Himanen J P, Mirza U A, Chait B T, Bookchin R M, Manning J M
The Rockefeller University, New York, New York 10021, USA.
J Biol Chem. 1996 Oct 11;271(41):25152-6. doi: 10.1074/jbc.271.41.25152.
As part of a comprehensive effort to map the most important regions of sickle hemoglobin that are involved in polymerization, we have determined whether two sites previously shown to be involved, Leu-88(beta) and Lys-95(beta), had additive effects when substituted. The former site is part of the hydrophobic pocket that binds Val-6(beta), the natural mutation of HbS, and the latter site is a prominent part of the hemoglobin exterior. A sickle hemoglobin triple mutant with three amino acid substitutions on the beta-chain, E6V/L88A/K95I, has been expressed in yeast and characterized extensively. Its oxygen binding curve, cooperativity, response to allosteric effectors, and the alkaline Bohr effect showed that it was completely functional. The polymer solubility of the deoxy triple mutant, measured by a new micromethod requiring reduced amounts of hemoglobin, was identical to that of the E6V(beta)/K95I(beta) mutant, i.e. when the K95I(beta) substitution was present on the same tetramer together with the naturally occurring E6V(beta) substitution, the L88A(beta) replacement had no additive effect on polymer inhibition. The results suggest that Lys-95(beta) on the surface of the tetramer and its complementary binding region on the adjoining tetramer are potential targets for the design of an effective antisickling agent.
作为绘制参与聚合反应的镰状血红蛋白最重要区域综合工作的一部分，我们已确定先前显示参与其中的两个位点，即β链上的Leu-88和Lys-95，在被取代时是否具有累加效应。前一个位点是结合β链第6位缬氨酸（HbS的天然突变位点）的疏水口袋的一部分，而后一个位点是血红蛋白表面的一个突出部分。一种在β链上有三个氨基酸取代的镰状血红蛋白三重突变体E6V/L88A/K95I已在酵母中表达并进行了广泛表征。其氧结合曲线、协同性、对别构效应剂的反应以及碱性玻尔效应表明它完全具有功能。通过一种需要较少血红蛋白量的新微方法测量的脱氧三重突变体的聚合物溶解度，与E6V(β)/K95I(β)突变体相同，即当K95I(β)取代与天然存在的E6V(β)取代存在于同一四聚体上时，L88A(β)替换对聚合物抑制没有累加效应。结果表明，四聚体表面的Lys-95(β)及其相邻四聚体上的互补结合区域是设计有效抗镰变剂的潜在靶点。
