Pou S, Cohen M S, Britigan B E, Rosen G M
Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710.
J Biol Chem. 1989 Jul 25;264(21):12299-302.
Using the spin trap, 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and an excess of dimethyl sulfoxide, we previously reported that in the absence of an exogenous iron catalyst, human neutrophils will not generate hydroxyl radical, manifested as the catalse-inhibitable methyl radical spin-trapped adduct, 2,2,5-trimethyl-1-pyrrolidinyloxy (DMPO-CH3) (Britigan, B. E., Rosen, G. M., Chai, Y., and Cohen, M. S. (1986) J. Biol. Chem. 261, 4426-4431). However, superoxide destroys the preformed hydroxyl radical spin-trapped adduct, 2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxy (DMPO-OH), and DMPO-CH3. The present study was undertaken to better resolve the limits of sensitivity of the spin-trapping method. Photolytically generated DMPO-CH3 and DMPO-OH slowly decomposed in the presence of a low flux (1 microM/min) of enzymatically (xanthine/xanthine oxidase)-generated superoxide, but more rapid decomposition of these adducts occurred with higher superoxide flux (5 microM/min). Inclusion of cysteine markedly increased the rate of DMPO-OH and DMPO-CH3 decomposition, masking the effect of superoxide alone. The addition of varying concentrations of superoxide dismutase did not lead to increased formation of DMPO-OH or DMPO-CH3, as should have occurred if these adducts were being destroyed by superoxide. As a positive control, we employed an iron-supplemented system with phorbol 12-myristate 13-acetate-stimulated neutrophils or xanthine/xanthine oxidase to generate DMPO-CH3. Addition of superoxide dismutase increased the magnitude of DMPO-CH3, primarily by increasing the rate of hydrogen peroxide formation, and to a lesser extent by prolonging the half-life of DMPO-CH3. Although spin-trapped adducts can be destroyed by a high concentration of superoxide, or by lower concentrations of superoxide in the presence of thiol-containing compounds, our results demonstrate that such decomposition does not interfere with the ability of the spin-trapping method to detect hydroxyl radical generated by human neutrophils. These data do not support the capacity of neutrophils to generate hydroxyl radical in the absence of an exogenous Haber-Weiss catalyst.
我们之前使用自旋捕获剂5,5-二甲基-1-吡咯啉-1-氧化物（DMPO）和过量的二甲基亚砜报道，在没有外源性铁催化剂的情况下，人类中性粒细胞不会产生羟基自由基，表现为可被过氧化氢酶抑制的甲基自由基自旋捕获加合物2,2,5-三甲基-1-吡咯烷氧基（DMPO-CH3）（布里蒂根，B.E.，罗森，G.M.，柴，Y.，和科恩，M.S.（1986年）《生物化学杂志》261，4426 - 4431）。然而，超氧化物会破坏预先形成的羟基自由基自旋捕获加合物2,2-二甲基-5-羟基-1-吡咯烷氧基（DMPO-OH）和DMPO-CH3。本研究旨在更好地解析自旋捕获方法的灵敏度极限。在低通量（1微摩尔/分钟）的酶促（黄嘌呤/黄嘌呤氧化酶）产生的超氧化物存在下，光解产生的DMPO-CH3和DMPO-OH会缓慢分解，但在较高的超氧化物通量（5微摩尔/分钟）下，这些加合物的分解更快。加入半胱氨酸显著增加了DMPO-OH和DMPO-CH3的分解速率，掩盖了单独超氧化物的作用。加入不同浓度的超氧化物歧化酶并没有导致DMPO-OH或DMPO-CH3形成增加，而如果这些加合物是被超氧化物破坏的话应该会出现这种情况。作为阳性对照，我们使用了一个铁补充系统，用佛波醇12-肉豆蔻酸酯13-乙酸酯刺激中性粒细胞或黄嘌呤/黄嘌呤氧化酶来产生DMPO-CH3。加入超氧化物歧化酶增加了DMPO-CH3的量，主要是通过增加过氧化氢的形成速率，在较小程度上是通过延长DMPO-CH
