Interspecies hybrid HbS: Complete neutralization of Val6(beta)-dependent polymerization of human beta-chain by pig alpha-chains

Interspecies hybrid HbS (alpha(2)(P)beta(2)(S)), has been assembled in vitr o from pig alpha-globin and human beta(S)-chain. The alpha(2)(P)beta(2)(S) retains normal tetrameric structure (alpha(2)beta(2)) of human Hb and an O- 2 affinity comparable to that of HbS in 50 mM Hepes buffer; but, its O-2 af finity is slightly higher than that of HbS in the presence of allosteric ef fecters (chloride, DPG and phosphate). The H-1-NMR spectroscopy detected di stinct differences between the heme environments and alpha(1)beta(1) interf aces of pig Hb and HbS, while their alpha(1)beta(2) interfaces appear very similar. The interspecies hybrid alpha(2)(H)beta(2)(P) resembles pig Hb; th e pig beta-chain dictated the conformation of the heme environment of the h uman alpha-subunit, and to the alpha(1)beta(1) interfaces of the hybrid. In the alpha(2)(P)beta(2)(S) hybrid, beta(S)-chain dictated the conformation of human heme environment to the pig alpha-chain in the hybrid; but the con formation of alpha(1)beta(1) interface of this hybrid is close to, but not identical to that of HbS. On the other hand, the alpha(1)beta(2) interface conformation is identical to that of HbS. More important, the alpha(2)(P)be ta(2)(S) does not polymerize when deoxygenated; pig alpha-chain completely neutralizes the beta(S)-chain dependent polymerization. The polymerization inhibitory propensity of pig alpha-chain is higher when it is present in th e cis alpha(P)beta(S) dimer relative to that in a trans alpha(P)beta(A) dim er. The semisynthetically generated chimeric pig-human and human-pig alpha- chains by exchanging the alpha(1-30) segments of human and pig alpha-chains have established that the sequence differences of pig alpha(31-141) segmen t can also completely neutralize the polymerization. Comparison of the elec trostatic potential energy landscape of the alpha-chain surfaces of HbS and alpha(2)(P)beta(2)(S) suggests that the differences in electrostatic poten tial energy surfaces on the alpha-chain of alpha(2)(P)beta(2)(S) relative t o that in HbS, particularly the ones involving CD region, E-helix and EF-co rner of pig alpha-chain are responsible for the polymerization neutralizati on activity. The pig and human-pig chimeric alpha-chains can serve as bluep rints for the design of a new generation of variants of alpha-chain(s) suit able for the gene therapy of sickle cell disease. (C) 2000 Academic Press.