A spectrophotometric method for the determination of proteins damaged by oxidized lipids
A spectrophotometric method for the determination of proteins damaged by oxidized lipids
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DOI：
10.1006/abio.1998.2758
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发表时间：
1998-09-10
影响因子：
2.9
通讯作者：
Zamora, R
中科院分区：
文献类型：
--
作者：
Hidalgo, FJ;Alaiz, M;Zamora, R
优化了埃利希反应，以确定作为脂质过氧化和氧化应激的结果产生的吡咯化蛋白质。该程序包括在受控酸度和温度下用对-（二甲氨基）苯甲醛处理修饰的蛋白质，并测定相对于在不存在试剂的情况下获得的空白产生的加合物。以ε-N-吡咯基正亮氨酸（Pnl）为标准计算埃利希加合物的消光系数为35，000 M-1 cm（-1）。在0.16-20 μ M Pnl范围内，响应是线性的和可重复的。将该测定法应用于测定牛血清白蛋白、牛Cu-球蛋白、牛γ-球蛋白及其混合物中的吡咯含量，与ImM的4，5（E）-环氧-2（E）-庚烯醛孵育过夜，获得与蛋白质碱性水解后通过毛细管电泳测定Pnl的结果相似的结果。该方法也适用于吡咯测定牛血浆蛋白与epoxylenals，hydroxylenals，脂质过氧化氢，和脂质过氧化的次级产品，或氧化与Fe 3 +/抗坏血酸。所有这些处理产生血浆蛋白的吡咯化，并且所有埃利希加合物给出非常相似的吸收光谱，除了在用羟基烯醛处理中产生的吸收光谱。上述结果表明，蛋白质吡咯化是脂质过氧化过程和氧化应激的正常结果，并且埃利希加合物可以有效地确定这种吡咯化，（C）1998学术出版社。
The Ehrlich reaction was optimized to determine pyrrolized proteins produced as a consequence of lipid peroxidation and oxidative stress. The procedure consisted of the treatment of the modified protein with p-(dimethylamino)benzaldehyde at a controlled acidity and temperature, and the determination of adducts produced against the blank obtained in the absence of the reagent. The extinction coefficient of Ehrlich adducts was calculated by using epsilon-N-pyrrolylnorleucine (Pnl) as standard and was 35,000 M-1 cm(-1). The response was linear and reproducible within the range 0.16-20 mu M Pnl. The assay was applied to determination of pyrrole content in bovine serum albumin, bovine cu-globulins, bovine gamma-globulins, and mixtures of them, incubated overnight with 1 mM of 4,5(E)-epoxy-2(E)-heptenal, obtaining results similar to those from determination of Pnl by capillary electrophoresis after basic hydrolysis of the protein. The method was also applied to pyrrole determination in bovine plasma proteins either incubated with epoxyalkenals, hydroxyalkenals, lipid hydroperoxides, and secondary products of lipid peroxidation, or oxidized with Fe3+/ascorbate. All these treatments produced pyrrolization of plasma proteins and all Ehrlich adducts gave very similar absorbance spectra with the exception of that produced in the treatment with hydroxyalkenals. The above results suggest that protein pyrrolization is a normal consequence of the lipid peroxidation process and of oxidative stress, and that Ehrlich adducts may be valid to determine this pyrrolization, (C) 1998 Academic Press.