A method for biotin labeling of biologically active oligogalacturonides using a chemically stable hydrazide linkage

Oligogalacturonides (oligomers of alpha-1,4-D-galacturonic acid) with degrees of polymerization (DP) between 8 and 16 were labeled with biotin using a rapid and simple two-reaction protocol that yields a stable oligogalacturonide derivative. In the first reaction biotin-x-hydrazide was coupled to the anomeric carbon of the reducing galacturonic acid residue by a hydrazone linkage. Carbohydrate-hydrazone linkages such as these have been widely used to label a variety of biomolecules. However, we show herein that the oligogalacturonide-hydrazone linkage is hydrolyzed in water. In the second reaction the hydrazone linkage was reduced with sodium cyanoborohydride to form a stable hydrazide. The stability of hydrazide-linked oligogalacturonides was confirmed using high-performance anion-exchange chromatography (HPAEC). The biotin and uronic acid content of the HPAEC fractions was determined using quantitative colorimetric microplate assays. Electrospray mass spectrometry and 1H NMR spectroscopy were used to confirm the structure of the HPAEC-purified biotin-derivatized oligogalacturonides. Biotin-derivatized oligogalacturonides will be useful in studies of the biological functions of oligogalacturonides.