Adachi K, Reddy L R, Surrey S
Division of Hematology, University of Pennsylvania School of Medicine, Children's Hospital of Philadelphia, Pennsylvania 19104.
J Biol Chem. 1994 Dec 16;269(50):31563-6.
Characterization of the hydrophobic EF acceptor pocket involving Phe-beta 85 and Leu-beta 88 as well as the Val-beta 6 donor site is critical for understanding the polymerization of deoxy Hb S. Glu substitutions 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 polymerization compared with Hb S. Critical concentrations for polymerization of Hb SF beta 85E and Hb SL beta 88E were 2.4- and 7-fold higher, respectively, than that of Hb S, while the value for Hb SL beta 88E was intermediate between those previously reported for Hb SL beta 88A and Hb SL beta 88F (Adachi, K., Konitzer, P., Paulraj, C. G., and Surrey, S. (1994) J. Biol. Chem. 269, 17477-17480). Kinetics of polymerization of Glu-beta 85 and Glu-beta 88 deoxy Hb S tetramers were biphasic at lower hemoglobin concentrations like deoxy Hb SL beta 88A, suggesting formation of two types of polymers during polymerization. The time required to form half the total amount of polymer (t1/2) for deoxy Hb SF beta 85E was 10-fold shorter than that for deoxy Hb SL beta 88E. In addition, t1/2 for deoxy Hb SF beta 85E was 2.5-fold shorter, while that for Hb SL beta 88E was 4-fold longer than deoxy Hb SL beta 88A at equivalent concentrations. These results suggest that hydrophobicity of the amino acid at beta 88 appears more critical than that at beta 85 in the acceptor pocket for Val-beta 6. Furthermore, stereospecificity of the acceptor pocket in addition to hydrophobicity of beta 88 are critical for stable hydrophobic interactions with Val-beta 6 during deoxy Hb S polymerization.
对涉及β85位苯丙氨酸、β88位亮氨酸的疏水EF受体口袋以及β6位缬氨酸供体位点的表征，对于理解脱氧血红蛋白S的聚合至关重要。在血红蛋白S中β85或β88位进行了谷氨酸取代，并在酵母中表达，以评估在血红蛋白S聚合过程中受体口袋疏水性的作用。与血红蛋白S相比，这两种取代都导致四聚体稳定性降低、氧亲和力增加以及聚合受到抑制。血红蛋白SFβ85E和血红蛋白SLβ88E聚合的临界浓度分别比血红蛋白S高2.4倍和7倍，而血红蛋白SLβ88E的值介于先前报道的血红蛋白SLβ88A和血红蛋白SLβ88F之间（足立健、科尼策、保罗拉杰、萨里，（1994年）《生物化学杂志》269卷，第17477 - 17480页）。谷氨酸 - β85和谷氨酸 - β88脱氧血红蛋白S四聚体的聚合动力学在较低血红蛋白浓度下呈双相，类似于脱氧血红蛋白SLβ88A，表明在聚合过程中形成了两种类型的聚合物。脱氧血红蛋白SFβ85E形成聚合物总量一半所需的时间（t1/2）比脱氧血红蛋白SLβ88E短10倍。此外，在等效浓度下，脱氧血红蛋白SFβ85E的t1/2短2.5倍，而血红蛋白SLβ88E的t1/2比脱氧血红蛋白SLβ88A长4倍。这些结果表明，在β6位缬氨酸的受体口袋中，β88位氨基酸的疏水性似乎比β85位的更关键。此外，除了β88位的疏水性外，受体口袋的立体特异性对于脱氧血红蛋白S聚合过程中与β6位缬氨酸的稳定疏水相互作用至关重要。
