Rosell Federico I, Mauk Marcia R, Mauk A Grant
Department of Biochemistry and Molecular Biology and Centre for Blood Research, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Biochemistry. 2005 Feb 15;44(6):1872-9. doi: 10.1021/bi0480077.
Thermal denaturation of the human hemopexin-heme complex was investigated under a variety of solution conditions to identify factors that influence heme release. The midpoint temperature for the transition between the folded and folded states, T(m), of the hemopexin-ferriheme complex exhibits a significant dependence on pH. When the pH is reduced from 7 to 5 (50 mM BisTris buffer and 50 mM NaCl), T(m) decreases by approximately 23 degrees C despite the relatively higher chloride concentration that tends to stabilize the protein. The thermal stability of the hemopexin-ferroheme complex was examined at pH 7.4 to yield a T(m) that is 3.2 degrees C lower than that of the hemopexin-ferriheme complex under identical conditions. The effect of transition metal ions, which hemopexin has recently been shown to bind [Mauk, M. R., Rosell, F. I., Lelj-Garolla, B., Moore, G. R., and Mauk, A. G. (2005) Biochemistry 44, XXXX-XXXX], was also considered. Cu(2+) and Zn(2+) had the greatest effect, reducing T(m) for the transition by 4.8 and 6.5 degrees C, respectively, relative to the value for the protein in the absence of metal ions [T(m) = 64.9 degrees C [10 mM sodium phosphate buffer (pH 7.4)]]. These metal ions also interfered significantly with the recovery of the native state from the unfolded protein when the protein on returning to 20 degrees C. The current results demonstrate how the conditions within the endosomes of hepatocytes (pH approximately 5.0, [Cl(-)] approximately 60 mM) and the potential presence of transition metal ions or heme iron reduction contribute to the membrane receptor-mediated process of heme release from hemopexin.
在多种溶液条件下研究了人血红蛋白结合珠蛋白 - 血红素复合物的热变性，以确定影响血红素释放的因素。血红蛋白结合珠蛋白 - 高铁血红素复合物折叠态与未折叠态转变的中点温度T(m)对pH有显著依赖性。当pH从7降至5（50 mM BisTris缓冲液和50 mM NaCl）时，尽管氯化物浓度相对较高，倾向于使蛋白质稳定，但T(m)仍降低约23℃。在pH 7.4下检测了血红蛋白结合珠蛋白 - 亚铁血红素复合物的热稳定性，得到的T(m)比相同条件下血红蛋白结合珠蛋白 - 高铁血红素复合物的T(m)低3.2℃。还考虑了过渡金属离子的影响，最近已证明血红蛋白结合珠蛋白能结合这些离子[Mauk, M. R., Rosell, F. I., Lelj - Garolla, B., Moore, G. R., and Mauk, A. G. (2005) Biochemistry 44, XXXX - XXXX]。相对于无金属离子时蛋白质的值[T(m) = 64.9℃ [10 mM磷酸钠缓冲液(pH 7.4)]]，Cu(2+)和Zn(2+)的影响最大，分别使转变的T(m)降低4.8℃和6.5℃。当蛋白质回到20℃时，这些金属离子也显著干扰了从未折叠蛋白恢复到天然状态的过程。目前的结果表明，肝细胞内体中的条件（pH约为5.0，[Cl(-)]约为60 mM）以及过渡金属离子的潜在存在或血红素铁的还原如何促进膜受体介导的血红素从血红蛋白结合珠蛋白释放的过程。
