Macfadyen A J, Reiter C, Zhuang Y, Beckman J S
Department of Pediatrics, Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, Alabama 35233, USA.
Chem Res Toxicol. 1999 Mar;12(3):223-9. doi: 10.1021/tx980253u.
Peroxynitrite (ONOO-) is a relatively stable oxidant produced by activated macrophages and neutrophils. To detect peroxynitrite, a novel human superoxide dismutase (SOD) trap was developed by substituting a tyrosine near the copper in the active site. The copper can catalyze nitration of this tyrosine by peroxynitrite. The nitrated tyrosine can serve as a reporter for peroxynitrite by measuring the extent of nitration with Western blots developed with a nitrotyrosine antibody. The new SOD mutant differs from bovine SOD whose sole tyrosine is far removed from the active site. Nitration of bovine SOD was second-order with respect to SOD concentration, whereas nitration of the new mutant SODs followed first-order kinetics with respect to peroxynitrite. The tyrosine SODs were used to assess whether peroxynitrite crosses erythrocyte membranes through the band 3 anion exchange protein. Tyrosine-containing SOD entrapped within normal human erythrocyte ghosts became nitrated in proportion to peroxynitrite concentration. The band 3 anion exchange protein inhibitors, phenyl isothiocyanate (PITC) and 4, 4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), inhibited up to 90% of the nitration. The erythrocyte membrane proteins, spectrin, band 3 anion exchange protein, and proteins 4.1 and 4.2, were also nitrated. Nitration of erythrocyte membrane proteins was also inhibited by PITC and DIDS. These data suggest that the band 3 anion exchange protein is the major route for the entry of peroxynitrite into erythrocytes. The ability of peroxynitrite to cross cell membranes can contribute to its toxicity by allowing access to intracellular target molecules.
过氧亚硝酸盐（ONOO-）是一种由活化的巨噬细胞和中性粒细胞产生的相对稳定的氧化剂。为了检测过氧亚硝酸盐，通过替换活性位点中靠近铜的酪氨酸，开发了一种新型的人超氧化物歧化酶（SOD）捕获剂。铜可以催化过氧亚硝酸盐对该酪氨酸的硝化作用。通过用硝基酪氨酸抗体进行蛋白质印迹法测量硝化程度，硝化的酪氨酸可以作为过氧亚硝酸盐的报告分子。新的SOD突变体与牛SOD不同，牛SOD唯一的酪氨酸远离活性位点。牛SOD的硝化作用相对于SOD浓度是二级反应，而新的突变体SOD的硝化作用相对于过氧亚硝酸盐遵循一级动力学。酪氨酸SOD被用于评估过氧亚硝酸盐是否通过带3阴离子交换蛋白穿过红细胞膜。包裹在正常人红细胞空壳内的含酪氨酸SOD会按照过氧亚硝酸盐浓度成比例地被硝化。带3阴离子交换蛋白抑制剂，异硫氰酸苯酯（PITC）和4,4'-二异硫氰酸根合芪-2,2'-二磺酸盐（DIDS），可抑制高达90%的硝化作用。红细胞膜蛋白，血影蛋白、带3阴离子交换蛋白以及蛋白4.1和4.2也被硝化。红细胞膜蛋白的硝化作用也被PITC和DIDS抑制。这些数据表明带3阴离子交换蛋白是过氧亚硝酸盐进入红细胞的主要途径。过氧亚硝酸盐穿过细胞膜的能力可通过使其能够接触细胞内靶分子而增加其毒性。
