Abstract
1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) is widely used as a crosslinker for fluorescence labeling of protein in the fields of biochemistry and food analysis. Many natural polysaccharides often contain some proteins or peptides that are very low in content but play a vital role in their biological function as well as technical applications. Determination of these low-content proteinaceous matters requires a highly sensitive and selective method. In this study, a methodological approach for investigations of the presence of proteinaceous material over the molar mass distribution (MD) of polysaccharides was developed using gum acacia (GA) as a model polysaccharide. EDC fluorescence-labeling method was modified by changing the pH (7, 9, and 11) of the solution for the analysis of low-content protein in food materials. Fluorescence spectroscopy and asymmetrical flow field-flow fractionation (AF4) were employed for characterizing the labeling efficiency and physiochemical properties of unlabeled and fluorescence-labeled GA. AF4 provided molar mass (M) and the radius of gyration (rG) of arabinogalactan (AG) and arabinogalactan protein complex (AGP) and determined the presence of proteinaceous matter over the MD. The labeling efficiencies of GA at pH 7, 9, and 11 determined by fluorescence spectroscopy were 56.5, 68.4, and 72.0%, respectively, with an increment of 15.5% when pH was increased from 7 to 11. The modified EDC fluorescence-labeling method allows highly sensitive and selective analysis of low-content proteinaceous matters and their distribution in natural polysaccharides.Graphical abstract
Highlights
Precolumn fluorescent labeling of protein is often used to increase the sensitivity and selectivity of their detection [1,2,3,4]
The influence of pH (7, 9, and 11) on the labeling efficiency of gum acacia (GA) was evaluated by batch fluorescence detector (FL) and coupling FL detector online with AF4-multi-angle light scattering (MALS)-dRI
The results showed that the labeling efficiency was greatly improved by increasing pH
Summary
Precolumn fluorescent labeling of protein is often used to increase the sensitivity and selectivity of their detection [1,2,3,4]. Primary amine (-NH2) and cysteine on the proteins content or prolonging the reaction time [11, 12]. It is necessary to increase the labeling efficiency and shorten the reaction time for sensitive and fast analysis of very lowcontent proteins in samples. It is reported that higher pH is conducive to the reaction of the intermediate and primary amine (step 2) [10, 13,14,15]. It is assumed that higher labeling efficiency and a faster reaction rate can be obtained at high pH in step 2
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