Wang Hao, Schoebel Stefan, Schmitz Florian, Dong Hansong, Hedfalk Kristina
Department of Plant Pathology, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, China.
Department and Chemistry and Molecular Biology, Gothenburg University, Box 462, 405 30 Göteborg, Sweden.
MethodsX. 2020 Feb 20;7:100816. doi: 10.1016/j.mex.2020.100816. eCollection 2020.
Hydrogen peroxide (HO) is an important signal molecule produced in animal and plant cells. The balance of HO between the intra- and extracellular space is regulated by integral membrane proteins, which thereby modulate signaling. Several methods have been established to analyze aquaporin mediated transport of HO in whole cells with the intrinsic limitation that the amount of protein responsible for a certain activity cannot be standardized. As a consequence, the quantification of the transport and specific activity is difficult to extract making it problematic to compare isoforms and mutated variants of one specific target. Moreover, in cell-based assays, the expression of the target protein may alter the physiological processes of the host cell providing a complication and the risk of misleading results. To improve the measurements of protein based HO transport, we have developed an assay allowing quantitative measurements.•Using purified aquaporin reconstituted in proteoliposomes, transport of HO can be accurately measured.•Inside the liposomes, HO catalyzes the reaction between Amplex Red and horseradish peroxidase (HRP) giving rise to the fluorescent product resorufin.•Analysing pure protein provides direct biochemical evidence of a specific transport excluding putative cellular background.
过氧化氢（H₂O₂）是动植物细胞中产生的一种重要信号分子。细胞内和细胞外空间之间的H₂O₂平衡由整合膜蛋白调节，从而调节信号传导。已经建立了几种方法来分析水通道蛋白介导的H₂O₂在全细胞中的转运，但存在固有限制，即负责特定活性的蛋白量无法标准化。因此，转运和比活性的定量难以提取，使得比较一个特定靶点的同工型和突变变体存在问题。此外，在基于细胞的测定中，靶蛋白的表达可能会改变宿主细胞的生理过程，带来复杂性和产生误导性结果的风险。为了改进基于蛋白的H₂O₂转运测量，我们开发了一种允许进行定量测量的测定方法。•使用重组在蛋白脂质体中的纯化水通道蛋白，可以准确测量H₂O₂的转运。•在脂质体内，H₂O₂催化Amplex Red与辣根过氧化物酶（HRP）之间的反应，产生荧光产物试卤灵。•分析纯蛋白提供了特定转运的直接生化证据，排除了假定的细胞背景。
