Cook Kristina M
Charles Perkins Centre, University of Sydney, Sydney, NSW, Australia.
Methods Mol Biol. 2019;1967:65-86. doi: 10.1007/978-1-4939-9187-7_5.
The redox potential of a protein disulphide bond is one of the most important factors for determining the role of a disulphide bond. Disulphide bonds can have a stabilizing role for the structure of a protein or they can play a functional role which can regulate protein bioactivity. Determining the redox potential of disulphides can help distinguish the functional from the structural disulphide bonds. In this chapter, two methods for determining the redox potential of a protein disulphide bond are described. The first method uses maleimide-biotin labeling of free cysteine thiols and western blot densitometry to determine the fraction of reduced disulphide bond under various redox-buffering conditions. The second method uses differential cysteine labeling and tandem mass spectrometry to determine the redox potential.
蛋白质二硫键的氧化还原电位是决定二硫键作用的最重要因素之一。二硫键对蛋白质结构可起到稳定作用，或者发挥调节蛋白质生物活性的功能作用。测定二硫键的氧化还原电位有助于区分功能性二硫键和结构性二硫键。在本章中，描述了两种测定蛋白质二硫键氧化还原电位的方法。第一种方法利用马来酰亚胺 - 生物素标记游离半胱氨酸硫醇，并通过蛋白质免疫印迹光密度测定法来确定在各种氧化还原缓冲条件下二硫键的还原分数。第二种方法利用差异半胱氨酸标记和串联质谱来测定氧化还原电位。
