Department of Sensor and Biomedical Technology, School of Electronics Engineering, Vellore Institute of Technology, Vellore, 632014, Tamil Nadu, India.
J Fluoresc. 2024 Sep;34(5):2229-2237. doi: 10.1007/s10895-023-03440-8. Epub 2023 Sep 20.
A detailed spectroscopic study (fluorescence, absorption, and lifetime) was conducted to gain insight into the nature of the binding interaction between fluorophore Hoechst33258 (H258) and jaundice marker Bilirubin (Br). The fluorescence emission of the H258 (Ex/Em = 340-502nm) showed a conc. dependent quenching in the presence of Br (1.25 M to 10 M). The Stern-Volmer constant demonstrated an upward curve depicting the occurrence of both static and dynamic quenching with an acquired value of K = 3.1x 10 M and biomolecular quenching rate constant K = 8.6 x 10 M S . The static quenching was evaluated using the sphere of action model and a sphere radius of 0.3nm indicated the presence of a static component in the quenching. The FRET analysis with overlap integral (J) = 1.4x10 M cm nm and Foster Radius(R ) = 26.82 Å with 59% efficiency suggested occurrence of dynamic quenching. Further studies with the time-resolved fluorescence also indicated the presence of dynamic quenching. The lifetime values of H258 reduced from 3.9ns to 0.5ns. Molecular docking studies further support both static and dynamic components in quenching. A non-covalent interaction of H258 with Br in the presence of HSA is predominantly characterized by H-bonding with residues Lys, Asn, Glu, Gln, and Br. The H258 and Br interaction was within the distance of 3.04 Å, which is in coherence with the sphere of action model (0.3nm) and Van-der-Waals along with hydrophobic interactions, which suggested both static and dynamic quenching. Thus, H258 can serve as an efficient fluorophore to monitor binding interactions and can be further exploited as a suitable probe for investigating conformational changes and detection of Br in subsequent studies.
进行了详细的光谱研究（荧光、吸收和寿命），以深入了解荧光染料 Hoechst33258（H258）与黄疸标志物胆红素（Br）之间结合相互作用的性质。H258 的荧光发射（Ex/Em = 340-502nm）在 Br（1.25M 至 10M）存在下表现出浓度依赖性猝灭。Stern-Volmer 常数显示出向上的曲线，描绘了静态和动态猝灭的发生，并获得了 K = 3.1x 10 M 和生物分子猝灭速率常数 K = 8.6 x 10 M S 。使用作用球模型评估静态猝灭，并且作用球半径为 0.3nm 表明猝灭中存在静态成分。通过荧光能量转移（FRET）分析，重叠积分（J）= 1.4x10 M cm nm 和福斯特半径（R ）= 26.82 Å ，效率为 59%，表明发生了动态猝灭。使用时间分辨荧光的进一步研究也表明存在动态猝灭。H258 的寿命值从 3.9ns 降低到 0.5ns。分子对接研究进一步支持猝灭中的静态和动态成分。H258 与 Br 在 HSA 存在下的非共价相互作用主要通过与残基 Lys、Asn、Glu、Gln 和 Br 的氢键来表征。H258 和 Br 的相互作用在 3.04 Å 的距离内，这与作用球模型（0.3nm）和范德华力以及疏水力一致，这表明存在静态和动态猝灭。因此，H258 可以用作监测结合相互作用的有效荧光染料，并可以在随后的研究中进一步用作研究构象变化和检测 Br 的合适探针。
