光谱学结合分子模拟技术研究槲皮素对黄嘌呤氧化酶的抑制作用

Study on the Inhibition of Quercetin on XOD Using Multi-Spectroscopic and Molecular Modeling Approaches

在pH 7.4Tris–Hcl缓冲体系中,利用紫外光谱法、荧光光谱法等多种光谱学方法并结合分子模拟技术研究了槲皮素对黄嘌呤氧化酶活性的抑制作用。结果表明,槲皮素能够有效抑制XOD活性,其半抑制浓度(IC50)为(2.74±0.04)×10-6 mol·L-1,接近于阳性对照别嘌呤醇的半抑制浓度[(2.69±0.02)×10-6 mol·L-1]。荧光光谱和圆二色谱显示,槲皮素能够有效猝灭XOD的内源性荧光,说明二者发生了相互作用,并诱导XOD的二级结构发生改变。Stern–Volmer方程和热力学分析表明,槲皮素与XOD形成基态复合物使XOD的内源荧光猝灭,且范德华力和氢键是两者结合反应的主要驱动力。分子模拟结果进一步证实槲皮素通过氢键结合到XOD活性中心的钼原子周围,阻碍了底物黄嘌呤进入XOD活性中心,从而抑制了XOD对黄嘌呤的催化活性,减少了尿酸的生成。

The inhibition of quercetin against XOD in Tris– Hcl(pH 7.4)was investigated by multi-spectroscopic methods including UV?vis absorption,fluorescence and circular dichroism(CD)combined with molecular docking.The flavonoid quercetin significantly inhibited XOD with an IC50 value of(2.74±0.04)×10-6 mol·L-1,which was little higher than that of allopurinol[(2.69 ± 0.02)×10-6 mol·L-1].Strong fluorescence quenching and secondary structure changes of XOD were observed,and Van der Waals forces and hydrogen bonds dominated the binding process on account of the negative enthalpy and entropy changes.Further molecular docking study validated that quercetin can bind to the site near molybdenum atomic(Mo)domain,occupying the catalytic center to avoid the entrance of the substrate xanthine,resulting in the inhibition of XOD activity.