Reactive carbonyl formation by oxidative and non-oxidative pathways
Reactive carbonyl formation by oxidative and non-oxidative pathways
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DOI：
10.2741/adams
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发表时间：
2001-08-01
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影响因子：
--
通讯作者：
Leeuwenburgh, C
中科院分区：
文献类型：
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作者：
Adams, S;Green, P;Leeuwenburgh, C
在体外和各种病理学中，分光光度法可作为自由基反应对蛋白质损伤的指示剂。我们研究了模型蛋白质和各种氧化和非氧化反应，以及血红蛋白、肌红蛋白和细胞色素c对蛋白质羰基水平的影响。我们发现，氧化和非氧化机制都会将羰基引入蛋白质中，为2，4-二硝基苯肼(DNPH)的定量提供了部分依据。牛血清白蛋白暴露在次氯酸、过氧亚硝酸盐和金属催化氧化等氧化情景中，表现出不同的、但增加的羰基水平。蛋白质与丙二醛、丙烯醛、乙醇醛和乙二醛等各种醛孵育的其他非氧化性修饰体系也会产生大量的羰基。此外，纯化的肌红蛋白、血红蛋白和细胞色素c在与DNPH相同的波长上显示出高吸光度。观察到的高水平是由于DNPH分析光谱附近的血红蛋白、肌红蛋白和细胞色素c的固有吸收。这些研究表明，羰基含量可能是由氧化和非氧化机制造成的，含血红素的化合物可能会影响羰基的量化。
The spectrophotometric protein carbonyl assay is used as an indicator of protein damage by free radical reactions in vitro and in a variety of pathologies. We investigated model proteins and a variety of oxidative and non-oxidative reactions, as well as what effects hemoglobin, myoglobin, and cytochrome c might have on levels of protein carbonyls. We show that oxidative as well as non-oxidative mechanisms introduce carbonyl groups into proteins, providing a moiety for quantification with 2,4-dinitrophenylhydrazine (DNPH). Bovine serum albumin exposed to oxidative scenarios, such as hypochlorous acid, peroxynitrite, and metal-catalyzed oxidation exhibited variable, but increased levels of carbonyls. Other non-oxidative modification systems, in which proteins are incubated with various aldehydes, such as malondialdehyde, acrolein, glycolaldehyde, and glyoxal also generated significant amounts of carbonyls. Furthermore, purified myoglobin, hemoglobin, and cytochrome c show high absorbance at the same wavelengths as DNPH. The high levels observed are due to the innate absorbance of hemoglobin, myoglobin, and cytochrome c near the assay spectra of DNPH. These studies show that carbonyl content could be due to oxidative as well as non-oxidative mechanisms and that heme-containing compounds may effect carbonyl quantification.