Inhibition of hemoglobin S polymerization in vitro by a novel 15-mer EF-helix, β73 histidine-containing peptide

Our mutational studies on Hb S showed that the Hb S beta 73His variant (beta 6Val and beta 73His) promoted polymerization, while Hb S beta 73Leu (beta 6Val and beta 73Leu) inhibited polymerization. On the basis of these results, we speculated that EF-helix peptides containing, beta 73His interact with Hb S and compete with Hb S, resulting in inhibition of Hb S polymerization. We, therefore, studied inhibitory effects of 15-, 11-, 7-, and 3- mer EF-helix peptides containing, beta 73His on Hb S polymerization. The delay time prior to Hb S 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 that the fiber elongation rate for Hb S polymers decreased with increasing concentration of the 15-mer His peptide. In contrast, the same 15-mer peptide containing beta 73Leu instead of His and peptides shorter than 11 amino acids containing beta 73His including His alone showed little effect on the kinetics of polymerization and elongation of polymers. Analysis by protein-chip arrays showed that only the 15-mer beta 73His peptide interacted with Hb S. CD spectra of the 15-mer beta 73His peptide did not show a specific helical structure; however, computer docking analysis suggested a lower energy for interaction of Hb S with the 15-mer beta 73His peptide compared to peptides containing other amino acids at this position. These results suggest that the 15-mer beta 73His peptide interacts with Hb S via the, beta 4Thr in the, beta(S)-globin chain in Hb S. This interaction may influence hydrogen bond interaction between beta 73Asp and beta 4Thr in Hb S polymers and interfere in hydrophobic interactions of beta 6Val, leading to inhibition of Hb S polymerization.