Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil.
Int J Biol Macromol. 2013 Aug;59:333-41. doi: 10.1016/j.ijbiomac.2013.04.070. Epub 2013 May 3.
Glossoscolex paulistus (HbGp) extracellular hemoglobin is a giant oligomeric protein. It is constituted by 144 heme containing subunits and non-heme structures (linkers), with a total molecular mass of 3.6MDa. AUC and DLS studies were developed for three HbGp forms, oxy-, met- and cyanomet-, at several pH values, in order to characterize the species in solution upon oligomeric dissociation. Isolated SEC fractions, trimer and dodecamer, are less stable as compared to the whole oxy-HbGp. The monomer d displays a large thermal stability up to 59°C. Hydrodynamic properties of the isolated subunits are very similar to those described for them in the whole protein, in the presence of urea or at pH 10.0. The degree of HbGp oligomeric dissociation, in alkaline pH, depends significantly on the iron oxidation state. Also on the ligand coordinated to the heme iron. Thus, at pH 8.0, the oxy-HbGp is partially dissociated, while the met-form is fully dissociated. The cyanomet-HbGp remains undissociated. Our present results show that the effect of pH on the HbGp oligomeric stability is similar to that associated to the urea-induced unfolding. Simultaneous use of AUC and DLS allowed the characterization of the species in the SEC fractions of isolated HbGp subunits.
巴西钩口线虫（HbGp）细胞外血红蛋白是一种巨大的寡聚蛋白。它由 144 个含有血红素的亚基和非血红素结构（连接子）组成，总分子量为 3.6MDa。为了研究寡聚体解离时溶液中的物种，在几个 pH 值下对三种 HbGp 形式（氧合、高铁和氰高铁）进行了 AUC 和 DLS 研究。与整个氧合 HbGp 相比，分离的 SEC 级分、三聚体和十二聚体的稳定性较差。单体 d 的热稳定性很大，高达 59°C。在存在脲或在 pH 10.0 时，分离的亚基的流体力学性质与在整个蛋白质中描述的非常相似。碱性 pH 下 HbGp 寡聚体解离的程度，很大程度上取决于铁的氧化态和配位到血红素铁的配体。因此，在 pH 8.0 时，氧合 HbGp 部分解离，而高铁形式完全解离。氰高铁 HbGp 保持不解离。我们目前的结果表明，pH 值对 HbGp 寡聚稳定性的影响与脲诱导的解折叠相关。AUC 和 DLS 的同时使用允许对分离的 HbGp 亚基 SEC 级分中的物种进行表征。
