Inhibition of Hb S Polymerization In Vitro by a Novel EF Helix ß73 His Containing Peptide.

Abstract

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.