Abstract Using polarized Excitation Emission Matrix (pEEM) spectroscopy to measure the intrinsic emission of proteins offers a potentially useful methodology for a wide variety of potential applications. However, the presence of Rayleigh light scatter causes significant problems when attempting to use Parallel Factor (PARAFAC) and for anisotropy calculations. The use of polarized Total Synchronous Fluorescence Spectroscopy (pTSFS) can minimize Rayleigh scatter and avoid the use of complex data correction methods. Here, we investigated for the first time the use of pTSFS and PARAFAC to analyze the intrinsic emission of an Immunoglobulin (IgG) type protein in its native state. To enable PARAFAC analysis however, TSFS data (which is not trilinear) must first be transformed into an EEM like layout (t-EEM) and this generated a region with no experimentally acquired information ( There were only subtle structural changes measured over the temperature range (15–35 °C) analyzed and PARAFAC only resolved two emitting components. A Trp emission component (average signal from all Trp present) which represented >92% of the explained variance, and a much weaker, mostly Tyr related emission with ~3% of the explained variance. The recovery of this Tyr component was only possible because pTSFS measurements were less contaminated by Rayleigh scattering. Changes in Tyr-to-Trp energy transfer rates caused by thermal motion were detected as an increase in Tyr contribution, which could not be resolved with the equivalent pEEM measurements due to light scatter contamination. The increased selectivity, sensitivity, and reproducibility of pTSFS measurements shows that this is a better option than pEEM for fluorescence emission based monitoring of protein structural change or lot-to-lot variance of IgG type proteins.

中文翻译：

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使用偏振全同步荧光光谱 (pTSFS) 和 PARAFAC 分析来表征内在蛋白质发射

摘要 使用极化激发发射矩阵 (pEEM) 光谱测量蛋白质的固有发射为各种潜在应用提供了一种潜在有用的方法。然而，当尝试使用平行因子 (PARAFAC) 和进行各向异性计算时，瑞利光散射的存在会导致严重的问题。使用偏振全同步荧光光谱 (pTSFS) 可以最大限度地减少瑞利散射并避免使用复杂的数据校正方法。在这里，我们首次研究了使用 pTSFS 和 PARAFAC 来分析天然状态下免疫球蛋白 (IgG) 型蛋白质的内在发射。然而，要启用 PARAFAC 分析，由于光散射污染，这无法通过等效的 pEEM 测量解决。pTSFS 测量增加的选择性、灵敏度和重现性表明，对于基于荧光发射的蛋白质结构变化或 IgG 型蛋白质批次间差异的监测，这是比 pEEM 更好的选择。