Fluorescence assay of the interaction between hemoglobin and the cytoplasmic domain of erythrocyte membrane band 3.

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 of band 3 (cdb3) in an oxygen dependent manner, with deoxyHb exhibiting significantly greater affinity for cdb3 than oxyHb, the deoxyHb-cdb3 interaction has been hypothesized to constitute the molecular switch for all O2-controlled erythrocyte processes. In this study, we describe a rapid and accurate method for quantitating the interaction of deoxyHb binding to cdb3. For this purpose, enhanced green fluorescent protein (eGFP) is fused to the COOH-terminus of cdb3, and the binding of Hb to the NH2-terminus of cdb3-eGFP is quantitated by Hb-mediated quenching of cdb3-eGFP fluorescence. As expected, the intensity of cdb3-eGFP fluorescence decreases only slightly following addition of oxyHb. However, upon deoxygenation of the same Hb-cdb3 solution, the fluorescence decreases dramatically (i.e. confirming that deoxyHb exhibits much greater affinity for cdb3 than oxyHb). Using this fluorescence quenching method, we not only confirm previously established characteristics of the Hb-cdb3 interaction, but also establish an assay that can be exploited to screen for inhibitors of the sickle Hb-cdb3 interaction that accelerates sickle Hb polymerization.