Division of Biomedical Sciences, University of the Highlands and Islands, Old Perth Road, Inverness, IV2 3JH, Scotland, UK; Edinburgh Napier University, Edinburgh, Scotland, UK.
Division of Biomedical Sciences, University of the Highlands and Islands, Old Perth Road, Inverness, IV2 3JH, Scotland, UK.
Free Radic Biol Med. 2022 Mar;182:73-78. doi: 10.1016/j.freeradbiomed.2022.02.019. Epub 2022 Feb 22.
New readily accessible systemic redox biomarkers are needed to understand the biological roles reactive oxygen species (ROS) play in humans because overtly flawed, technically fraught, and unspecific assays severely hamper translational progress. The antibody-linked oxi-state assay (ALISA) makes it possible to develop valid ROS-sensitive target-specific protein thiol redox state biomarkers in a readily accessible microplate format. Here, we used a maximal exercise bout to disrupt redox homeostasis in a physiologically meaningful way to determine whether the catalytic core of the serine/threonine protein phosphatase PP2A is a candidate systemic redox biomarker in human erythrocytes. We reasoned that: constitutive oxidative stress (e.g., haemoglobin autoxidation) would sensitise erythrocytes to disrupted ion homeostasis as manifested by increased oxidation of the ion regulatory phosphatase PP2A. Unexpectedly, an acute bout of maximal exercise lasting ~16 min decreased PP2A-specific reversible thiol oxidation (redox ratio, rest: 0.46; exercise: 0.33) without changing PP2A content (rest: 193 pg/ml; exercise: 191 pg/ml). The need for only 3-4 μl of sample to perform ALISA means PP2A-specific reversible thiol oxidation is a capillary-fingertip blood-compatible candidate redox biomarker. Consistent with biologically meaningful redox regulation, thiol reductant-inducible PP2A activity was significantly greater (+10%) at rest compared to exercise. We establish a route to developing new readily measurable protein thiol redox biomarkers for understanding the biological roles ROS play in humans.
新的易于获得的系统氧化还原生物标志物对于理解活性氧（ROS）在人类中的生物学作用是必要的，因为明显有缺陷、技术上复杂且非特异性的测定方法严重阻碍了转化研究的进展。抗体连接的氧化状态测定法（ALISA）使得以易于获得的微量滴定板格式开发有效的 ROS 敏感的靶特异性蛋白巯基氧化还原状态生物标志物成为可能。在这里，我们使用最大运动来以生理上有意义的方式破坏氧化还原平衡，以确定丝氨酸/苏氨酸蛋白磷酸酶 PP2A 的催化核心是否是人类红细胞中的候选系统氧化还原生物标志物。我们推断：组成性氧化应激（例如，血红蛋白自氧化）会使红细胞对离子稳态的破坏敏感，表现为离子调节磷酸酶 PP2A 的氧化增加。出乎意料的是，持续约 16 分钟的急性最大运动剧烈运动降低了 PP2A 特异性可逆巯基氧化（氧化还原比，休息时：0.46；运动时：0.33），而不改变 PP2A 含量（休息时：193 pg/ml；运动时：191 pg/ml）。仅需 3-4 μl 样品即可进行 ALISA 意味着 PP2A 特异性可逆巯基氧化是一种毛细管指尖血兼容的候选氧化还原生物标志物。与有意义的氧化还原调节一致，与运动相比，休息时可还原的巯基诱导的 PP2A 活性显著增加（+10%）。我们建立了一种开发新的易于测量的蛋白质巯基氧化还原生物标志物的方法，以了解 ROS 在人类中的生物学作用。
