I402G mutant was 235.8 min-1 and 6.9 min-1, respectively, suggesting higher enzyme activity of the recombinant BO. Conclusions The coordinating amino acids have important significance in maintaining the integrity of active centers and enzyme activities of recombinant BO and its mutants. The enzyme activities of the mutants I402G and C457S are much lower than those of recombinant BO, therefore they are not appropriate for diagnostic purpose. Ru-incorporation facilitates the formation of a new intact active center in C457S mutant, which therefore acquires enzyme activity.

参考文献/References:

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备注/Memo

备注/Memo:
收稿日期:2005-6-12。
基金项目:Guangdong Provincial Laboratory for Green Chemical Technology and by Japan Society for Promotion of Science
通讯作者:ZhangLei,PhD,associate professor,Fax:020-87110234
更新日期/Last Update: 1900-01-01