Stability of Maleimide-PEG and Mono-Sulfone-PEG Conjugation to a Novel Engineered Cysteine in the Human Hemoglobin Alpha Subunit.
Stability of Maleimide-PEG and Mono-Sulfone-PEG Conjugation to a Novel Engineered Cysteine in the Human Hemoglobin Alpha Subunit.
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DOI：
10.3389/fchem.2021.707797
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发表时间：
2021
影响因子：
5.5
通讯作者：
Reeder BJ
中科院分区：
文献类型：
--
作者：
Cooper CE;Bird M;Sheng X;Choi JW;Silkstone GGA;Simons M;Syrett N;Piano R;Ronda L;Bettati S;Paredi G;Mozzarelli A;Reeder BJ
为了使用血红蛋白基氧载体作为氧治疗剂或血液替代品，必须增加血红蛋白分子的大小以防止快速肾清除。一种常见的方法使用通过2-亚氨基硫杂环戊烷在表面赖氨酸处的反应产生的巯基的马来酰亚胺PEG化。然而，这产生了高度异质的分子混合物。我们最近设计了一种血红蛋白，在α亚基表面上有一个新的反应性半胱氨酸残基，产生一个单一的聚乙二醇化位点（β Cys 93 Ala/α Ala 19 Cys）。这使得能够通过马来酰亚胺-PEG进行均相PEG化，效率>80%，并且对蛋白质功能没有可辨别的影响。然而，马来酰亚胺-PEG加合物在体内通过逆迈克尔反应和与内源性硫醇物质的交叉缀合而发生解缀合。因此，我们将我们的马来酰亚胺-PEG加合物与使用对解缀合不太敏感的单砜-PEG产生的加合物进行了比较。单砜-PEG与α Ala 19 Cys血红蛋白发生反应，效率> 80%，尽管在较高的PEG：血红蛋白比例下观察到一些副反应;加合物与氧结合的亲和力和协同性与野生型血红蛋白相似。当与马来酰亚胺-PEG直接比较时，当在ImM还原型谷胱甘肽存在下在37°C下孵育7天时，单砜-PEG加合物显著更稳定。用单砜-PEG处理的血红蛋白保留> 90%的其缀合，而对于马来酰亚胺-PEG，< 70%的马来酰亚胺-PEG缀合物保持完整。尽管马来酰亚胺-PEG化对于作为氧治疗剂的急性治疗用途肯定是足够稳定的，但是对于旨在用于较长血管保留（数周-数月）的药物，诸如单砜-PEG的试剂可能更合适。
In order to use a Hemoglobin Based Oxygen Carrier as an oxygen therapeutic or blood substitute, it is necessary to increase the size of the hemoglobin molecule to prevent rapid renal clearance. A common method uses maleimide PEGylation of sulfhydryls created by the reaction of 2-iminothiolane at surface lysines. However, this creates highly heterogenous mixtures of molecules. We recently engineered a hemoglobin with a single novel, reactive cysteine residue on the surface of the alpha subunit creating a single PEGylation site (βCys93Ala/αAla19Cys). This enabled homogenous PEGylation by maleimide-PEG with >80% efficiency and no discernible effect on protein function. However, maleimide-PEG adducts are subject to deconjugation via retro-Michael reactions and cross-conjugation to endogenous thiol species in vivo. We therefore compared our maleimide-PEG adduct with one created using a mono-sulfone-PEG less susceptible to deconjugation. Mono-sulfone-PEG underwent reaction at αAla19Cys hemoglobin with > 80% efficiency, although some side reactions were observed at higher PEG:hemoglobin ratios; the adduct bound oxygen with similar affinity and cooperativity as wild type hemoglobin. When directly compared to maleimide-PEG, the mono-sulfone-PEG adduct was significantly more stable when incubated at 37°C for seven days in the presence of 1 mM reduced glutathione. Hemoglobin treated with mono-sulfone-PEG retained > 90% of its conjugation, whereas for maleimide-PEG < 70% of the maleimide-PEG conjugate remained intact. Although maleimide-PEGylation is certainly stable enough for acute therapeutic use as an oxygen therapeutic, for pharmaceuticals intended for longer vascular retention (weeks-months), reagents such as mono-sulfone-PEG may be more appropriate.