光谱法结合分子动力学模拟研究臭氧对肌红蛋白结构的作用机制

臭氧(O₃)是一种具有强氧化性作用的杀菌消毒剂，因其安全无害等特点已被广泛用于肉制品生产加工的减菌处理，但O₃减菌处理对红肉色泽具有较强的负面作用，且其作用机制尚缺乏研究。针对肌红蛋白（Mb）存在状态是决定红色肉色泽关键因素的基础，通过紫外-可见吸收光谱法、荧光光谱法和圆二色光谱法（CD）研究O₃作用下Mb的光谱特性变化，结合蛋白质氧化特征指标分析和分子动力学模拟技术探究O₃对Mb分子的作用效果与机制。光谱研究结果表明，O₃处理可使Mb的紫外-可见光谱图在412 nm左右处的铁卟啉环特征峰及540和580 nm附近的氧合肌红蛋白（OMb）特征峰的强度减弱，其中铁卟啉环特征峰发生蓝移；利用固定激发波长280 nm下测定Mb内源性荧光和同步荧光光谱表明O₃会降低Mb的荧光强度，增大铁卟啉基团贡献的荧光峰强度和造成酪氨酸残基荧光光谱特征峰的蓝移；O₃作用使Mb三维荧光光谱特征峰强度的下降及光散射强度的增加。以上变化推断出O₃会促进Mb的氧化，造成其氨基酸残基疏水基团裸露，使Mb所处微环境及其蛋白构象改变；CD分析表明O₃与肌红蛋白接触时间越久，蛋白质二级结构变化越明显，造成α-螺旋的含量下降，无规则卷曲增加。辅以检测不同强度O₃处理Mb的含量及性质的变化，可知O₃处理使OMb含量下降，高铁肌红蛋白（MMb）含量增加，同时O₃处理Mb的羰基含量增加和巯基含量下降，这也进一步证实O₃作用促进了Mb的氧化，此外，O₃处理Mb表面疏水性的增强，说明O₃造成Mb体系微环境的极性变化。分子动力学模拟结果显示O₃会提高Mb肽链的RMSD值，影响Mb肽链的稳定性，减弱铁卟啉环与Mb肽链的相互作用；RMSF结果表明Mb活性口袋附近氨基酸残基的变化较大；蛋白质二级结构分析与光谱学试验研究结果一致，Mb的α-螺旋的含量下降，无规则卷曲增加。总而言之，O₃可作用于Mb的氨基酸残基，导致蛋白质二级结构和疏水性改变，并发生蛋白氧化及铁卟啉环暴露，进而引起红色肉色泽发生改变。该研究可为生鲜红肉护色技术制定等提供一定理论依据。

Spectroscopic Method Combined with Molecular Dynamics Simulation to Study the Action Mechanism of Ozone on Myoglobin Structure

Ozone (O₃) has been widely used for reducing bacteria in fresh meat. However, O₃ treatment has a negative impact on the red meat color, and the action mechanism of O₃ on red meat color is still lack of research. The existence of myoglobin (Mb) is the basis for determining the key factors of red meat color. Therefore, the spectroscopic characteristics of myoglobin (Mb) under O₃ were analyzed by UV-Vis absorption spectroscopy, fluorescence spectroscopy and circular dichroism (CD) spectroscopy. Moreover, the protein oxidation characteristics and molecular dynamics simulation were used to explore the effect and mechanism of O₃ on Mb molecule. The results of spectroscopic studies show that the O₃ treatment can decrease the intensity peak of the iron porphyrin ring at about 412 nm and the characteristic peak of oxygenated myoglobin (OMb) near 540 and 580 nm in the ultraviolet-visible spectrum of Mb. The characteristic peak of the iron porphyrin ring blue-shifted. It also caused changes in the endogenous fluorescence and synchronous fluorescence spectra of Mb measured at a fixed excitation wavelength of 280 nm, indicating that the fluorescence intensity of Mb was reduced by O₃ and the fluorescence peak intensity contributed by the iron porphyrin group was increased and it also caused a blue shift in the characteristic peak of the fluorescence spectrum of the tyrosine residue. The characteristic peak intensity of the three-dimensional fluorescence spectrum decreased and the light scattering intensity increased. It was concluded that O₃ would cause the protein oxidation of Mb, the exposure of hydrophobic group of the amino acid residues in Mb and the conformation change of the protein. The CD spectroscopy results show that the longer the contact time between O₃ and myoglobin, the more obvious the change of protein secondary structure, resulting in a decrease in the content of α-helix and an increase in random curl. Combined with the chemical detection on the content and characteristics of Mb, it shows that O₃ caused the decrease of OMb content, and the increase of MMb, carbonyl and sulfhydryl content, indicating that O₃ treatments could lead to the protein oxidation. Moreover, O₃ treatments increased the hydrophobicity of protein surface, indicating it resulted in the polarity change of the microenvironment of the protein system. Molecular dynamics simulation results show that O₃ can increase the RMSD value of Mb peptide chain, affect the stability of Mb peptide chain, and weaken the interaction between porphyrin ring and Mb peptide chain. The change in RMSF value Mb peptide chain discovered that amino acid residues of Mb near the active pocket changed obviously; Molecular dynamics simulations of protein structural changes were consistent with the results of spectroscopic experiments, namely, the alpha-helix in Mb decreased and the irregular curl increased after O₃ treatment. In conclusion, O₃ treatment could interact with the residues of Mb, led to the changes in the secondary structure and the hydrophobicity of Mb, and brought on the oxidation of protein and the exposure of iron porphyrin ring, therefore resulting in the change of red meat color. This study can provide theoretical basis for the color protection of red meats.