Aggarwal Soumya, Tanwar Neetu, Singh Ankit, Munde Manoj
School of Physical Sciences, Jawaharlal Nehru University, New Delhi110067, India.
ACS Omega. 2022 Nov 4;7(45):41044-41057. doi: 10.1021/acsomega.2c04419. eCollection 2022 Nov 15.
The insulin-protamine interaction is at the core of the mode of action in many insulin formulations (Zn + insulin + protamine) and to treat diabetes, in which protamine is added to the stable form of hexameric insulin (Zn-insulin). However, due to the unavailability of quantitative data and a high-resolution structure, the binding mechanism of the insulin-protamine complex remains unknown. In this study, it was observed that Zn-insulin experiences destabilization as observed by the loss of secondary structure in circular dichroism (CD), and reduction in thermal stability in melting study, upon protamine binding. In isothermal titration calorimetry (ITC), it was found that the interactions were mostly enthalpically driven. This is in line with the positive Δ value (+880 cal mol), indicating the role of hydrophilic interactions in the complex formation, with the exposure of hydrophobic residues to the solvent, which was firmly supported by the 8-anilino-1-naphthalene sulfonate (ANS) binding study. The stoichiometry () value in ITC suggests the multiple insulin molecules binding to the protamine chain, which is consistent with the picture of the condensation of insulin in the presence of protamine. Atomic force microscopy (AFM) suggested the formation of a heterogeneous Zn-insulin-protamine complex. In fluorescence, Zn-insulin experiences strong Tyr quenching, suggesting that the location of the protamine-binding site is near Tyr, which is also supported by the molecular docking study. Since Tyr is critical in the stabilization of insulin self-assembly, its interaction with protamine may impair insulin's self-association ability and thermodynamic stability while at the same time promoting its flexible conformation desired for better biological activity.
胰岛素与鱼精蛋白的相互作用是许多胰岛素制剂（锌 + 胰岛素 + 鱼精蛋白）作用方式的核心，用于治疗糖尿病，其中鱼精蛋白被添加到六聚体胰岛素（锌 - 胰岛素）的稳定形式中。然而，由于缺乏定量数据和高分辨率结构，胰岛素 - 鱼精蛋白复合物的结合机制仍然未知。在本研究中，观察到锌 - 胰岛素在与鱼精蛋白结合后，通过圆二色性（CD）中二级结构的丧失以及熔解研究中热稳定性的降低，出现了去稳定化。在等温滴定量热法（ITC）中，发现相互作用主要由焓驱动。这与正的Δ值（+880 cal mol）一致，表明亲水相互作用在复合物形成中的作用，同时疏水残基暴露于溶剂中，这得到了8 - 苯胺基 - 1 - 萘磺酸盐（ANS）结合研究的有力支持。ITC中的化学计量比（）值表明多个胰岛素分子与鱼精蛋白链结合，这与在鱼精蛋白存在下胰岛素凝聚的情况一致。原子力显微镜（AFM）表明形成了异质的锌 - 胰岛素 - 鱼精蛋白复合物。在荧光实验中，锌 - 胰岛素经历了强烈的酪氨酸淬灭，表明鱼精蛋白结合位点位于酪氨酸附近，分子对接研究也支持这一点。由于酪氨酸在胰岛素自组装的稳定中起关键作用，它与鱼精蛋白的相互作用可能会损害胰岛素的自缔合能力和热力学稳定性，同时促进其具有更好生物活性所需的灵活构象。
