Identification of modified tryptophan residues in apolipoprotein B-100 derived from copper ion-oxidized low-density lipoprotein.
Identification of modified tryptophan residues in apolipoprotein B-100 derived from copper ion-oxidized low-density lipoprotein.
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源自铜离子氧化低密度脂蛋白的载脂蛋白 B-100 中修饰色氨酸残基的鉴定。
DOI：
10.1021/bi991464g
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发表时间：
1999
期刊：
影响因子：
2.9
通讯作者：
Smith,CV
中科院分区：
文献类型：
--
作者：
Yang,C;Gu,ZW;Yang,M;Lin,SN;Siuzdak,G;Smith,CV
低密度脂蛋白（LDL）的氧化修饰可能有助于动脉粥样硬化的发病机制。虽然LDL脂质成分的氧化产物已被广泛研究，但对载脂蛋白B-100的氧化产物知之甚少。为了确定特定的氧化修饰，我们氧化LDL中存在的Cu 2+，处理DNPH，沉淀和脱脂的蛋白质，消化的蛋白质与胰蛋白酶，并分析了肽的高效液相色谱法。我们分离出9种肽，其在365 nm处表现出可测量的吸光度，这是来自DNPH的腙的特征，并且在来自未氧化的LDL的肽中未观察到。出乎意料的是，我们在未用DNPH处理的Cu 2+氧化的LDL中获得了在365 nm处具有吸光度的相同肽。N-末端序列分析和质谱分析表明，从Cu 2+氧化的LDL中分离的肽都含有犬尿氨酸残基，取代了天然载脂蛋白中的Trp残基。我们在Cu 2+氧化的LDL中观察到的产物谱与在体外由HOCl或髓过氧化物酶氧化的LDL中观察到的谱显著不同，并且在Cu 2+催化的LDL氧化中Trp优先氧化为犬尿氨酸与用HOCl或髓过氧化物酶氧化LDL后观察到的产物形成对比。迄今为止，我们的研究支持工作假设，即蛋白质氧化的特定产物是足够独特的，可以开发为LDL和其他毒性和疾病中生物分子氧化机制的生物标志物。
Oxidative modifications of low-density lipoproteins (LDL) may contribute to the pathogenesis of atherosclerosis. Although the oxidation products of the lipid components of LDL have been studied extensively, less is known about the oxidation products of the apoprotein, apolipoprotein B-100. To identify the specific oxidative modifications, we oxidized LDL in the presence of Cu2+, treated with DNPH, precipitated and delipidated the protein, digested the protein with trypsin, and analyzed the peptides by high-performance liquid chromatography. We isolated nine peptides that exhibited measurable absorbance at 365 nm, which is characteristic of hydrazones derived from DNPH and is not observed in peptides derived from unoxidized LDL. Unexpectedly, we obtained the same peptides with absorbance at 365 nm in Cu2+-oxidized LDL not treated with DNPH. N-terminal sequence analyses and mass spectrometry indicated that the peptides isolated from the Cu2+-oxidized LDL all contained kynurenine residues in place of Trp residues found in the native apoprotein. The product profile we observed in Cu2+-oxidized LDL was remarkably different from the profiles observed in LDL oxidized by HOCl or myeloperoxidase in vitro, and the preferential oxidation of Trp to kynurenine in Cu2+-catalyzed oxidation of LDL contrasts with the products observed following oxidation of LDL with HOCl or myeloperoxidase. Our studies to date support the working hypothesis that the specific products of protein oxidation are sufficiently distinct to be developed as biomarkers of proposed mechanisms of oxidation of LDL and biological molecules in other toxicities and diseases.