TY - JOUR
T1 - Tetrathiatriarylmethyl radical with a single aromatic hydrogen as a highly sensitive and specific superoxide probe
AU - Liu, Yangping
AU - Song, Yuguang
AU - De Pascali, Francesco
AU - Liu, Xiaoping
AU - Villamena, Frederick A.
AU - Zweier, Jay L.
PY - 2012/12/1
Y1 - 2012/12/1
N2 - Superoxide (O2•-) 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 O 2•- 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, O 2•- preferentially reacts with CT02-H at this site with production of the diamagnetic quinone methide via oxidative dehydrogenation. Upon reaction with O2•-, CT02-H loses its EPR signal and this EPR signal decay can be used to quantitatively measure O2•-. 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 O2•- detection method. More than five-fold higher reactivity of CT02-H for O2•- 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 O2•- as evidenced by its inertness to other oxidoreductants. The O2•- 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 O2•- 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 O2 •-, 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 O2•- generation and was shown to provide sensitive detection of extracellular O 2•- 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 O 2•- by either EPR or HPLC methods.
AB - Superoxide (O2•-) 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 O 2•- 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, O 2•- preferentially reacts with CT02-H at this site with production of the diamagnetic quinone methide via oxidative dehydrogenation. Upon reaction with O2•-, CT02-H loses its EPR signal and this EPR signal decay can be used to quantitatively measure O2•-. 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 O2•- detection method. More than five-fold higher reactivity of CT02-H for O2•- 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 O2•- as evidenced by its inertness to other oxidoreductants. The O2•- 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 O2•- 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 O2 •-, 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 O2•- generation and was shown to provide sensitive detection of extracellular O 2•- 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 O 2•- by either EPR or HPLC methods.
KW - Electron paramagnetic resonance spectroscopy
KW - Free radicals
KW - Oxygen free radicals
KW - Reactive oxygen species
KW - Spin trapping
KW - Trityl radical
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U2 - 10.1016/j.freeradbiomed.2012.09.011
DO - 10.1016/j.freeradbiomed.2012.09.011
M3 - Article
C2 - 23000244
AN - SCOPUS:84868660867
SN - 0891-5849
VL - 53
SP - 2081
EP - 2091
JO - Free Radical Biology and Medicine
JF - Free Radical Biology and Medicine
IS - 11
ER -