Modification of EDC method for increased labeling efficiency and characterization of low-content protein in gum acacia using asymmetrical flow field-flow fractionation coupled with multiple detectors

Abstract

Abstract1-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

Fluorescence Quenching of Xanthene Dyes during Amide Bond Formation Using DMTMM

“…EDC functions as a coupling agent by activating available carboxylic acid groups to form an o-acylisourea intermediate, which prepares compounds for coupling with amines. 23 Our data suggests that EDC may react with the open carboxylic acid form of FAM in a manner that forces it to maintain its fluorescent state, accounting for the increased fluorescence observed after 24 h. In comparison, NHS has no inherent reactivity with carboxylic acids, and thus, it is not surprising that it exerts no detectable influence over fluorescence. 24 Interestingly, while DMTMM activates carboxylic acids in a manner similar to EDC, we observe a decrease in fluorescence rather than an increase.…”

“…As noted earlier, the interconversion between the closed, non-fluorescent forms and the opened, carboxylic acid forms of many of the xanthene dyes studied is sensitive to environmental conditions. EDC functions as a coupling agent by activating available carboxylic acid groups to form an o-acylisourea intermediate, which prepares compounds for coupling with amines . Our data suggests that EDC may react with the open carboxylic acid form of FAM in a manner that forces it to maintain its fluorescent state, accounting for the increased fluorescence observed after 24 h. In comparison, NHS has no inherent reactivity with carboxylic acids, and thus, it is not surprising that it exerts no detectable influence over fluorescence .…”