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首页> 外文期刊>Biochemistry >Role of Heme in the Unfolding and Assembly of Myoglobin
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Role of Heme in the Unfolding and Assembly of Myoglobin

机译：血红素在肌红蛋白展开和组装中的作用

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The unfolding of wild-type holomyoglobin in the ferric state (metMb) appears to be a simple two-state process, even though hemichrome spectra are often observed and apoMb denaturation involves an intermediate. To resolve these discrepancies, we measured GuHCl-induced, equilibrium unfolding of five sperm whale metMb variants, which were selected to examine the relative importance of apoglobin stability and hemin affinity. Combined analysis of CD, Trp fluorescence, and Soret absorbance titration curves for all the variants requires a six-state mechanism containing native (N), intermediate (I), and unfolded (U) states of apoMb and their hemin-bound counterparts, NH (holoMb), IH, and UH, respectively. The unfolding parameters for the apoMbs were obtained in independent experiments and then fixed in the analysis of the holoprotein data, where only the affinities of the apoglobin states for hemin were allowed to vary. This cofactor binding analysis applies generally to all globins and led to three specific conclusions. (1) The stability of holo-metMb is determined primarily by the high affinity (K-d similar to 10(-13) M) of native apoMb (N) for hemin. (2) The partially unfolded intermediate with hemin bound (IH) has a hemichrome spectrum indicative of a bis-histidyl axial coordination and is seen clearly when the stability of the N state or its affinity for hemin is reduced. (3) Although the affinity of the intermediate for hemin (Kd similar to 10(-11) M) is similar to 100-fold lower than that for the native state, free hemin can bind to it and promote the assembly of the holoprotein.
机译：即使经常观察到半彩色光谱并且apoMb变性涉及一个中间物，在铁态（metMb）上野生型全血红蛋白的展开似乎是一个简单的两态过程。为了解决这些差异，我们测量了5种抹香鲸metMb变体的GuHCl诱导的平衡展开，选择这些变体来检查载脂蛋白稳定性和血红素亲和力的相对重要性。所有变体的CD，Trp荧光和Soret吸光度滴定曲线的组合分析需要六种状态的机制，其中包含apoMb的天然（N），中间（I）和未折叠（U）状态以及与血红素结合的对应物NH （holoMb），IH和UH。在独立的实验中获得了载脂蛋白Mb的解折叠参数，然后在全蛋白质数据分析中进行了固定，其中仅改变了载脂蛋白状态对血红素的亲和力。该辅助因子结合分析通常适用于所有球蛋白，并得出三个具体结论。 （1）holo-metMb的稳定性主要取决于天然apoMb（N）对血红素的高亲和力（K-d类似于10（-13）M）。 （2）与血红素结合（IH）的部分展开的中间体具有指示双组氨酸轴向配位的半彩色光谱，并且当N状态的稳定性或其对血红素的亲和力降低时可以清楚地看到。 （3）尽管中间体对血红素的亲和力（Kd类似于10（-11）M）比天然状态低约100倍，但游离血红素可与其结合并促进全蛋白的组装。

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