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

Abstract

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.

Myosin IIA interacts with the spectrin-actin membrane skeleton to control red blood cell membrane curvature and deformability

“…To characterize the distribution of NMIIA in RBCs, we immunostained fixed, biconcave RBCs for the NMIIA motor domain and stained F-actin with phalloidin. The low abundance of NMIIA in RBCs [∼6,200 molecules per cell (5,17)], the high concentration of fluorescence-quenching hemoglobin (30), and the small sizes of RBCs [8 μm in diameter × 2 μm thick (31)] make imaging RBC NMIIA challenging. Therefore, we imaged RBCs using sensitive, superresolution AiryScan confocal fluorescence microscopy (32).…”

On the Biophysical Investigation of Sulfamethazine‐Hemoglobin Binding and the Resulting Adverse Effects of Antibiotics

“…where F(λ) is the emission intensity of the donor at wavelength λ and ϵ(λ) is the absorption coefficient of the acceptor at λ. In this case, k 2 =2/3, n=1.36 and φ=0.062 for Hb [37,38] . The parameters obtained using equation 5–6, [39,40] were, J=3.83 10 −15 cm 3 .…”

“…In this case, k 2 = 2/3, n = 1.36 and ϕ = 0.062 for Hb. [37,38] The parameters obtained using equation 5-6, [39,40] were, J = 3.83 �10 À 15 cm 3 . L mol À 1 , R 0 = 2.78 nm, E = 0.1424 and r = 3.75 nm.…”

Cation Homeostasis in Red Cells From Patients With Sickle Cell Disease Heterologous for HbS and HbC (HbSC Genotype)

“…Physiological regulation of KCC is also complex ( Gibson and Ellory, 2003 ), with evidence for cascades of protein kinases and phosphatases ( Cossins et al, 1994 ), acting on both serine–threonine and tyrosine residues, impacting on transporter activity. This enzymatic regulation is probably key to the differences in response to O 2 ( Gibson et al, 1994 , Merciris et al, 2001 ), perhaps interacting with Hb at the level of the red cell membrane ( Sega et al, 2012 , Sega et al, 2015 ). This aspect, however, remains to be fully elucidated.…”