Habib Ahsan, Tabata Masaaki
Department of Chemistry, Faculty of Science and Engineering, Saga University, 1 Honjo-machi, Saga 840-8502, Japan.
J Inorg Biochem. 2004 Nov;98(11):1696-702. doi: 10.1016/j.jinorgbio.2004.07.005.
Oxidative DNA damage was investigated by free radicals generated from HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) buffer, which is widely used in biochemical or biological studies, in the presence of Au(III). The effect of free radicals on the DNA damage was ascertained by gel electrophoresis, electron spin resonance (ESR) spectroscopy and circular dichroism (CD) spectroscopy. ESR results indicated the generation of nitrogen-centered cationic free radicals from the HEPES in the presence of Au(III) which cause the DNA damage. No ESR spectra were observed for phosphate, tris(hydroxymethyl)aminomethane (Tris-HCl) and acetate buffers in the presence of Au(III) or for HEPES buffer in the presence of other metal ions such as Mn(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II) and Pd(II) or Au(III)(TMPyP) and Pd(II)(TMPyP), where H(2)(TMPyP) denotes tetrakis(1-methylpyridium-4-yl)porphyrin. Consequently, no DNA damage was observed for these buffer agents (e.g., phosphate, Tris-HCl or acetate) in the presence of Au(III) or for HEPES in the presence of other metal ions or the metalloporphyrins mentioned above. No detectable inhibitory effect on the DNA damage was observed by using the typical scavengers of reactive oxygen species (ROS) ()OH, O(2)(-) and H(2)O(2). This non-inhibitory effect indicated that no reactive oxygen species were generated during the incubation of DNA with HEPES and Au(III). The drastic change in CD spectra from positive ellipticity to negative ellipticity approximately at 270 nm with increasing concentration of Au(III) also indicated the significant damage of DNA. Only HEPES or Au(III) itself did not damage DNA. A mechanism for the damaging of DNA is proposed.
在金（III）存在的情况下，研究了由广泛用于生化或生物学研究的HEPES（2-[4-(2-羟乙基)-1-哌嗪基]乙磺酸）缓冲液产生的自由基对DNA的氧化损伤。通过凝胶电泳、电子自旋共振（ESR）光谱和圆二色性（CD）光谱确定自由基对DNA损伤的影响。ESR结果表明，在金（III）存在下，HEPES产生了以氮为中心的阳离子自由基，导致DNA损伤。在金（III）存在下，磷酸盐、三（羟甲基）氨基甲烷（Tris-HCl）和乙酸盐缓冲液，或在其他金属离子如锰（II）、铁（III）、钴（II）、镍（II）、铜（II）、锌（II）和钯（II）或金（III）（四甲基吡啶卟啉）和钯（II）（四甲基吡啶卟啉）存在下的HEPES缓冲液，均未观察到ESR光谱，其中H₂（四甲基吡啶卟啉）表示四（1-甲基吡啶-4-基）卟啉。因此，在金（III）存在下，这些缓冲剂（如磷酸盐、Tris-HCl或乙酸盐），或在上述其他金属离子或金属卟啉存在下的HEPES，均未观察到DNA损伤。使用活性氧（ROS）（·OH、O₂⁻和H₂O₂）的典型清除剂，未观察到对DNA损伤有可检测的抑制作用。这种非抑制作用表明，在DNA与HEPES和金（III）孵育期间未产生活性氧。随着金（III）浓度的增加，CD光谱在约270nm处从正椭圆率急剧变为负椭圆率，这也表明DNA受到了显著损伤。仅HEPES或金（III）本身不会损伤DNA。提出了一种DNA损伤的机制。
