基于拉曼光谱的羊毛二硫键及构象含量分析

Analysis of disulfide bonds and conformational content of wool based on Raman spectroscopy

拉曼光谱可提供快速、简单、可重复且无损伤的定性分析。采用激光共聚焦显微拉曼光谱仪对羊毛的二硫键以及构象进行了无损分析。研究激光波长、激光强度、扫描时间、物镜大小、形态结构对羊毛微观结构测量结果的影响,优化了激光拉曼光谱对羊毛的测试条件;分别采用超声波、还原以及氧化等不同方法处理纤维,并对比分析羊毛二硫键以及构象变化。结果表明,羊毛较优的拉曼测量条件为激发波长785 nm、扫描时间20 s、激光强度50 mW和50倍物镜观察。通过对比不同方法处理后纤维的拉曼光谱图可发现:超声波处理羊毛后β折叠构象含量降低;二硫苏糖醇处理羊毛后在2569 cm^(-1)处出现了巯基的吸收峰,羊毛二硫键相对含量降低至28.9%,二硫键由分子内的GGG构型转变为分子间的GGT和TGT构型,蛋白质大分子由α螺旋构象转化为β折叠构象;过氧化氢处理羊毛后在1044 cm^(-1)处产生新的S—O吸收峰,β折叠构象含量降低。

Objective Analysis of disulfide bonds content and its conformation is essential to study the microstructural changes in wool.Some of the currently used chemical testing methods are both time-consuming and labor-intensive,and also cause damage to the fiber during testing,together with inaccurate results sometimes.Raman spectroscopy analysis provides fast,simple,reproducible,and non-destructive and qualitative analysis,and this research proposes to analyze disulfide bonds and conformation content of wool using Roman spectroscopy.Method To investigate the effect of different methods such as ultrasound,reduction and oxidation on the disulfide bonds and conformational changes of wool.The disulfide bonds and the conformation of wool were analyzed non-destructively using laser confocal microscopic Raman spectroscopy.The effects of laser wavelength,laser intensity,scanning time,objective size,and morphological structure on the measurement results of wool fibers were analyzed in detail.The testing conditions of laser Raman spectroscopy on wool fibers were optimized.A comparative analysis of the disulfide bonds and conformational changes of wool fibers was also carried out.Results The results showed that the better Raman measurement conditions for wool fibers were excitation wavelength of 785 nm,laser intensity of 50 mW,scanning time of 20 s and 50×objective observation.The macromolecular structure of wool was not significantly changed by changing morphology.From the comparison of Raman spectra of fibers treated by different methods,it was found that the content of β-folding conformation decreased after ultrasonic treatment of wool fibers.The absorption peak of sulfhydryl group appeared at 2569 cm^(-1) after dithiothreitol treatment of wool fibers.The relative content of disulfide bonds in wool fibers was decreased to 28.9%,and the disulfide bonds were transformed from the intramolecular GGG configuration to the intermolecular GGT and TGT configurations.The protein macromolecules were converted fromα-helical conformation toβ-folded conformation.The treatment of wool fibers with hydrogen peroxide produced a new S—O absorption peak at 1044 cm^(-1),and the content ofβ-folded conformation decreased.Conclusion Wool fibers were measured by laser confocal Raman spectroscopy,and the best test conditions were obtained by analyzing the Raman spectra at laser wavelength 785 nm,scanning time 20 s,laser intensity 50 mW and 50×objective.The macromolecular structure of wool was not significantly changed by changing morphology.After optimizing the test conditions,ultrasonic treatment and DTT reduction treatment were compared and analyzed.Raman spectra of wool fibers treated with H 2O 2 oxidation showed that the Raman spectra of fibers treated by different methods had a good response.Among them,the reduction treatment has the greatest influence on the disulfide bond and conformation of the fiber.The results show that Raman spectroscopy has great advantages such as simplicity,reproducibility,and being non-destructive compared with conventional wool disulfide bonds and conformational measurements.