A Method for Biotin Labeling of Biologically Active Oligogalacturonides Using a Chemically Stable Hydrazide Linkage
✍ Scribed by Brent L. Ridley; Mark D. Spiro; John Glushka; Peter Albersheim; Alan Darvill; Debra Mohnen
- Publisher
- Elsevier Science
- Year
- 1997
- Tongue
- English
- Weight
- 168 KB
- Volume
- 249
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
nent polyanionic structural component of plant cell Oligogalacturonides (oligomers of a-1,4-D-galacturo-walls (8). Homogalacturonan is cleaved by plant and nic acid) with degrees of polymerization (DP) between microbial enzymes such as a-1,4-endopolygalacturo-8 and 16 were labeled with biotin using a rapid and nase and a-1,4-endopectate lyase. During this process simple two-reaction protocol that yields a stable oligooligogalacturonides with the required degree of polygalacturonide derivative. In the first reaction biotinmerization (DP) 2 for biological activity may be formed x-hydrazide was coupled to the anomeric carbon of (9)(10)(11). The treatment of various tissues from a diverthe reducing galacturonic acid residue by a hydrazone gent group of dicotyledonous plants with oligogalacturlinkage. Carbohydrate-hydrazone linkages such as onides at concentrations as low as 0.1 mM elicits various these have been widely used to label a variety of bioresponses such as rapid changes in ion flux at the molecules. However, we show herein that the oligogaplasma membrane, regulation of tobacco explant morlacturonide-hydrazone linkage is hydrolyzed in waphogenesis, and induction of a variety of defense reter. In the second reaction the hydrazone linkage was sponses including the accumulation of phytoalexins (2, reduced with sodium cyanoborohydride to form a sta-12, 13). In most bioassays the oligogalacturonides with ble hydrazide. The stability of hydrazide-linked oligomaximal biological activity have DPs between 10 and galacturonides was confirmed using high-perfor-15, but smaller or larger oligomers can be the most mance anion-exchange chromatography (HPAEC).
active in certain bioassays (2,14). In this report we
The biotin and uronic acid content of the HPAEC fracdescribe a method to label biologically active oligogations was determined using quantitative colorimetric lacturonides.
microplate assays. Electrospray mass spectrometry
Carbohydrates derivatized with labels that can be and 1 H NMR spectroscopy were used to confirm the detected easily at low concentrations have been used structure of the HPAEC-purified biotin-derivatized oligogalacturonides. Biotin-derivatized oligogalactur-to study the biosynthesis and deposition of structural onides will be useful in studies of the biological func-carbohydrates (15-17) and to identify putative oligotions of oligogalacturonides.