Analysis of fluorophore-labeled human cytochrome b5

Cytochrome b5 (b5) is a small accessory protein involved in several reactions. The interaction of b5 with its partner proteins, e.g., cytochrome P450 enzymes, has received much attention and has been the subject of numerous studies. In order to obtain further insights into protein-protein interactions of b5, we modified it with a green fluorophore, Alexa 488, which has a maleimide linker. The labeling was applied to both WT (without Cys) and the T70C mutant. This study revealed labeled sites in WT b5 and confirmed the Cys-selective labeling in the T70C mutant.细胞色素b5（Cytochrome b5，简称b5）是一类参与多种生化反应的小型辅助蛋白。b5与其伴侣蛋白（如细胞色素P450酶（cytochrome P450 enzymes））之间的相互作用已受到广泛关注，且成为诸多研究的核心主题。为深入探究b5的蛋白质-蛋白质相互作用机制，我们采用带有马来酰亚胺连接臂的绿色荧光基团Alexa 488对其进行修饰。本次标记实验同时覆盖野生型（WT，不含半胱氨酸残基）与T70C突变体。本研究鉴定出野生型b5的标记位点，并验证了T70C突变体中半胱氨酸选择性标记的有效性。