Tetrathiatriarylmethyl radical with a single aromatic hydrogen as a highly sensitive and specific superoxide probe.
Tetrathiatriarylmethyl radical with a single aromatic hydrogen as a highly sensitive and specific superoxide probe.
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DOI：
10.1016/j.freeradbiomed.2012.09.011
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发表时间：
2012-12-01
影响因子：
7.4
通讯作者：
Zweier, Jay L.
中科院分区：
文献类型：
--
作者：
Liu, Yangping;Song, Yuguang;De Pascali, Francesco;Liu, Xiaoping;Villamena, Frederick A.;Zweier, Jay L.
关键词：
超氧化物在正常生理和疾病中起着关键作用;然而，由于现有检测方法的灵敏度和/或特异性有限，其测量仍然具有挑战性。我们证明，四硫代三芳基甲基（TAM）自由基与一个单一的芳香族氢（CT 02-H）可以作为一个高度敏感和特异性的探针。CT 02-H是完全取代的TAM基团CT-03（芬兰三苯甲基）的类似物，由于其芳香氢而具有电子顺磁共振（EPR）双峰信号。由于氢的中性性质和可忽略的空间位阻，在该位点优先与CT 02-H反应，通过氧化脱氢产生抗磁性醌甲基化物。在与反应时，CT 02-H失去其EPR信号，并且该EPR信号衰减可用于定量测量。这伴随着颜色从绿色变为紫色，醌甲基化物产物在540 nm处表现出独特的UV-Vis吸光度（ε = 15，900 M−1 cm−1），提供了额外的检测方法。结果表明，CT 02-H的反应性比CT-03高5倍以上，其二级速率常数为1.7 × 104 M−1 s−1，而CT-03为3.1 × 103 M−1 s−1。CT 02-H对其他氧化还原剂表现出高特异性，这一点可以通过其对其他氧化还原剂的惰性来证明。通过CT 02-H从黄嘌呤/黄嘌呤氧化酶检测到的生成速率与通过细胞色素c还原测量的生成速率一致，但检测灵敏度高10至100倍。EPR检测CT 02-H可在120 min内测量极低的通量，检测限为0.34 nM/min。HPLC串联电化学检测用于定量检测稳定的醌甲基化物产物，是一种高灵敏度和特异性的测量方法，灵敏度限约为2 × 10−13 mol（10 nM，20 μl进样体积）。基于其EPR谱的O2依赖性线宽增宽，CT 02-H还能够同时测量O2浓度和生成，并且显示出对由甲萘醌或缺氧/复氧刺激的内皮细胞中的细胞外生成提供灵敏的检测。因此，CT 02-H是一种独特的探针，可为EPR或HPLC方法的测量提供非常高的灵敏度和特异性。
Superoxide plays crucial roles in normal physiology and disease; however, its measurement remains challenging because of the limited sensitivity and/or specificity of prior detection methods. We demonstrate that a tetrathiatriarylmethyl (TAM) radical with a single aromatic hydrogen (CT02-H) can serve as a highly sensitive and specific probe. CT02-H is an analogue of the fully substituted TAM radical CT-03 (Finland trityl) with an electron paramagnetic resonance (EPR) doublet signal due to its aromatic hydrogen. Owing to the neutral nature and negligible steric hindrance of the hydrogen, preferentially reacts with CT02-H at this site with production of the diamagnetic quinone methide via oxidative dehydrogenation. Upon reaction with , CT02-H loses its EPR signal and this EPR signal decay can be used to quantitatively measure . This is accompanied by a change in color from green to purple, with the quinone methide product exhibiting a unique UV–Vis absorbance (ε =15,900 M−1 cm−1) at 540 nm, providing an additional detection method. More than five-fold higher reactivity of CT02-H for relative to CT-03 was demonstrated, with a second-order rate constant of 1.7 × 104 M−1 s−1 compared to 3.1 × 103 M−1 s−1 for CT-03. CT02-H exhibited high specificity for as evidenced by its inertness to other oxidoreductants. The generation rates detected by CT02-H from xanthine/xanthine oxidase were consistent with those measured by cytochrome c reduction but detection sensitivity was 10- to 100-fold higher. EPR detection of CT02-H enabled measurement of very low flux with a detection limit of 0.34 nM/min over 120 min. HPLC in tandem with electrochemical detection was used to quantitatively detect the stable quinone methide product and is a highly sensitive and specific method for measurement of , with a sensitivity limit of ~2 × 10−13 mol (10 nM with 20-μl injection volume). Based on the O2-dependent linewidth broadening of its EPR spectrum, CT02-H also enables simultaneous measurement of O2 concentration and generation and was shown to provide sensitive detection of extracellular generation in endothelial cells stimulated either by menadione or with anoxia/reoxygenation. Thus, CT02-H is a unique probe that provides very high sensitivity and specificity for measurement of by either EPR or HPLC methods.
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影响因子：
64.8
作者：
Chen, Chun-An;Wang, Tse-Yao;Varadharaj, Saradhadevi;Reyes, Levy A.;Hemann, Craig;Talukder, M. A. Hassan;Chen, Yeong-Renn;Druhan, Lawrence J.;Zweier, Jay L.
通讯作者：
Zweier, Jay L.
影响因子：
3.3
作者：
Kuppusamy, P;Wang, PH;Zweier, JL
通讯作者：
Zweier, JL
影响因子：
3.9
作者：
Kutala, VK;Parinandi, NL;Kuppusamy, P
通讯作者：
Kuppusamy, P
DOI：
10.1073/pnas.0510977103
发表时间：
2006-05-16
影响因子：
11.1
作者：
Kussmaul, Lothar;Hirst, Judy
通讯作者：
Hirst, Judy
DOI：
10.1073/pnas.91.8.3388
发表时间：
1994-04-12
影响因子：
11.1
作者：
KUPPUSAMY, P;CHZHAN, M;ZWEIER, JL
通讯作者：
ZWEIER, JL