Engineering tyrosine electron transfer pathways decreases oxidative toxicity in hemoglobin: implications for blood substitute design.
Engineering tyrosine electron transfer pathways decreases oxidative toxicity in hemoglobin: implications for blood substitute design.
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DOI：
10.1042/bcj20160243
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发表时间：
2016-10-01
期刊：
影响因子：
--
通讯作者：
Cooper CE
中科院分区：
文献类型：
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作者：
Silkstone GG;Silkstone RS;Wilson MT;Simons M;Bülow L;Kallberg K;Ratanasopa K;Ronda L;Mozzarelli A;Reeder BJ;Cooper CE
基于血红蛋白（Hb）的氧载体（HBOC）已被设计用于取代或增强红细胞的氧携带能力。然而，由于与内在血红素介导的氧化毒性和一氧化氮（NO）清除有关的不良副作用，临床结果通常令人失望。氧化还原活性酪氨酸残基可以促进内源性抗氧化剂和氧化铁酰血红素之间的电子转移。在Hb的α-亚基（Y42）上存在合适的残基，但在β-亚基（F41）的同源位置不存在残基。因此，我们用酪氨酸代替了这个残基（βF41Y, Hb Mequon）。在脂质体中加入铁（met） Hb后，通过共轭二烯和单线态氧形成测量，βF41Y突变对脂质过氧化的内在速率没有影响。然而，在抗坏血酸生理水平存在时，βF41Y显著降低了这些比率。此外，在加入脂质过氧化氢过氧十八烯二酸后，β-亚基的血红素损伤在βF41Y中减慢了5倍。NO双加氧酶活性降低20%，亚硝酸盐还原酶活性提高一倍，提高了βF41Y的NO生物利用度。高铁血红蛋白的内在血红素损失率在β-亚基中增加了一倍，而在α-亚基中保持不变。我们得出结论，在血红蛋白中添加氧化还原活性酪氨酸突变，能够从血浆抗氧化剂中转移电子，降低血红素介导的氧化反应活性，提高NO的生物利用度。这类突变作为HBOC产物的一个组成部分，具有减少不良副作用的潜力。
Hemoglobin (Hb)-based oxygen carriers (HBOC) have been engineered to replace or augment the oxygen-carrying capacity of erythrocytes. However, clinical results have generally been disappointing due to adverse side effects linked to intrinsic heme-mediated oxidative toxicity and nitric oxide (NO) scavenging. Redox-active tyrosine residues can facilitate electron transfer between endogenous antioxidants and oxidative ferryl heme species. A suitable residue is present in the α-subunit (Y42) of Hb, but absent from the homologous position in the β-subunit (F41). We therefore replaced this residue with a tyrosine (βF41Y, Hb Mequon). The βF41Y mutation had no effect on the intrinsic rate of lipid peroxidation as measured by conjugated diene and singlet oxygen formation following the addition of ferric(met) Hb to liposomes. However, βF41Y significantly decreased these rates in the presence of physiological levels of ascorbate. Additionally, heme damage in the β-subunit following the addition of the lipid peroxide hydroperoxyoctadecadieoic acid was five-fold slower in βF41Y. NO bioavailability was enhanced in βF41Y by a combination of a 20% decrease in NO dioxygenase activity and a doubling of the rate of nitrite reductase activity. The intrinsic rate of heme loss from methemoglobin was doubled in the β-subunit, but unchanged in the α-subunit. We conclude that the addition of a redox-active tyrosine mutation in Hb able to transfer electrons from plasma antioxidants decreases heme-mediated oxidative reactivity and enhances NO bioavailability. This class of mutations has the potential to decrease adverse side effects as one component of a HBOC product.