Morita Yoshitsugu, Saito Asuka, Yamaguchi Jun, Komatsu Teruyuki
Department of Applied Chemistry, Faculty of Science and Engineering, Chuo University, 1-13-27 Kasuga, Bunkyo-ku Tokyo 112-8551 Japan
RSC Chem Biol. 2020 Jul 13;1(3):128-136. doi: 10.1039/d0cb00056f. eCollection 2020 Aug 1.
The allosteric O release of haemoglobin (Hb) allows for efficient O delivery from the lungs to the tissues. However, allostery is weakened in Hb-based O carriers because the chemical modifications of the Lys- and Cys-β93 residues prevent the quaternary transition of Hb. In this paper, we describe the synthesis and O binding properties of a recombinant Hb [rHb(βK120C)]-albumin heterotrimer that maintains sufficient Hb allostery. The rHb(βK120C) core, with two additional cysteine residues at the symmetrical positions on its protein surface, was expressed using yeast cells. The mutations did not influence either the O binding characteristics or the quaternary transition of Hb. Maleimide-activated human serum albumins (HSAs) were coupled with rHb(βK120C) at the two Cys-β120 positions, yielding the rHb(βK120C)-HSA trimer, in which the Cys-β93 residues were unreacted. Molecular dynamics simulation demonstrated that the HSA moiety does not interact with the amino acid residues around the haem pockets and the αβ surfaces of the rHb(βK120C) core, the alteration of which retards Hb allostery. Circular dichroism spectroscopy demonstrated that the quaternary transition between the relaxed (R) state and the tense (T) state of the Hb core occurred upon both the association and dissociation of O. In phosphate-buffered saline solution (pH 7.4) at 37 °C, the rHb(βK120C)-HSA trimer exhibited a sigmoidal O equilibrium curve with the O affinity and cooperativity identical to those of native Hb ( = 12 Torr, = 2.4). Moreover, we observed an equal Bohr effect and 2,3-diphosphoglycerate response in the rHb(βK120C)-HSA trimer compared with naked Hb.
血红蛋白（Hb）的变构性氧释放使得氧气能够从肺部高效输送至组织。然而，基于血红蛋白的氧载体中的变构作用会被削弱，因为赖氨酸和半胱氨酸β93残基的化学修饰会阻止血红蛋白的四级结构转变。在本文中，我们描述了一种重组血红蛋白[rHb(βK120C)]-白蛋白异源三聚体的合成及其氧结合特性，该三聚体保持了足够的血红蛋白变构性。rHb(βK120C)核心在其蛋白质表面的对称位置有两个额外的半胱氨酸残基，通过酵母细胞进行表达。这些突变既不影响血红蛋白的氧结合特性，也不影响其四级结构转变。马来酰亚胺活化的人血清白蛋白（HSA）在两个半胱氨酸β120位置与rHb(βK120C)偶联，生成rHb(βK120C)-HSA三聚体，其中半胱氨酸β93残基未发生反应。分子动力学模拟表明，HSA部分不与rHb(βK120C)核心血红素口袋周围以及αβ表面的氨基酸残基相互作用，而这些氨基酸残基的改变会阻碍血红蛋白的变构作用。圆二色光谱表明，在氧气结合和解离时，血红蛋白核心的松弛（R）态和紧张（T）态之间会发生四级结构转变。在37℃的磷酸盐缓冲盐水溶液（pH 7.4）中，rHb(βK120C)-HSA三聚体呈现出S形的氧平衡曲线，其氧亲和力和协同性与天然血红蛋白相同（P50 = 12 Torr，n = 2.4）。此外，与裸露的血红蛋白相比，我们在rHb(βK120C)-HSA三聚体中观察到了相同的玻尔效应和2,3-二磷酸甘油酸反应。
