Lukyanov Konstantin A, Belousov Vsevolod V
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, Moscow 117997, Russia.
Biochim Biophys Acta. 2014 Feb;1840(2):745-56. doi: 10.1016/j.bbagen.2013.05.030. Epub 2013 May 29.
Life is a constant flow of electrons via redox couples. Redox reactions determine many if not all major cellular functions. Until recently, redox processes remained hidden from direct observation in living systems due to the lack of adequate methodology. Over the last years, imaging tools including small molecule probes and genetically encoded sensors appeared, which provided, for the first time, an opportunity to visualize and, in some cases, quantify redox reactions in live cells. Genetically encoded fluorescent redox probes, such as HyPer, rxYFP and roGFPs, have been used in several models, ranging from cultured cells to transgenic animals, and now enough information has been collected to highlight advantages and pitfalls of these probes.
In this review, we describe the main types of genetically encoded redox probes, their essential properties, advantages and disadvantages. We also provide an overview of the most important, in our opinion, results obtained using these probes. Finally, we discuss redox-dependent photoconversions of GFP and other prospective directions in redox probe development.
Fluorescent protein-based redox probes have important advantages such as high specificity, possibility of transgenesis and fine subcellular targeting. For proper selection of a redox sensor for a particular model, it is important to understand that HyPer and roGFP2-Orp1 are the probes for H2O2, whereas roGFP1/2, rxYFP and roGFP2-Grx1 are the probes for GSH/GSSG redox state. Possible pH changes should be carefully controlled in experiments with HyPer and rxYFP.
Genetically encoded redox probes are the only instruments allowing real-time monitoring of reactive oxygen species and thiol redox state in living cells and tissues. We believe that in the near future the palette of FP-based redox probes will be expanded to red and far-red parts of the spectrum and to other important reactive species such as NO, O2 and superoxide. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.
生命是通过氧化还原对进行的电子持续流动过程。氧化还原反应决定了许多（即便不是所有）主要的细胞功能。直到最近，由于缺乏适当的方法，氧化还原过程在生命系统中仍无法直接观察到。在过去几年中，出现了包括小分子探针和基因编码传感器在内的成像工具，首次为在活细胞中可视化以及在某些情况下量化氧化还原反应提供了机会。基因编码的荧光氧化还原探针，如HyPer、rxYFP和roGFP，已在从培养细胞到转基因动物等多种模型中使用，现在已收集到足够的信息来突出这些探针的优点和缺陷。
在本综述中，我们描述了基因编码氧化还原探针的主要类型、其基本特性、优点和缺点。我们还概述了我们认为使用这些探针获得的最重要结果。最后，我们讨论了GFP的氧化还原依赖性光转换以及氧化还原探针开发中的其他潜在方向。
基于荧光蛋白的氧化还原探针具有重要优点，如高特异性、转基因可能性和精细的亚细胞靶向性。对于为特定模型正确选择氧化还原传感器，重要的是要了解HyPer和roGFP2-Orp1是用于检测H2O2的探针，而roGFP1/2、rxYFP和roGFP2-Grx1是用于检测GSH/GSSG氧化还原状态的探针。在使用HyPer和rxYFP的实验中，应仔细控制可能的pH变化。
基因编码氧化还原探针是唯一能够实时监测活细胞和组织中活性氧和硫醇氧化还原状态的工具。我们相信，在不久的将来，基于荧光蛋白的氧化还原探针的种类将扩展到光谱的红色和远红部分，以及其他重要的活性物质，如NO、O2和超氧化物。本文是名为“研究活性氧的当前方法——利弊与膜蛋白生物物理学”的特刊的一部分。客座编辑：克里斯汀·温特伯恩。
