Rehman Md Tabish, Shamsi Hira, Khan Asad U
Medical Microbiology and Molecular Biology Laboratory, Interdisciplinary Biotechnology Unit, Aligarh Muslim University , Aligarh, Uttar Pradesh, 202 002, India.
Mol Pharm. 2014 Jun 2;11(6):1785-97. doi: 10.1021/mp500116c. Epub 2014 May 6.
The mechanism of interaction between imipenem and HSA was investigated by various techniques like fluorescence, UV.vis absorbance, FRET, circular dichroism, urea denaturation, enzyme kinetics, ITC, and molecular docking. We found that imipenem binds to HSA at a high affinity site located in subdomain IIIA (Sudlow's site I) and a low affinity site located in subdomain IIA.IIB. Electrostatic interactions played a vital role along with hydrogen bonding and hydrophobic interactions in stabilizing the imipenem.HSA complex at subdomain IIIA, while only electrostatic and hydrophobic interactions were present at subdomain IIA.IIB. The binding and thermodynamic parameters obtained by ITC showed that the binding of imipenem to HSA was a spontaneous process (ΔGD⁰(D)= -32.31 kJ mol(-1) for high affinity site and ΔGD⁰(D) = -23.02 kJ mol(-1) for low affinity site) with binding constants in the range of 10(4)-10(5) M(-1). Spectroscopic investigation revealed only one binding site of imipenem on HSA (Ka∼10(4) M(-1)). FRET analysis showed that the binding distance between imipenem and HSA (Trp-214) was optimal (r = 4.32 nm) for quenching to occur. Decrease in esterase-like activity of HSA in the presence of imipenem showed that Arg-410 and Tyr-411 of subdomain IIIA (Sudlow's site II) were directly involved in the binding process. CD spectral analysis showed altered conformation of HSA upon imipenem binding. Moreover, the binding of imipenem to subdomain IIIA (Sudlow's site II) of HSA also affected its folding pathway as clear from urea-induced denaturation studies.
通过荧光、紫外可见吸收、荧光共振能量转移（FRET）、圆二色性、尿素变性、酶动力学、等温滴定量热法（ITC）和分子对接等多种技术研究了亚胺培南与人类血清白蛋白（HSA）之间的相互作用机制。我们发现亚胺培南与位于亚结构域IIIA（Sudlow位点I）的高亲和力位点以及位于亚结构域IIA.IIB的低亲和力位点结合。在亚结构域IIIA稳定亚胺培南-HSA复合物时，静电相互作用与氢键和疏水相互作用共同发挥了至关重要的作用，而在亚结构域IIA.IIB仅存在静电和疏水相互作用。ITC获得的结合和热力学参数表明，亚胺培南与HSA的结合是一个自发过程（高亲和力位点的ΔGD⁰(D)= -32.31 kJ mol(-1)，低亲和力位点的ΔGD⁰(D) = -23.02 kJ mol(-1)），结合常数在10(4)-10(5) M(-1)范围内。光谱研究表明亚胺培南在HSA上只有一个结合位点（Ka∼10(4) M(-1)）。FRET分析表明亚胺培南与HSA（色氨酸-214）之间的结合距离对于发生猝灭是最佳的（r = 4.32 nm）。在亚胺培南存在下HSA酯酶样活性的降低表明亚结构域IIIA（Sudlow位点II）的精氨酸-410和酪氨酸-411直接参与了结合过程。圆二色光谱分析表明亚胺培南结合后HSA的构象发生了改变。此外，从尿素诱导的变性研究可以清楚地看出，亚胺培南与HSA亚结构域IIIA（Sudlow位点II）的结合也影响了其折叠途径。
