Fluorescence quenching is widely used to obtain association constants between proteins and ligands. This methodology is based on assumption that ground‐state complex between protein and ligand is responsible for quenching. Here, we call the attention about the risk of using the temperature criterion for decision of applying or not fluorescence quenching data to measure association constants. We demonstrated that hydrophobic effect can be the major force involved in the interaction and, as such, superposes the well‐established rationalization that host/guest complexation is weakened at higher temperatures due to loss of translational and rotational degrees of freedom. To do so, the complexation of bovine serum albumin with octyl gallate was studied by steady‐state, time‐resolved fluorescence spectroscopy and isothermal titration calorimetry. The results clearly demonstrated the complexation, even though the Stern‐Volmer constant increased at higher temperatures (1.6 × 10⁴ and 4.1 × 10⁵ mol⁻¹ L at 20°C and 40°C), which could suggest a simple dynamic process and not complexation. The entropy‐driven feature of the interaction was demonstrated by the unfavorable enthalpy (∆H ° = 104.4 kJmol⁻¹) but favorable entropy (∆S ° = 447.5 Jmol⁻¹ K⁻¹). The relevance of the ligand hydrophobicity was also evaluated by comparing ascorbic acid and its ester ascorbyl palmitate. Docking simulations showed a higher number of hydrophobic contacts and lower energy poses for the esters, confirming the experimental results. In conclusion, the well‐established rationalization that host/guest complexation is weakened at higher temperatures is not straightforward for protein‐ligand interactions. Hence, the temperature effect for a decision between static and dynamic quenching and its use to decide if a complexation at ground state is taking place between ligand and protein should not be used.

中文翻译：

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白蛋白中没食子酸辛酯的熵驱动结合：未能应用温度效应来区分动态和静态荧光猝灭。

荧光猝灭被广泛用于获得蛋白质和配体之间的关联常数。该方法基于蛋白质和配体之间的基态复合物负责淬灭的假设。在这里，我们提请注意使用温度标准来决定是否应用荧光猝灭数据来测量关联常数的风险。我们证明疏水效应可能是相互作用的主要力量，因此，叠加了公认的合理性，即由于平移和旋转自由度的丧失，宿主/客体络合在较高温度下减弱。为此，通过稳态、时间分辨荧光光谱和等温滴定量热法研究了牛血清白蛋白与没食子酸辛酯的络合。⁴和 4.1 × 10 ⁵  mol ^-1 L 在 20°C 和 40°C)，这可能表明一个简单的动态过程而不是络合。相互作用的熵驱动特征由不利的焓（Δ H ° = 104.4 kJmol ^-1）但有利的熵（Δ S ° = 447.5 Jmol ^-1 K ^-1）。还通过比较抗坏血酸及其抗坏血酸棕榈酸酯来评估配体疏水性的相关性。对接模拟显示酯类有更多的疏水性接触和更低的能量姿势，证实了实验结果。总之，宿主/客体复合在较高温度下减弱的公认合理化对于蛋白质-配体相互作用并不简单。因此，不应使用温度效应来决定静态和动态淬灭之间的关系，以及用于决定配体和蛋白质之间是否发生基态络合的用途。