ROLE OF BETA-87 THR IN THE BETA-6 VAL ACCEPTOR SITE DURING DEOXY HB-SPOLYMERIZATION

Three new Hb S variants containing beta 87 Leu, Trp, or Asp instead of Thr were expressed in yeast in order to further define the role of th e beta 87 position in stability and polymerization of deoxy Hb S. Prev ious studies showed that hydrophobicity at beta 85 Phe and beta 88 Leu is critical for stabilization of hemoglobin. Results with the three H b S beta 87 variants, however, showed minimal differences in stability , suggesting that beta 87 amino acid hydrophobicity is not critical fo r stabilization of hemoglobin. Polymerization properties of the varian ts in the deoxy form, however, were affected by the beta 87 amino acid . Polymerization of Hb S beta 87 Thr --> Leu and Hb S beta 87 Thr --> Trp was preceded by a delay time like Hb S, while Hb S beta 87 Thr --> Asp did not show a delay time. In addition, changes in time required for half polymer formation (Tin) as a function of hemoglobin concentra tion for Hb S beta 87 Thr --> Asp were similar to that for beta 87 Thr --> Gln. Hb S beta 87 Thr --> Leu polymerized at a lower hemoglobin c oncentration than Hb S while beta 87 Thr --> Trp and Hb S beta 87 Thr --> Asp required much higher hemoglobin concentrations for polymer for mation. Critical concentration required for deoxy Hb S beta 87 Thr --> Asp polymerization was 6- and 2.3-fold greater than that for Hb S bet a 85 Phe --> Glu and Hb S beta 88 Leu --> Glu, respectively. These res ults suggest that even though beta 87 Thr is not a direct interaction site for beta 6 Val in deoxy Hb S polymers, it does play a critical ro le in formation of the hydrophobic acceptor pocket which then promotes protein-protein interactions facilitating formation of stable nuclei and polymers of deoxy Hb S.