Khan J, Brennand D M, Bradley N, Gao B, Bruckdorfer R, Jacobs M
Department of Pharmacology, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, U.K.
Biochem J. 1998 Mar 1;330 ( Pt 2)(Pt 2):795-801. doi: 10.1042/bj3300795.
The modification of tyrosine residues in proteins to 3-nitrotyrosine by peroxynitrite or other potential nitrating agents has been detected in biological systems that are subject to oxidative stress. A convenient semi-quantitative method has been developed to assay nitrated proteins in biological fluids and homogenates using a competitive ELISA developed in our laboratory. This assay selectivity detected 3-nitro-l-tyrosine residues in a variety of peroxynitrite-treated proteins (BSA, human serum albumin (HSA), alpha1-antiprotease inhibitor, pepsinogen and fibrinogen) and also in a nitrated peptide, but had a low affinity for free 3-nitro-L-tyrosine and 3-chloro-L-tyrosine. The IC50 values for the inhibition of antibody binding by different nitrated proteins were in the range 5-100 nM, suggesting that the antibody discriminated between nitrotyrosine residues in different environments. The presence of nitrotyrosine in plasma proteins was detected by Western blot analysis and quantified by the ELISA. A concentration of 0. 12+/-0.01 microM nitro-BSA equivalents was measured in the proteins of normal plasma which was increased in peroxynitrite-treated plasma and was elevated in inflammatory conditions. HSA and low-density lipoprotein (LDL) isolated from plasma contained 0.085+/-0.04 and 0. 03+/-0.006 nmol nitro-BSA equivalents/mg protein, respectively. Comparison of the level of nitration in peroxynitrite-treated HSA and LDL in the presence and absence of plasma indicates that nitration and presumably oxidation is inhibited by plasma antioxidants. The presence of nitrotyrosine in LDL is consistent with previous reports implicating peroxynitrite in the oxidative modification of lipoproteins and the presence of a low concentration of oxidized LDL in the blood.
在遭受氧化应激的生物系统中，已检测到过氧亚硝酸盐或其他潜在硝化剂将蛋白质中的酪氨酸残基修饰为3-硝基酪氨酸。我们实验室开发了一种便捷的半定量方法，使用竞争性酶联免疫吸附测定法（ELISA）来检测生物体液和匀浆中的硝化蛋白质。该测定法可选择性地检测多种经过氧亚硝酸盐处理的蛋白质（牛血清白蛋白（BSA）、人血清白蛋白（HSA）、α1-抗蛋白酶抑制剂、胃蛋白酶原和纤维蛋白原）以及硝化肽中的3-硝基-L-酪氨酸残基，但对游离的3-硝基-L-酪氨酸和3-氯-L-酪氨酸亲和力较低。不同硝化蛋白质抑制抗体结合的半数抑制浓度（IC50）值在5 - 100 nM范围内，这表明该抗体能够区分不同环境中的硝基酪氨酸残基。通过蛋白质印迹分析检测血浆蛋白中硝基酪氨酸的存在，并通过ELISA进行定量。在正常血浆蛋白中测得的硝基-BSA当量浓度为0.12±0.01微摩尔，在过氧亚硝酸盐处理的血浆中升高，在炎症条件下也升高。从血浆中分离出的HSA和低密度脂蛋白（LDL）分别含有0.085±0.04和0.03±0.006纳摩尔硝基-BSA当量/毫克蛋白质。比较有血浆和无血浆存在时过氧亚硝酸盐处理的HSA和LDL中的硝化水平表明，血浆抗氧化剂可抑制硝化作用以及可能的氧化作用。LDL中硝基酪氨酸的存在与先前报道一致，这些报道表明过氧亚硝酸盐参与脂蛋白的氧化修饰以及血液中低浓度氧化LDL的存在。
