POLE OF HYDROPHOBICITY OF PHENYLALANINE BETA-85 AND LEUCINE BETA-88 IN THE ACCEPTOR POCKET FOR VALINE BETA-6 DURING HEMOGLOBIN-S POLYMERIZATION

Characterization of the hydrophobic EF acceptor pocket involving Phe-b eta 85 and Leu-beta 88 as well as the Val-beta 6 donor site is critica l for understanding the polymerization of deoxy Hb S, Glu substitution s at beta 85 or beta 88 in Hb S were made and expressed in yeast in an effort to evaluate the role of hydrophobicity in the acceptor pocket during polymerization of Hb S, Both substitutions result in decreased tetramer stability, increases in oxygen affinity, and inhibition in po lymerization compared with Kb S, Critical concentrations for polymeriz ation of Hb S-F beta 85E and Hb S-L beta 88E were 2.4- and 7-fold high er, respectively, than that of Hb S, while the value for Hb S-L beta 8 8E was intermediate between those previously reported for Hb S-L beta 88A and Hb S-L beta 88F (Adachi, K,, Konitzer, P., Paulraj, C, G,, and Surrey, S, (1994) J. Biol, Chem. 269, 17477-11480), Kinetics of polym erization of Glu-beta 85 and Glu-beta 88 deoxy Hb S tetramers were bip hasic at lower hemoglobin concentrations like deoxy Hb S-L beta 88A: s uggesting formation of two types of polymers during polymerization, Th e time required to form half the total amount of polymer (t(1/2)) for deoxy Hb S-F beta 85E was 10-fold shorter than that for deoxy Hb S-L b eta 88E, In addition, t(1/2) for deoxy Hb S-F beta 85E was 2.5-fold sh orter, while that for Hb S-L beta 88E was 4-fold longer than deoxy Hb S-L beta 88A,t equivalent concentrations, These results suggest that h ydrophobicity of the amino acid at beta 88 appears more critical than that at beta 85 in the acceptor pocket for Val-beta 6, Furthermore, st ereospecificity of the acceptor pocket in addition to hydrophobicity o f beta 88 are critical for stable hydrophobic interactions with Val-be ta 6 during deoxy Pb S polymerization.