HbS-Savaria: the anti-polymerization effect of a single mutation in human alpha-chains

Recombinant alpha-Savaria globin (alpha(S49R)) was assembled with beta(S) chains by the alloplex intermediate pathway to generate tetrameric rHbS-Sarvaria (alpha (2) (S49R) beta (2) (E6V) ) that exhibited normal O(2) affinity and co-operatively at pH 7.4. Allosteric effectors, 2,3-DPG, L35, and NaCl increased O(2) affinity by 15%. Bohr effects were similar for rHbS-Savaria and HbS (0.38 +/- 0.025 vs. 0.46 +/- 0.03, respectively). The C(SAT) of HbS increased from 16.7 +/- 0.8 to 27.0 +/- 1.0 g/dL. Co-polymerization demonstrated inhibition predominantly by the Cis-dimer. Molecular modeling indicated that the positive charge at alpha-49 generated a strong anion-binding site and reduced flexibility of the CD-region by restricting movement in the E and F helices. The molecular distance between Arg-49 and Asn-78 in the neighboring double strand decreased, and electrostatic repulsion between the inter-double strands increased, resulting in inhibition of polymerization. The Savaria mutation may be useful for the design of super-inhibitory alpha-chains and gene therapy of sickle cell anemia.