Fatima Sadaf, Sen Priyankar, Sneha P, Priyadoss C George
Genes and Proteins Lab, Department of Biotechnology, Jamia Millia Islamia, New Delhi, 110025, India.
Centre for Bioseparation Technology, VIT University, Vellore, 632014, India.
Appl Biochem Biotechnol. 2017 May;182(1):82-96. doi: 10.1007/s12010-016-2312-4. Epub 2016 Nov 16.
The bindings of detemir [LysB29(Nε-tetradecanoyl)des(B30)-insulin] with two highly homologous albumins, HSA (human serum albumin) and BSA (bovine serum albumin), were investigated through CD, spectrofluorophotometry, and molecular docking analysis. The absence of any tryptophanyl residue in detemir makes albumin binding study possible by exclusive tryptophanyl spectral quenching at 340 nm (λem = 296 nm). The interactions found to be static (Kq > 10 M s) with Stern-Volmer constants ≈10 M. The observed ΔG that was negative in all cases concludes the reactions were spontaneous. Domains I and III of an albumin unfold with 5.0 M urea at pH 7.4, although domain II remains intact. Significant decreases in ΔH and ΔS were due to unfolding explicit that detemir binding may involve domains I and III of albumins. Temperature-dependent changes in binding were higher in HSA than BSA but after unfolding such changes were very less, further indicating the role of domains I and III in detemir binding. Pro28 and Tyr26 of insulin were found to be interacting with Arg114 and Val116 of HSA domain I, while myristate segment of detemir binds to Lys519 of domain III. Interactions seem to be predominantly hydrophobic and entropy driven. Although detemir binds to albumin through myristate, the peptide part shows involvement in binding.
通过圆二色光谱（CD）、荧光分光光度法和分子对接分析，研究了地特胰岛素[赖氨酰B29（Nε-十四烷酰）去（B30）胰岛素]与两种高度同源的白蛋白，即人血清白蛋白（HSA）和牛血清白蛋白（BSA）的结合情况。地特胰岛素中不存在任何色氨酸残基，这使得通过在340 nm（发射波长λem = 296 nm）处仅由色氨酸光谱猝灭来进行白蛋白结合研究成为可能。发现相互作用是静态的（猝灭常数Kq > 10¹² M⁻¹ s⁻¹），斯特恩-沃尔默常数约为10⁵ M⁻¹。在所有情况下观察到的吉布斯自由能变化ΔG均为负值，这表明反应是自发的。在pH 7.4条件下，5.0 M尿素可使白蛋白的结构域I和III展开，而结构域II保持完整。焓变ΔH和熵变ΔS的显著降低是由于展开过程所致，这明确表明地特胰岛素的结合可能涉及白蛋白的结构域I和III。与BSA相比，HSA中结合的温度依赖性变化更高，但展开后这种变化非常小，这进一步表明结构域I和III在地特胰岛素结合中的作用。发现胰岛素的Pro28和Tyr26与HSA结构域I的Arg114和Val116相互作用，而地特胰岛素的肉豆蔻酸片段与结构域III的Lys519结合。相互作用似乎主要是由疏水作用和熵驱动的。虽然地特胰岛素通过肉豆蔻酸与白蛋白结合，但肽段部分也参与了结合。
