Department of Food Science, University of Wisconsin, 1605 Linden Drive, Madison, WI 53706, USA.
J Chromatogr A. 2012 Dec 28;1270:330-3. doi: 10.1016/j.chroma.2012.10.070. Epub 2012 Nov 6.
A chromatographic method for the analysis of whey protein isolate (WPI)-dextran glycates was developed in this work that is useful for quantification of sample purity and concentration, and as a sample-preparation method for subsequent analysis by gel electrophoresis (SDS-PAGE) and laser-light scattering. Glycation was by the Maillard reaction between WPI and dextran of 3 different sizes. Glycate fractions from each dextran were collected and analyzed by fluorescent and glycoprotein staining of gels, bicinchoinic acid protein assay, and static and dynamic laser light scattering. The weight-average molecular mass of the glycates was 27-34 kDa (from 3.5 kDa dextran), 32-39 kDa (from 10 kDa dextran), and 250-270 kDa (from 150 kDa dextran). The new method was used to characterize the kinetics of the glycation reaction, which followed a reversible pseudo first-order model. The kinetics of decomposition of the purified glycate by hydrolysis was also examined. The new method is rapid (25 min) and quantitative, and is the first chromatographic method for direct analysis of WPI-dextran glycation products.
本工作开发了一种用于乳清蛋白分离物（WPI）-葡聚糖糖基化产物分析的色谱方法，该方法可用于定量分析样品纯度和浓度，以及作为凝胶电泳（SDS-PAGE）和激光光散射后续分析的样品制备方法。糖基化是通过 WPI 和 3 种不同大小的葡聚糖之间的美拉德反应进行的。从每种葡聚糖中收集糖基化产物，并通过凝胶的荧光和糖蛋白染色、双辛可宁酸蛋白测定法以及静态和动态激光光散射进行分析。糖基化物的重均分子量为 27-34 kDa（来自 3.5 kDa 葡聚糖）、32-39 kDa（来自 10 kDa 葡聚糖）和 250-270 kDa（来自 150 kDa 葡聚糖）。该新方法用于表征糖基化反应的动力学，该反应遵循可逆的拟一级模型。还研究了通过水解纯化的糖基化物的分解动力学。该新方法快速（25 分钟）且定量，是直接分析 WPI-葡聚糖糖基化产物的第一种色谱方法。
