新型重组血红蛋白，rHb（β N108Q），具有低氧亲和力、高协同性以及抗自氧化稳定性。

Novel recombinant hemoglobin, rHb (beta N108Q), with low oxygen affinity, high cooperativity, and stability against autoxidation.

作者信息

Tsai C H, Fang T Y, Ho N T, Ho C

机构信息

Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, USA.

出版信息

Biochemistry. 2000 Nov 14;39(45):13719-29. doi: 10.1021/bi001116a.

DOI:10.1021/bi001116a

PMID:11076511

Abstract

Using our Escherichia coli expression system, we have constructed rHb (beta N108Q), a new recombinant hemoglobin (rHb), with the amino acid substitution located in the alpha(1)beta(1) subunit interface and in the central cavity of the Hb molecule. rHb (beta N108Q) exhibits low oxygen affinity, high cooperativity, enhanced Bohr effect, and slower rate of autoxidation of the heme iron atoms from the Fe(2+) to the Fe(3+) state than other low-oxygen-affinity rHbs developed in our laboratory, e.g., rHb (alpha V96W) and rHb (alpha V96W, beta N108K). It has been reported by Olson and co-workers [Carver et al. (1992) J. Biol. Chem. 267, 14443-14450; Brantley et al. (1993) J. Biol. Chem. 268, 6995-7010] that the substitution of phenylalanine for leucine at position 29 of myoglobin can inhibit autoxidation in myoglobin and at position 29 of the alpha-chain of hemoglobin can lower NO reaction in both the deoxy and the oxy forms of human normal adult hemoglobin. Hence, we have further introduced this mutation, alpha L29F, into beta N108Q. rHb (alpha L29F, beta N108Q) is stabilized against auto- and NO-induced oxidation as compared to rHb (beta N108Q), but exhibits lower oxygen affinity at pH below 7.4 and good cooperativity as compared to Hb A. Proton nuclear magnetic resonance (NMR) studies show that rHb (beta N108Q) has similar tertiary structure around the heme pockets and quaternary structure in the alpha(1)beta(1) and alpha(1)beta(2) subunit interfaces as compared to those of Hb A. The tertiary structure of rHb (alpha L29F, beta N108Q) as measured by (1)H NMR, especially the alpha-chain heme pocket region (both proximal and distal histidyl residues), is different from that of CO- and deoxy-Hb A, due to the amino acid substitution at alpha L29F. (1)H NMR studies also demonstrate that rHb (beta N108Q) can switch from the R quaternary structure to the T quaternary structure without changing ligation state upon adding an allosteric effector, inositol hexaphosphate, and reducing the temperature. On the basis of its low oxygen affinity, high cooperativity, and stability against autoxidation, rHb (beta N108Q) is considered a potential candidate for the Hb-based oxygen carrier in a blood substitute system.

摘要

利用我们的大肠杆菌表达系统，我们构建了rHb（β N108Q），这是一种新的重组血红蛋白（rHb），其氨基酸取代位于α(1)β(1)亚基界面以及Hb分子的中央腔内。与我们实验室开发的其他低氧亲和力rHb，如rHb（α V96W）和rHb（α V96W，β N108K）相比，rHb（β N108Q）表现出低氧亲和力、高协同性、增强的玻尔效应，并且血红素铁原子从Fe(2+)态自动氧化为Fe(3+)态的速率较慢。Olson及其同事 [Carver等人（1992年）《生物化学杂志》267卷，14443 - 14450页；Brantley等人（1993年）《生物化学杂志》268卷，6995 - 7010页] 报道，肌红蛋白第29位的亮氨酸被苯丙氨酸取代可抑制肌红蛋白的自动氧化，而血红蛋白α链第29位的相同取代可降低人正常成人血红蛋白脱氧和氧合形式下的NO反应。因此，我们进一步将此突变，即α L29F，引入β N108Q中。与rHb（β N108Q）相比，rHb（α L29F，β N108Q）对自动氧化和NO诱导的氧化更稳定，但在pH低于7.4时表现出较低的氧亲和力，并且与Hb A相比具有良好的协同性。质子核磁共振（NMR）研究表明，与Hb A相比，rHb（β N108Q）在血红素口袋周围具有相似的三级结构，在α(1)β(1)和α(1)β(2)亚基界面具有相似的四级结构。通过(1)H NMR测量，rHb（α L29F，β N108Q）的三级结构，特别是α链血红素口袋区域（近端和远端组氨酸残基），由于α L29F处的氨基酸取代，与CO - 和脱氧 - Hb A不同。(1)H NMR研究还表明，添加变构效应剂肌醇六磷酸并降低温度时，rHb（β N108Q）可以在不改变配体状态的情况下从R四级结构转变为T四级结构。基于其低氧亲和力、高协同性以及对自动氧化的稳定性，rHb（β N108Q）被认为是血液替代系统中基于血红蛋白的氧载体的潜在候选者。