POLYMERIZATION OF RECOMBINANT HB S-KEMPSEY (DEOXY-R STATE) AND HB S-KANSAS (OXY-T STATE)

In order to investigate the role of the R (relaxed) to T (tense) structural transition in facilitating polymerization of deoxy-Hb S, we have engineered and expressed two Hb S variants which destabilize either T state (Hb S-Kempsey, alpha(2) beta(2)(Val-6, Asn-99)). R State structures (Hb S-Kansas, alpha(2) beta(2)(Val-6, Thr-102)), Polymerization of deoxy-Hb S-Kempsey, which shows high oxygen affinity and increased dimer dissociation, required about 2- and Ci fold higher hemoglobin concentrations than deoxy-fm S for polymerization in low and high phosphate concentrations, and its kinetic pattern of polymerization was biphasic, In contrast, oxy- or CO Hb S-Kansas, which shows low oxygen affinity and increased dimer dissociation, polymerized at a slightly higher critical concentration than that required for polymerization of deoxy-Hb S in both low and high phosphate buffers, Polymerization of oxy- and CO Kb S Kansas was linear and showed no delay time, which is similar to oversaturated oxy- or CO Hb S, These results suggest that nuclei formation, which occurs during the delay time prior to deoxy-Hb S polymerization, does not occur in T state oxy-Hb S-Kansas, even though the critical concentration for polymerization of T state oxy-Hb S-Kansas is similar to that of T state deoxy Hb S.