Fluorescent labeling of pectic oligosaccharides with 2-aminobenzamide and enzyme assay for pectin |
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Authors: | Ishii Tadashi Ichita Junji Matsue Hajime Ono Hiroshi Maeda Ikuko |
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Institution: | Forestry and Forest Products Research Institute, PO Box 16, Tsukuba, Norin Kenkyu, Danchi-nai, Ibaraki 305-8687, Japan. tishii@ffpri.affrc.go.jp |
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Abstract: | Oligogalacturonides oligomers composed of (1-->4)-linked alpha-D-galactosyluronic acid residues] with degrees of polymerization (DP) from 1 to 10, and a tri-, penta-, and heptasaccharide generated from the backbone of rhamnogalacturonan I (RG-I) were labeled at their reducing ends using aqueous 2-aminobenzamide (2AB) in the presence of sodium cyanoborohydride in over 90% yield. These derivatives were analyzed by high-performance anion-exchange chromatography (HPAEC) and structurally characterized by electrospray-ionization mass spectrometry (ESIMS) and by 1H and 13C NMR spectroscopy. The 2AB-labeled oligogalacturonides and RG-I oligomers are fragmented by endo- and exo-polygalacturonase and by Driselase, respectively. 2AB-labeled oligogalacturonide is an exogenous acceptor for galacturonosyltransferase of transferring galacturonic acid from UDP-GalA. Thus, the 2AB-labeled oligogalacturonides and RG-I oligomers are useful for studying enzymes involved in pectin degradation and biosynthesis and may be of value in determining the biological functions of pectic fragments in plants. |
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Keywords: | 2-Aminobenzamide Electrospray-ionization mass spectrometry Fluorescent labeling Galacturonosyltransferase NMR Pectin Polygalacturonase |
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