Piperine (PIP) is the most active main component in pepper. The interaction of small molecules with biomolecules leads to structural and functional changes. In this study, the binding mechanism and antioxidant activity of PIP with hemoglobin (Hb) are presented using spectroscopic and computational methods. Results showed that the redox activity of PIP on Hb showed concentration dependence. Fluorescence and isothermal titration calorimetric experiments showed that the Hb–PIP system had a static quenching mechanism at a single binding site. The addition of PIP caused a slight perturbation to the secondary structure of Hb by structural analysis. The structural stability of the Hb–PIP binding system was demonstrated by molecular dynamics simulations, and molecular docking and thermodynamic constants confirmed that the electrostatic interaction force was dominant in the energy contribution of the system. Research results are conducive to the potential use of PIP in related meat products.

胡椒碱（PIP）是胡椒中最活跃的主要成分。小分子与生物分子的相互作用导致结构和功能的变化。在这项研究中，使用光谱和计算方法介绍了 PIP 与血红蛋白 (Hb) 的结合机制和抗氧化活性。结果表明，PIP对Hb的氧化还原活性呈浓度依赖性。荧光和等温滴定量热实验表明，Hb-PIP 系统在单个结合位点具有静态猝灭机制。通过结构分析，添加 PIP 对 Hb 的二级结构造成了轻微的扰动。通过分子动力学模拟证明了 Hb-PIP 结合系统的结构稳定性，分子对接和热力学常数证实了静电相互作用力在系统的能量贡献中占主导地位。研究结果有利于PIP在相关肉制品中的潜在应用。