Our mutational studies on HbS showed that the HbS β73His variant (β6Val and β73His) promoted polymerization, while HbS β73Leu (β6Val and β73Leu) inhibited polymerization. Based on these results, we speculated that EF-helix peptides containing β73His interact with β4Thr in HbS and compete with HbS, resulting in inhibition of HbS polymerization. We, therefore, studied inhibitory effects of 15-, 11-, 7-and 3-mer EF-helix peptides containing β73His on HbS polymerization. The delay time prior to HbS polymerization increased only in the presence of the 15-mer His peptide; the higher the amount, the longer the delay time. DIC image analysis also showed fiber elongation rate for HbS polymers decreased with increasing concentration of the 15-mer His peptide. In contrast, the same 15-mer-peptide containing β73Leu instead of His and peptides shorter than 11 amino acids containing β73His including His alone showed little effect on kinetics of polymerization and elongation of polymers. Analysis by protein-chip arrays showed that only the 15-mer β73His peptide interacted with HbS. CD spectra of the 15-mer β73His peptide did not show a specific helical structure, however, computer docking analysis suggested a lower energy for interaction of HbS with the 15-mer β73His peptide compared to peptides containing other amino acids at this position. These results suggest that the 15-mer β73His peptide interacts with HbS via the β4Thr in the β S -globin chain in HbS. This interaction may influence hydrogen bond interaction between β73Asp and β4Thr in HbS polymers and interfere in hydrophobic interactions of β6 Val leading to inhibition of HbS polymerization.Hb S is a naturally occurring mutation of human tetrameric hemoglobin in which the β subunits have a hydrophobic Val in place of a negatively charged Glu at the β6 position. The consequence of this mutation is that solubility of Hb S decreases when oxy Hb S loses oxygen. When deoxy Hb S becomes oversaturated deoxy Hb S assembles into long, multi-stranded fibers under physiological conditions (1,2). Fiber formation is characterized by a delay time prior to polymerization, which is explained by homogeneous and heterogeneous nucleation. During oversaturated Hb S conditions deoxy Hb S monomers form very small polymers by homogeneous nucleation, and these polymers grow by the end addition of hemoglobin molecules in solution. The surface of these growing fibers also can serve as heterogeneous nucleation sites for further growth of additional polymers (1,3). The polymer then assembles into 14-stranded fibers, which finally form a viscous gel. Intracellular polymers or fibers cause reduction in red blood cell deformability (sickling), leading to obstruction of flow in the microcirculation, thus creating vasooclusion and a wide array of physiological problems including episodes of painful crises (1,4).Computational refinements of x-ray-determined crystal structures clarified the details of many of the axial and lateral contacts in Hb S polymers (5). These results and properties of th...
Sickle hemoglobin polymerization involves not only ß6 Val in a largely hydrophobic acceptor pocket but other contact sites. We recently found that the ß73 His Hb S variant (ß6 Val and ß73 His) promoted polymerization compared to deoxy Hb S, while ß73 Leu Hb S (ß6 Val and ß73 Leu) inhibited polymerization like deoxy Hb C-Harlem (ß6 Val and ß73 Asn). In fact, ß73 Asp in Hb S makes a hydrogen bond with ß4 Thr in deoxy Hb S polymers, and this is a unique position to promote or inhibit polymerization by amino acid change [Adachi et al., Biochemistry (2003), 42, 4476]. Kinetics of polymerization, solubility and minimum concentration required for polymerization of the ß73 Hb S variants were affected by ß73 amino acid (inhibition of polymerization: His < <Asp <<Asn < Leu). Inhibition of Hb S ß73 Leu polymerization compared to Hb S may be caused by weakening of the hydrogen bond interaction between the hydroxyl group of ß4 Thr and the ß73 amino acid like Hb C-Harlem. Furthermore, kinetics of polymerization of 1:1 Hb S/Hb A ß73 His mixtures were enhanced compared to AS mixtures, while Hb S/Hb A ß73 Leu mixtures showed inhibitory effects similar to FS mixtures. These results suggest that the Hb A ß73His variant promotes Hb S polymerization almost as efficiently as Hb S ß73 His, and that the ß73 His in Hb A and Hb S variants strengthens the hydrogen bond with ß4 Thr, which helps facilitate formation of domains and 14-stranded fibers. Based on these results, we chemically synthesized a 15-mer EF helix peptide containing ß73 His (Lys-Lys-Val-Leu-Gly-Ala-Phe-Ser-His-Gly-Leu-Ala-His-Leu-Asp) and evaluated effects of this peptide on Hb S polymerization. DIC analysis in 1.0 M phosphate buffer showed peptide-induced inhibition of fiber elongation rate for deoxy-Hb S which increased linearly with increasing amounts of peptide (e.g., a 5-fold molar excess of peptide resulted in a 6-fold decrease in fiber elongation rate). Solubility of deoxy Hb S increased linearly with increasing peptide (e.g., a 5-fold molar excess of peptide increased solubility by 1.2-fold). The delay time prior to polymerization of deoxy Hb S in a high phosphate buffer also was increased significantly in the presence of this peptide. In contrast, the same 15-mer peptide containing ß73 Leu instead of His showed no effect on solubility or kinetics of polymerization, suggesting that the ß73 His peptide specifically affects the A-helix of Hb S containing ß4 Thr just like the ß73 His Hb S variant. This and possibly other peptides interrupting the ß73-ß4 interaction which result in inhibition of deoxy Hb S polymerization may be good candidates for inhibitors of Hb S polymerization.
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