Abstract
Hemoglobin function can be modulated by the red cell membrane but some mechanistic details are incomplete. For example, the 43-kDa chymotryptic fragment of the cytoplasmic portion of red cell membrane Band 3 protein and its corresponding N-terminal 11-residue synthetic peptide lower the oxygen affinity of hemoglobin but effects on cooperativity are unclear. Using highly purified preparations, we also find a lowered Hill coefficient (n values <2) at subequivalent ratios of Band 3 fragment or of synthetic peptide to Hb, resulting in an oxygen affinity that is moderately decreased and a partially hyperbolic shape for the O2 binding curve. Both normal HbA and sickle HbS display this property. Thus, the determinant responsible for the Hb cooperativity decreases by the 43-kDa fragment resides within its first 11 N-terminal residues. This effect is observed in the absence of chloride and is reversed by its addition. As effector to Hb ratios approach equivalence or with saturating chloride normal cooperativity is restored, and oxygen affinity is further lowered because the shape of the oxygen binding curve becomes completely sigmoidal. The relative efficiencies of 2,3-diphosphoglycerate (DPG), the 43-kDa Band 3 fragment, and the 11-residue synthetic peptide in lowering cooperativity are very similar. The findings are explained based on the stereochemical mechanism of cooperativity because of two populations of T-state hemoglobin tetramers, one with bound effector and the other with free (Perutz, M. F. (1989) Q. Rev. Biophys. 22, 139-237). As a result of this property, hemoglobin at the membrane inner surface in contact with the N-terminal region of Band 3 could preferentially bind O2 at low oxygen tension and then release it upon saturation with 2,3-diphosphoglycerate in the interior of the red cell. Membrane modulation of hemoglobin oxygen affinity has particularly interesting implications for the polymerization of hemoglobin S in the sickle red cell.
Highlights
Hemoglobin function can be modulated by the red cell membrane but some mechanistic details are incomplete
A functional consequence of this interaction was to lower the oxygen affinity of hemoglobin, but no effect on cooperativity was reported in the presence of the peptide a reduced n value was observed in the presence of the intact Band 3 chymotryptic fragment
The allosteric regulatory molecules DPG,1 chloride, and protons facilitate O2 release by binding preferentially to the the oxy (R-state) and deoxy (T-state), e.g. DPG binds very efficiently to a single site on deoxy-Hb (15) whereas chloride binds to several sites, some of which are shared with DPG but others are not (16 –20)
Summary
In this report we present evidence for a similar lowered cooperativity for HbA and HbS in the presence of subequivalent amounts, relative to Hb, of either the purified 43-kDa chymotryptic cytoplasmic fragment of membrane Band 3 or a synthetic 11-residue peptide corresponding to its N terminus sequence. Because the decreased cooperativity occurs with a moderate decrease in oxygen affinity in contrast to a greater decrease in the presence of saturating DPG, we consider the possibility that the cooperativity differences for the hemoglobin in direct contact with Band 3 in the red cell membrane and the hemoglobin that is intracellular and saturated with DPG could have a role in O2 transport because of a gradient of Hb tetramers with varying degrees of O2 saturation In this context, Band 3 cytoplasmic fragment might be considered an allosteric regulator of hemoglobin function
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