The aim of this study was to analyze the binding interactions between a common antihypertensive drug (ramipril, R) and the widely distributed plant flavonoid quercetin (Q), in the presence of human serum albumin (HSA). From the observed fluorescence spectra of the (HSA + R) system we can assume that ramipril is also one of the Site 3 ligands—similar to fusidic acid—the binding of which has been proven by RTG crystallography. Our claim is supported by near-UV CD spectroscopy, microscale themophoresis and molecular modeling. The presence of R slightly inhibited the subsequent binding of Q to HSA and, on the contrary, the pre-incubation of HSA with Q caused a stronger binding of R, most likely due to allosteric interactions. At high concentrations, R is also able to displace Q from its binding site. The dissociation constant KD for the binding of R is more than hundredfold larger than for Q, which means that R is a very weak binder to HSA. The knowledge of qualitative and quantitative parameters of R, as well as the methods used in this study, are important for future research into HSA binding. This study shows the importance of implementing other methods for KD determination. Microscale thermophoresis has proved to be a novel, practical and accurate method for KD determination on HSA, especially in cases when fluorescence spectroscopy is unable to produce usable results.

中文翻译：

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雷米普利和槲皮素对人血清白蛋白的结合相互作用分析：一种新的亲和力评价方法

本研究的目的是分析一种常见的抗高血压药物（雷米普利，R）与广泛分布的植物类黄酮槲皮素（Q）在人血清白蛋白（HSA）存在下的相互作用。从观察到的 (HSA + R) 系统的荧光光谱，我们可以假设雷米普利也是位点 3 配体之一——类似于夫西地酸——其结合已通过 RTG 晶体学证明。我们的主张得到了近紫外 CD 光谱、微尺度热泳和分子建模的支持。R 的存在略微抑制了 Q 与 HSA 的后续结合，相反，HSA 与 Q 的预孵育导致 R 的更强结合，这很可能是由于变构相互作用。在高浓度下，R 还能够从其结合位点置换 Q。R 结合的解离常数 KD 比 Q 大一百倍以上，这意味着 R 是 HSA 的非常弱的结合。R 的定性和定量参数的知识以及本研究中使用的方法对于未来对 HSA 结合的研究很重要。本研究显示了实施其他方法进行 KD 测定的重要性。微量热泳已被证明是一种新颖、实用且准确的 HSA KD 测定方法，尤其是在荧光光谱无法产生可用结果的情况下。本研究显示了实施其他方法进行 KD 测定的重要性。微型热泳已被证明是一种新颖、实用且准确的 HSA KD 测定方法，尤其是在荧光光谱无法产生可用结果的情况下。本研究显示了实施其他方法进行 KD 测定的重要性。微型热泳已被证明是一种新颖、实用且准确的 HSA KD 测定方法，尤其是在荧光光谱无法产生可用结果的情况下。