Dai Qingyuan, Wang Huiqin, Zhu Xiuling, Hornung Polyanna Silveira, Zhang Yuru, Hu Wenxuan, Lin Anqi, Yao Anyi, Beta Trust
School of Biological and Food Engineering, Anhui Polytechnic University, Beijing Middle Road, Wuhu 241000, China.
Wuhu Green Food Industry Research Institute Co., Ltd., Wuwei Economic Development Zone, Wuhu 238326, China.
Foods. 2025 May 30;14(11):1952. doi: 10.3390/foods14111952.
The functional properties of proteins are closely related to their structure and conformation. The effects of glycosylation and pH on the structural and conformational changes in whey protein isolate (WPI) were investigated using multispectral technology. More and higher-molecular-weight molecules of WPI-dextran conjugates (WDCs) with increased degrees of glycosylation (DGs) in SDS-PAGE occurred at the expense of band intensities of α-lactalbumin, β-lactoglobulin, and bovine serum albumin. The higher wavenumber shift in FTIR peaks of WPI after glycosylation in the Amide I, II, and III regions and the decrease in its intensity occurred. The maximum absorption wavelength (λ) of UV-Vis spectra of WPI before and after glycosylation in the range of 260-290 nm showed no significant difference in a pH range of 2.0-10.0. Moreover, the UV-Vis absorption intensities of WDCs at λ around 278 nm were highly and positively correlated with their DGs. The λ and intensities of total intrinsic fluorescence spectra of Tyr and Trp residues in WDCs with an increase in DGs had an obvious redshift and decrease, respectively. Although the intensities of synchronous fluorescence spectra of individual Tyr or Trp residues in WDCs with an increase in DGs also gradually decreased, the λ of the former and latter had a blueshift and redshift, respectively. UV-Vis absorption and fluorescence spectroscopies indicated that the changes in the λ and intensity of WPI were closely related to the protonation states of carbonyl groups and free amino groups and the degree of glycosylation. This work may be beneficial for understanding the structural and conformational changes in proteins by measuring the microenvironment around Tyr and/or Trp residues in proteins using UV-Vis absorption and synchronous fluorescence spectroscopies, providing a promising technique for quantitatively monitoring the degree of glycosylation (DG) in a rapid and practical way without any chemical reagents using UV-Vis absorption spectroscopy.
蛋白质的功能特性与其结构和构象密切相关。利用多光谱技术研究了糖基化和pH对乳清分离蛋白（WPI）结构和构象变化的影响。在SDS-PAGE中，随着糖基化程度（DG）增加，WPI-葡聚糖缀合物（WDCs）出现更多且分子量更高的分子，这是以α-乳白蛋白、β-乳球蛋白和牛血清白蛋白条带强度降低为代价的。糖基化后WPI在酰胺I、II和III区域的FTIR峰波数发生更高的位移且强度降低。在2.0 - 10.0的pH范围内，糖基化前后WPI的紫外可见光谱在260 - 290 nm范围内的最大吸收波长（λ）无显著差异。此外，WDCs在约278 nm处的紫外可见吸收强度与其DG高度正相关。随着DG增加，WDCs中Tyr和Trp残基的总固有荧光光谱的λ和强度分别有明显的红移和降低。尽管随着DG增加，WDCs中单个Tyr或Trp残基的同步荧光光谱强度也逐渐降低，但前者的λ发生蓝移，后者的λ发生红移。紫外可见吸收和荧光光谱表明，WPI的λ和强度变化与羰基和游离氨基的质子化状态以及糖基化程度密切相关。这项工作可能有助于通过使用紫外可见吸收和同步荧光光谱测量蛋白质中Tyr和/或Trp残基周围的微环境来理解蛋白质的结构和构象变化，为使用紫外可见吸收光谱以快速实用的方式在无需任何化学试剂的情况下定量监测糖基化程度（DG）提供了一种有前景的技术。
