用黄素腺嘌呤二核苷酸（FAD）结合物重构脱辅基葡萄糖氧化酶用于孕酮的生物传感

Reconstitution of apoglucose oxidase with FAD conjugates for biosensoring of progesterone.

作者信息

Posthuma-Trumpie Geertruida A, van den Berg Willy A M, van de Wiel Dirk F M, Schaaper Wim M M, Korf Jakob, van Berkel Willem J H

机构信息

University of Groningen, University Medical Centre Groningen, School of Behavioural and Cognitive Neuroscience, Department of Psychiatry, Hanzeplein 1, 9713 EZ Groningen, The Netherlands.

出版信息

Biochim Biophys Acta. 2007 Jul;1774(7):803-12. doi: 10.1016/j.bbapap.2007.04.009. Epub 2007 May 1.

DOI:10.1016/j.bbapap.2007.04.009

PMID:17544346

Abstract

The reconstitution of Aspergillus niger apoglucose oxidase (apoGOx) with FAD conjugates for biosensoring of progesterone was investigated. ApoGOx prepared by partial unfolding of the protein under acidic conditions consisted of reconstitutable monomers (50+/-10%), reconstitutable dimers (20+/-10%) and irreversibly aggregated oligomers (30+/-20%). Incubation of monomeric apoGOx with FAD or N(6)-(6-aminohexyl)-FAD (ahFAD) restored glucose oxidase (GOx) activity and induced dimerization with stoichiometric incorporation of FAD. N(6)-(6-aminohexyl)-FAD progesterone conjugates also induced dimerization. However, holoenzyme reconstitution required relatively high concentrations of apoprotein and was dependent on the type of conjugate. Restoration to 25-50% of the original enzyme activity was obtained. Binding of the FAD-progesterone conjugates might hinder the closure of a protein lid needed for dimer formation. Our results illustrate the prospects of FAD conjugates in sensitive detection of progesterone in biological matrices in a biosensor based on the recombination of apoGOx with progesterone-conjugated FAD.

摘要

研究了用FAD缀合物重组黑曲霉脱辅基葡萄糖氧化酶（apoGOx）用于孕酮生物传感的情况。在酸性条件下通过蛋白质部分展开制备的apoGOx由可重构单体（50±10%）、可重构二聚体（20±10%）和不可逆聚集的寡聚体（30±20%）组成。将单体apoGOx与FAD或N(6)-(6-氨基己基)-FAD（ahFAD）孵育可恢复葡萄糖氧化酶（GOx）活性，并诱导二聚化，同时化学计量地掺入FAD。N(6)-(6-氨基己基)-FAD孕酮缀合物也诱导二聚化。然而，全酶重组需要相对高浓度的脱辅基蛋白，并且取决于缀合物的类型。恢复到了原始酶活性的25 - 50%。FAD - 孕酮缀合物的结合可能会阻碍形成二聚体所需的蛋白质盖子的闭合。我们的结果说明了基于apoGOx与孕酮共轭FAD重组的生物传感器中，FAD缀合物在生物基质中灵敏检测孕酮的前景。