Disulfide-Linked Dinitroxides for Monitoring Cellular Thiol Redox Status through Electron Paramagnetic Resonance Spectroscopy
Disulfide-Linked Dinitroxides for Monitoring Cellular Thiol Redox Status through Electron Paramagnetic Resonance Spectroscopy
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DOI：
10.1021/acs.biochem.5b00531
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发表时间：
2015-12-01
期刊：
影响因子：
2.9
通讯作者：
Kao, Joseph P. Y.
中科院分区：
文献类型：
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作者：
Legenzov, Eric A.;Sims, Stephen J.;Kao, Joseph P. Y.
细胞内巯基二硫化物氧化还原平衡对细胞健康至关重要，并且可能是癌症对化疗和放疗反应的关键决定因素。因此，评估细胞内巯基二硫化物平衡的能力不仅可用于预测癌症对治疗的反应性，而且可用于评估疾病易感性。生物学中硫醇的测定依赖于比色法或荧光法，这两种方法都需要紫外可见光子，而紫外可见光子不会穿透人体。低频电子顺磁共振成像（EPRI）是一种新兴的磁成像技术，使用无线电波，穿透身体很好。因此，结合定制的成像剂，EPRI提供了在体内成像生理的机会。在这项研究中，我们已经制备了水溶性和膜渗透性的二硫键连接的二氮氧化物，在自然同位素丰度，并与D，N-15-取代。巯基如谷胱甘肽裂解二硫化物，用简单的双分子动力学，产生单体氮氧化物物种，在EPR谱的独特变化。利用D，N-15-取代二硫键-二氮氧化物和EPR光谱，我们获得了定量估计可访问的细胞内巯基在培养的人淋巴细胞。我们的估计是在良好的协议与公布的测量。这表明在体内进行巯基二硫键平衡的EPRI是可行的。最后，我们讨论了限制探针分子的设计，这将是有用的在体内的巯基氧化还原状态的EPRI。
Intracellular thioldisulfide redox balance is crucial to cell health, and may be a key determinant of a cancers response to chemotherapy and radiation therapy. The ability to assess intracellular thioldisulfide balance may thus be useful not only in predicting responsiveness of cancers to therapy, but in assessing predisposition to disease. Assays of thiols in biology have relied on colorimetry or fluorimetry, both of which require UV-visible photons, which do not penetrate the body. Low-frequency electron paramagnetic resonance imaging (EPRI) is an emerging magnetic imaging technique that uses radio waves, which penetrate the body well. Therefore, in combination with tailored imaging agents, EPRI affords the opportunity to image physiology within the body. In this study, we have prepared water-soluble and membrane-permeant disulfide-linked dinitroxides, at natural isotopic abundance, and with D,N-15-substitution. Thiols such as glutathione cleave the disulfides, with simple bimolecular kinetics, to yield the monomeric nitroxide species, with distinctive changes in the EPR spectrum. Using the D,N-15-substituted disulfide-dinitroxide and EPR spectroscopy, we have obtained quantitative estimates of accessible intracellular thiol in cultured human lymphocytes. Our estimates are in good agreement with published measurements. This suggests that in vivo EPRI of thioldisulfide balance is feasible. Finally, we discuss the constraints on the design of probe molecules that would be useful for in vivo EPRI of thiol redox status.