胆红素氧化酶及其变异体在胆红素血症评价中的应用

南方医科大学学报 ›› 2005, Vol. 25 ›› Issue (11): 1337-1340.

• • 下一篇

胆红素氧化酶及其变异体在胆红素血症评价中的应用

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

出版日期:2005-11-20 发布日期:2005-11-20
基金资助:
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

Value of bilirubin oxidase and its mutants in the diagnosis of hyperbilirubinemia

ZHANG Lei¹, ZHANG Xiao², LUO Zhi-Ying³

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

Online:2005-11-20 Published:2005-11-20

摘要/Abstract

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k_cat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 k_cat 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.

中图分类号:

张雷¹, 张晓², 罗智颖³. 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, 25(11): 1337-1340.

ZHANG Lei¹, ZHANG Xiao², LUO Zhi-Ying³. Value of bilirubin oxidase and its mutants in the diagnosis of hyperbilirubinemia[J]. Journal of Southern Medical University, 2005, 25(11): 1337-1340.

参考文献

[1] Liu YR, Zhu B, Wang XF, et al. Bilirubin as a potent antioxidant suppresses experimental autoimmune encephalomyelitis-implication for the role of oxidative stress in the development of multiple-sclerosis[J]. J Neuroimmunol, 2003, 139(1): 27-35.
[2] Kwak JY, Takeshige K, Cheung BS, et al. Bilirubin inhibits the activation of superoxide-producing NADPH oxidase in a neutrophil cell free-system[J]. Biochim Biophys Acta, 1991, 1076(2): 369-73.
[3] Hsia DYY, Allen FH, Gellis SS, et al. Erythroblastosis fetalis Ⅲ.Studies of serum bilirubin in relation to Kernicterus[J]. N Engl J Med, 1952, 247(2): 668-71.
[4] Hansen TWR, Allen JW, Tommarello S. Oxidation of bilirubin in the brain-further characterization of a potentially protective mechanism[J]. Mol Genet Metab, 1999, 68(1): 404-9.
[5] Ostrow JD., Pascolo L, Tiribeli C. Reassessment of the unbound concentration of unconjugated bilirubin in relation to neurotoxicity in vitro[J]. Pediat Res, 2003, 54(1), 98-104.
[6] McDonagh AF, Lightner DA. Like a shriveled blood orange-bilirubin, jaundice and phototherapy[J]. Pediatrics, 1985, 75(2): 443-5.
[7] Hansen TWR. Mechanisms ofbilirubin toxicity-clinical implication[J]. Clin Perinatol, 2002, 29940: 765.
[8] Hansen TWR, Allen JW. Oxidation of bilirubin by brain mitochondrial membranes-dependence on cell type and postnatal age[J].Biochem Mol Med, 1997, 60(1): 155-60.
[9] Shimizu A, Kwon JH, Sakurai T, et al. Myrothecium verrucaria bilirubin oxidase and its mutants for potential copper ligands[J].Biochem, 1999, 38(6): 3034-42.
[10] Kosaka A, Yamamoto C, Morishita Y, et al. Enzymatic determination of bilirubin fractions in serum[J]. Clin Biochem, 1987, 20(6):451-8.
[11] Andreu Y, Galban J, de Marcos S, et al. Determination of directbilirubin by a fluorimetric-enzymatic method based on bilirubin oxidase[J]. Fresenius J Anal Chem, 2000, 368(5): 516-21.
[12] Dai J, Parry DM, Krahn J. Transcutaneous bilirubinometry: its role in the assessment of neonatal jaundice[J]. Clin Biochem, 1997, 30(1): 1-9.
[13] Bhutani VK, Johnson LH, Gourley G. Measuring bilirubin through the skin[J]. Pediatrics 2003, 111(4): 919.
[14] Ozkan H, Oren H, Duman N, et al. Dermal bilirubin kinetics during phototherapy in term neonates[J]. Acta Paediatr, 2003, 92(5): 577-81.
[15] Ostrow JD, Pascolo L, Shapiro SM, et al. New concepts in bilirubin encephalopathy[J]. Eur J Clin Invest, 2003, 33(6): 988-97.
[16] Sakurai T, Zhan L, Fujita T, et al. Authentic and recombinant bilirubin oxidases are in different resting forms[J]. Biosci Biotechnol Biochem, 2003, 67(5): 1157-9.
[17] Sakurai T, Tanaka K, Kitagawa R, et al. Construction of a new expression system of bilirubin oxidase and its mutants to explore the dioxygen reduction mechanism[J]. J Inorg Biochem, 2003, 96(1): 223.
[18] Shimizu A, Sakurai T. Yamaguchi S, et al. Mutation of bilirubin oxidase by protein engineering for elucidating electron pathway[J]. J Inorg Biochem, 1997, 67(1): 56.

胆红素氧化酶及其变异体在胆红素血症评价中的应用

Value of bilirubin oxidase and its mutants in the diagnosis of hyperbilirubinemia

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 3

编辑推荐

Metrics

本文评价

[1]	邹外一，许多荣，苏畅，陈媚，李娟，罗绍凯 . 动态观察慢性髓性白血病患者β-catenin 变化与细胞遗传学疗效的关系[J]. 南方医科大学学报, 2010, 30(08): 1868-1873.
[2]	黄韬¹, 孙竞¹, 刘俊², 刘启发¹, 孟凡义¹. 双重滤过血浆成分分离法用于ABO血型主要不合非亲缘骨髓移植[J]. 南方医科大学学报, 2005, 25(11): 1438-1440.
[3]	关红梅, 王会丽, 魏雪. 不同面积脾栓塞治疗特发性血小板减少性紫癜及其对脾功能的影响[J]. 南方医科大学学报, 2004, 24(03): 343-344.