微過氧化酵素催化Isoluminol氧化之化學發光及應用

Chemiluminescence of Microperoxidase-Catalyzed Oxidation of Isoluminol and Its Applications

王秋燕(Chiu-Yen Wang)

指導教授 ： 林萬寅

國立臺灣大學/理學院/化學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.00226

摘要

微過氧化酵素（Fe(III)-MP8）經由胃蛋白酶 (pepsin) 及胰蛋白酶 (trypsin) 水解馬心臟酵素c後純化所得，仍然留由從14-21的八個胺基酸殘鏈（14Cys-Ala-Gln-Cys-His-Thr-Val-21Glu），此胺基酸殘鏈 分別以兩個Cys上的硫醚鍵與血基質環相接，且有18His作為血基質中心金屬離子的鄰近配位基（proximal ligand）。

研究中，利用止流儀利用微過氧化酵素對isoluminol化學發光的催化增強效果，並進一步比較不同氧化劑H2O2與mCPBA對化學發光造成的差異。而存在增強劑如，guanidine hydrochloride (GdnHCl) 或sodium chloride 藉由加速化學發光反應使MP8-isoluminol發光訊號增加。另外，我們也對pH值，氧化劑 (H2O2 或mCPBA) 和isoluminol濃度作探討。

最後，我們將所發展的化學發光系統進一步應用於偵測抗氧化劑如uric acid, thioamine及trolox等，線性範圍1 m M ~ 10 m M。由於抗氧化劑會清除化學發光反應中所產生的自由基，導致其化學發光訊號的抑制。並且在H2O2-MP8-isoluminol發光系統中 (pH 12.0) ，存在ascorbic acid導致微過氧化酵素與H2O2反應所形成中間體增加使化學發光訊號增強。將其應用於定量維他命C藥丸中ascorbic acid含量不受其他物質干擾，線性範圍10 m M ~ 100 m M。在mCPBA-MP8-isoluminol 發光系統中，存在10倍的ascorbic acid仍可選擇性偵測uric acid濃度，線性範圍1 m M ~ 10 m M。

關鍵字

微過氧化酵素

並列摘要

Microperoxidase-8 (MP-8) is a heme-containing octapeptide obtained from proteolytic digestion of horse heart cytochrome c with pepsin and trypsin. MP-8 retains residues 14-21 (14Cys-Ala-Gln-Cys-His- Thr-Val-Glu21) of the cytochrome c protein. The heme c remains linked to the octapeptide through two thioether bonds, with 18His serving as a fixed proximal ligand.

In this study, we have used the stopped-flow technique to study the chemiluminescence (CL) of MP-catalyzed oxidation of isoluminol with the oxidant H2O2 or mCPBA. Moreover, the presence of enchancers such as guanidine hydrochloride (GdnHCl) or sodium chloride dramatically increases the CL emission of the MP8-isoluminol system. The enchancement in CL is due to the acceleration of the CL cycle. We also investigated the effects of pH, concentrations of oxidant (H2O2 or mCPBA) and isoluminol.

We apply the enhanced MP8-isoluminol-H2O2 or mCPBA CL systems to the determination of antioxidants such as uric acid, thioamine and trolox in the range of 1 m M ~ 10 m M. The antioxidants can scavenge the radicals involved in CL reaction, leading to a decreased CL emission. The detection of ascorbic acid from 10 m M ~ 100 m M in vitamin C tablets was based on the enhanced CL signal for the MP8-isoluminol-H2O2 by increasing the intermediate of microperoxidase react with H2O2 at pH 12.0. And the mCPBA-MP8-isoluminol system can be used to selective determination uric acid from 1 m M ~ 10 m M even though in presence of 10 fold of ascorbic acid as interference species.

並列關鍵字

microperoxidase

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