Greilberger Joachim, Greilberger Michaela, Wintersteiger Reinhold, Zangger Klaus, Herwig Ralf
Otto Loewi Research Center for Vascular Biology, Immunology and Inflammation, Division of Physiological Chemistry, Medical University of Graz, 8010 Graz, Austria.
Schwarzl Medical Center, Institute of Scientific Laboratory, 8301 Graz, Austria.
Antioxidants (Basel). 2021 Sep 21;10(9):1501. doi: 10.3390/antiox10091501.
The generation of peroxynitrite (ONOO) is associated with several diseases, including atherosclerosis, hypertension, neurodegeneration, cancer, inflammation, and sepsis. Alpha-ketoglutarate (αKG) is a known potential highly antioxidative agent for radical oxidative species such as peroxides. The question arises as to whether αKG is also a potential scavenger of ONOO and a potential protector against ONOO-mediated nitration of proteins. NMR studies of 1 mM αKG in 100 mM phosphate-buffered saline at pH 7.4 and pH 6.0 were carried out in the presence or absence of a final concentration of 2 mM ONOO. An ONOO-luminol-induced chemiluminescence reaction was used to measure the scavenging function of several concentrations of αKG; quantification of αKG was performed via spectrophotometric enzymatic assay of αKG in the absence or presence of 0, 1, or 2 mM ONOO. The nitration of tyrosine residues on proteins was measured on ONOO-treated bovine serum albumin (BSA) in the presence or absence of 0-24 mM αKG by an ELISA technique using a specific anti-IgG against nitro-tyrosine. The addition of ONOO to αKG led to the formation of succinic acid and nitrite at pH 7.0, but not at pH 6.0, as αKG was stable against ONOO. The absorbance of enzymatically estimated αKG at the time point of 30 min was significantly lower in favour of ONOO (1 mM: 0.21 ± 0.03, 2 mM: 0.12 ± 0.05 vs. 0 mM: 0.32 ± 0.02; < 0.001). The luminol technique showed an inverse logarithmic correlation of the ONOO and αKG concentrations ( = -2 × 10 ln() + 1 × 10; = 0.99). The usage of 4 mM αKG showed a significant reduction by nearly half in the chemiluminescence signal (284,456 ± 29,293 cps, < 0.001) compared to the control (474,401 ± 18,259); for 20 and 200 mM αKG, there were further reductions to 163,546 ± 26,196 cps ( < 0.001) and 12,658 ± 1928 cps ( < 0.001). Nitrated tyrosine residues were estimated using the ELISA technique. A negative linear correlation was obtained by estimating nitrated tyrosine residues in the presence of αKG ( = 0.94): a reduction by half of nitrated tyrosine was estimated using 12 mM αKG compared to the control (326.1 ± 39.6 nmol vs. 844.5 ± 128.4 nmol; < 0.001).
过氧亚硝酸盐（ONOO）的产生与多种疾病相关，包括动脉粥样硬化、高血压、神经退行性变、癌症、炎症和败血症。α-酮戊二酸（αKG）是一种已知的对过氧化物等自由基氧化物种具有高度抗氧化作用的潜在物质。问题在于αKG是否也是ONOO的潜在清除剂以及是否能潜在地保护蛋白质免受ONOO介导的硝化作用。在pH 7.4和pH 6.0的100 mM磷酸盐缓冲盐水中，在存在或不存在终浓度为2 mM ONOO的情况下，对1 mM αKG进行了核磁共振研究。使用ONOO-鲁米诺诱导的化学发光反应来测量几种浓度αKG的清除功能；通过在不存在或存在0、1或2 mM ONOO的情况下对αKG进行分光光度酶法测定来对αKG进行定量。使用针对硝基酪氨酸的特异性抗IgG的ELISA技术，在存在或不存在0 - 24 mM αKG的情况下，对经ONOO处理的牛血清白蛋白（BSA）上酪氨酸残基的硝化作用进行测量。在pH 7.0时，将ONOO添加到αKG中会导致琥珀酸和亚硝酸盐的形成，但在pH 6.0时不会，因为αKG对ONOO稳定。在30分钟时间点，酶促估计的αKG的吸光度在存在ONOO的情况下显著降低（1 mM：0.21±0.03，2 mM：0.12±0.05 vs. 0 mM：0.32±0.02；<0.001）。鲁米诺技术显示ONOO和αKG浓度呈负对数相关（= -2×10 ln() + 1×10；= 0.99）。与对照（474,401±18,259）相比，使用4 mM αKG时化学发光信号显著降低近一半（284,456±29,293 cps，<0.001）；对于20和200 mM αKG，进一步降低至163,546±26,196 cps（<0.001）和12,658±1928 cps（<0.001）。使用ELISA技术估计硝化的酪氨酸残基。通过在存在αKG的情况下估计硝化的酪氨酸残基获得负线性相关（= 0.94）：与对照相比，使用12 mM αKG时估计硝化酪氨酸减少了一半（326.1±39.6 nmol vs. 844.5±128.4 nmol；<0.001）。
