This work aims at adopting an Electron Paramagnetic Resonance (EPR) spectroscopic technique to help understanding protein–phenolic conjugation and final functionalities relationship as well as the underlying structural basis of antioxidant and antibacterial dual functionalities. Specifically, lysozyme (Lys) was conjugated with two natural phenolic acids, i.e. rosmarinic acid (RA) and gentisic acid (GA, our previous work) with obviously different molecular features. Lys-RA displayed 8.6- and 4.0-times enhanced antioxidant stoichiometry compared to the native Lys and ones with GA, respectively, due to the stronger antioxidant activity of RA. However, RA conjugation mitigated both enzymatic and antibacterial activities of Lys-RA conjugates. Such inhibition effect is attributed to the greater structural and surface property changes of Lys upon conjugating with RA. Furthermore, the polyphenol conjugation related structural basis of disturbance, reactivity and selectivity were explored via site-directed spin labeling (SDSL)–EPR. A dynamic picture of reactivity and selectivity of phenolics conjugation on Lys was proposed.

这项工作旨在采用电子顺磁共振 (EPR) 光谱技术来帮助理解蛋白质-酚类共轭和最终功能关系以及抗氧化和抗菌双重功能的潜在结构基础。具体来说，溶菌酶（Lys）与两种天然酚酸，即迷迭香酸（RA）和龙胆酸（GA，我们之前的工作）结合，具有明显不同的分子特征。由于 RA 的抗氧化活性更强，与天然 Lys 和具有 GA 的 Lys-RA 相比，Lys-RA 的抗氧化化学计量分别提高了 8.6 倍和 4.0 倍。然而，RA 缀合减轻了 Lys-RA 缀合物的酶促和抗菌活性。这种抑制作用归因于 Lys 在与 RA 结合后更大的结构和表面性质变化。此外，通过定点自旋标记 (SDSL)-EPR 探索了多酚共轭相关的干扰、反应性和选择性的结构基础。提出了酚类缀合对 Lys 的反应性和选择性的动态图。