圆二色和等温滴定微量热技术研究人血清白蛋白与锌离子的结合机制

摘要

目的：体外利用圆二色和等温滴定微量热技术获得人血清白蛋白（HSA）与锌离子（Zn2+）相互作用的二级结构信息与热力学参数，认识Zn2+与HSA之间的相互作用机制，为探索并发挥微量元素在人体内的作用潜力提供科学依据。方法：采用圆二色技术和等温滴定微量热技术；HSA浓度设置为由低到高的梯度，分别为0.025 mmol·L-1、0.05 mmol·L-1、0.1 mmol·L-1、0.2 mmol·L-1，Zn2+浓度5 mmol·L-1。结果：圆二色检测到不同浓度的HSA与Zn2+相互作用后二级结构发生变化，由低到高浓度的HSA与Zn2+结合后α-螺旋含量随之降低，当HSA浓度为0.2 mmol·L-1时α-螺旋含量表现特殊；进而所进行的等温滴定微量热实验获得一整套有关分子相互作用的热力学信息，包括结合常数（Kb）、反应的化学计量数（N）、熵（ΔS）和焓（ΔH），发现当ZnSO4（mmol·L-1）׃HSA（mmol·L-1）=5׃0.2时，有两类结合位点。不同浓度的HSA分别与Zn2+结合，表现出不同构象变化，特别当HSA浓度达到0.2 mmol·L-1时，α-螺旋含量急剧降低，蛋白质疏水性减弱，肽链伸展。在进一步的等温滴定微量热实验中，发现ZnSO4（mmol·L-1）׃HSA（mmol·L-1）=5׃0.2的条件下，ZnSO4-HSA有两类结合位点，即吸热位点和放热位点。结论：揭示HSA上的吸热结合位点对Zn2+有高特异性，但非结合优先性。这一结果为Zn2+在未来成为打开HSA上吸热位点的“钥匙”提供可行性依据。%This article was aimed to study the mechanism of interaction between human serum albumin (HSA) and zinc ions, in order to provide the information on the secondary structure modification of HSA and the thermodynamics parameters using circular dichroism (CD) and isothermal titration calorimetry (ITC). CD and ITC were applied in the study. The concentration of HSA were 0.025 mmol·L-1, 0.05 mmol·L-1, 0.1 mmol·L-1, and 0.2 mmol·L-1, respectively. The concentration of zinc ion was 5 mmol·L-1. The CD analysis revealed that the secondary structure modification of HSA differed depending on the concentration of HAS. And the content ofα-helix was inversely proportional to the concentration of HSA. When the concentration of HSA was 0.2 mmol·L-1, the content ofα-helix was special. A series of thermodynamics parameters including association constants (Kb), stoichiometry (N), entropy (ΔS) and enthalpy (ΔH) were investigated by ITC analysis. And two types of binding sites were observed when ZnSO4(mmol·L-1)׃HSA (mmol·L-1)= 5׃0.2. The secondary structure modification of HSA interacting with zinc ions depended on the concentration of HSA, a dramatic reduction ofα-helix was detected when the concentration of HSA attained 0.2 mmol·L-1. And the protein hydrophobicity reduction and peptide chains expansion were equally observed at this concentration. The ITC analysis also revealed endothermic and exothermic binding sites in the ZnSO4-HSA interaction when ZnSO4 (mmol·L-1)׃HSA (mmol·L-1)= 5׃0.2, indicating that the endothermic sites were specific but not preferential for zinc ions interactions. These results provided theoretical supports for the application of Zn2+ to open the endothermic sites of HSA.