Lah Jurij, Prislan Iztok, Krzan Blaz, Salobir Mateja, Francky Andrej, Vesnaver Gorazd
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Askerceva 5, 1000 Ljubljana, Slovenia.
Biochemistry. 2005 Oct 25;44(42):13883-92. doi: 10.1021/bi0512952.
Human erythropoietin (EPO) is a glycoprotein hormone considered to be the principal regulator of red blood cell formation. Although its recombinant version (rEPO) has been widely used for treatment of various anemias and its biological effects are relatively well-known, we know little about its biophysical properties and their relation to its structure. To gain a fuller understanding of the structural and functional properties of rEPO on the molecular level we followed its thermal and urea-induced unfolding at different pH (3.1-9.4) and urea concentrations (0-8 M) using spectropolarimetry, UV absorption, intrinsic emission fluorescence, and differential scanning calorimetry. Our results show that under a variety of conditions rEPO undergoes thermal or urea-induced denaturation that may be considered as a reversible two-state process characterized by unusually high (thermal) or moderate (urea-induced) extent of the residual structure. The highest thermal stability of the protein observed in aqueous solutions at physiological pH appears to be due to the largest difference in the extent of structure in the denatured and native state at this pH. The comparison between experimentally determined energetics of rEPO denaturation and its structure-based calculations indicates that the parametrization of thermodynamic quantities in terms of changes in solvent accessible nonpolar and polar surface areas resulting from protein unfolding can be successfully used provided that these changes are estimated from combination of experimentally determined deltaC(o)p and deltaH(o) values and not calculated from the structure of the protein's folded and assumingly fully unfolded state.
人促红细胞生成素（EPO）是一种糖蛋白激素，被认为是红细胞生成的主要调节因子。尽管其重组版本（rEPO）已被广泛用于治疗各种贫血，且其生物学效应相对为人熟知，但我们对其生物物理性质及其与结构的关系了解甚少。为了在分子水平上更全面地了解rEPO的结构和功能特性，我们使用旋光光谱法、紫外吸收、内源发射荧光和差示扫描量热法，在不同pH值（3.1 - 9.4）和尿素浓度（0 - 8 M）下跟踪其热诱导和尿素诱导的去折叠过程。我们的结果表明，在各种条件下，rEPO会发生热诱导或尿素诱导的变性，这可以被视为一个可逆的两态过程，其特征是残余结构的程度异常高（热诱导）或中等（尿素诱导）。在生理pH值的水溶液中观察到的蛋白质最高热稳定性，似乎是由于在此pH值下变性态和天然态结构程度的最大差异。实验测定的rEPO变性能量与其基于结构的计算之间的比较表明，只要这些变化是根据实验测定的ΔC(o)p和ΔH(o)值的组合估算出来的，而不是从蛋白质折叠态和假定的完全去折叠态的结构计算出来的，那么根据蛋白质去折叠导致的溶剂可及非极性和极性表面积变化对热力学量进行参数化就可以成功使用。
