Graduate School of Comprehensive Human Sciences, University of Tsukuba.
Faculty of Pharmacy, Hue University of Medicine and Pharmacy, Hue University.
Biol Pharm Bull. 2022;45(6):798-802. doi: 10.1248/bpb.b22-00080.
Redox-active quinones generate reactive oxygen species (ROS) through their redox cycling with electron donors. Hydrogen peroxide (HO) causes S-oxidation of proteins and is associated with activation of the redox signaling pathway and/or toxicity (Chem. Res. Toxicol., 30, 2017, Kumagai et al.). In the present study, we developed a convenient assay based on a combination of an enzyme-linked immunosorbent assay and a biotin-PEAC-maleimide assay and used it to determine protein S-oxidation by ROS during redox cycling of 9,10-phenanthrenequinone (9,10-PQ) and pyrroloquinoline quinone (PQQ). S-Oxidation of proteins in a mouse liver supernatant was detected during reaction of 9,10-PQ or PQQ with electron donors such as dithiothreitol or reduced nicotinamide adenine dinucleotide phosphate (NADPH), whereas cellular protein oxidation was not observed in the absence of electron donors. These results suggest that the developed assay is useful for the detection of S-oxidation of proteins.
氧化还原活性醌通过与电子供体的氧化还原循环产生活性氧 (ROS)。过氧化氢 (HO) 引起蛋白质的 S-氧化，并与氧化还原信号通路的激活和/或毒性有关 (Chem. Res. Toxicol., 30, 2017, Kumagai 等人)。在本研究中，我们开发了一种基于酶联免疫吸附测定和生物素-PEAC-马来酰亚胺测定相结合的方便测定法，并使用该方法来确定在 9,10-菲醌 (9,10-PQ) 和吡咯并喹啉醌 (PQQ) 的氧化还原循环过程中 ROS 对蛋白质的 S-氧化作用。在 9,10-PQ 或 PQQ 与电子供体（如二硫苏糖醇或还原型烟酰胺腺嘌呤二核苷酸磷酸 (NADPH)）反应时，可检测到小鼠肝上清液中蛋白质的 S-氧化，而在没有电子供体的情况下则未观察到细胞内蛋白质氧化。这些结果表明，所开发的测定法可用于检测蛋白质的 S-氧化。
