Fluorescence assay of the interaction between hemoglobin and the cytoplasmic domain of erythrocyte membrane band 3.
作者： Martiana F. Sega , Haiyan Chu , John A. Christian , Philip S. Low
DOI: 10.1016/J.BCMD.2015.07.004
关键词: Oxygen tension 、 Binding site 、 Hemoglobin 、 Quenching (fluorescence) 、 Band 3 、 Green fluorescent protein 、 Chemistry 、 Fluorescence 、 Plasma protein binding 、 Biochemistry
摘要: Oxygen tension has emerged as a potent regulator of multiple erythrocyte properties, including glucose metabolism, cell volume, ATP release, and cytoskeletal organization. Because hemoglobin (Hb)1 binds to the cytoplasmic domain band 3 (cdb3) in an oxygen dependent manner, with deoxyHb exhibiting significantly greater affinity for cdb3 than oxyHb, deoxyHb-cdb3 interaction been hypothesized constitute molecular switch all O2-controlled processes. In this study, we describe rapid accurate method quantitating binding cdb3. For purpose, enhanced green fluorescent protein (eGFP) is fused COOH-terminus cdb3, Hb NH2-terminus cdb3-eGFP quantitated by Hb-mediated quenching fluorescence. As expected, intensity fluorescence decreases only slightly following addition oxyHb. However, upon deoxygenation same Hb-cdb3 solution, dramatically (i.e. confirming that exhibits much oxyHb). Using method, not confirm previously established characteristics interaction, but also establish assay can be exploited screen inhibitors sickle accelerates polymerization.
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