Department of Chemistry and the Leicester Institute of Structural & Chemical Biology, University of Leicester, Leicester, LE1 7RH, United Kingdom.
School of Chemistry, University of Bristol, Bristol, BS8 1TS, United Kingdom.
Anal Biochem. 2019 May 1;572:45-51. doi: 10.1016/j.ab.2019.02.021. Epub 2019 Feb 23.
Accumulating evidence suggests a new role for cellular heme as a signalling molecule, in which interactions with target proteins are more transient than found with traditionally-defined hemoproteins. To study this role, a precise method is needed for determining the heme-binding affinity (or dissociation constant, K). Estimates of K are commonly made following a spectrophotometric titration of an apo-protein with hemin. An impediment to precise determination is, however, the challenge in discriminating between the Soret absorbance for the product (holo-protein) and that for the titrant (hemin). An altogether different approach has been used in this paper to separate contributions made by these components to absorbance values. The pure component spectra and concentration profiles are estimated by a multivariate curve-resolution (MCR) algorithm. This approach has significant advantages over existing methods. First, a more precise determination of K can be made as concentration profiles for all three components (apo-protein/holo-protein/hemin) are determined and can be simultaneously fitted to a theoretical-binding model. Second, an absorption spectrum for the holo-protein is calculated. This is a unique advantage of MCR and attractive for investigating proteins in which the nature of heme binding has not, hitherto, been characterised because the holo-protein spectrum provides information on the interaction.
越来越多的证据表明，细胞血红素在作为信号分子方面发挥了新的作用，其与靶蛋白的相互作用比传统定义的血红素蛋白更为短暂。为了研究这种作用，需要一种精确的方法来确定血红素结合亲和力（或解离常数，K）。通常通过用血红素对脱辅基蛋白进行分光光度滴定来估计 K。然而，精确测定的一个障碍是，区分产物（全蛋白）和滴定剂（血红素）的 Soret 吸光度存在困难。在本文中，采用了一种完全不同的方法来分离这些成分对吸光度值的贡献。通过多变量曲线分辨率（MCR）算法估计纯组分光谱和浓度分布。与现有方法相比，该方法具有显著优势。首先，可以更精确地确定 K，因为可以确定所有三个组分（脱辅基蛋白/全蛋白/血红素）的浓度分布，并可以同时拟合到理论结合模型中。其次，计算出全蛋白的吸收光谱。这是 MCR 的独特优势，对于研究迄今为止尚未表征血红素结合性质的蛋白质具有吸引力，因为全蛋白光谱提供了关于相互作用的信息。
