Fluorescent labeling of pectic oligosaccharides with 2-aminobenzamide and enzyme assay for pectin.
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.
Ishii
Tadashi
T
Forestry and Forest Products Research Institute, PO Box 16, Tsukuba, Norin Kenkyu, Danchi-nai, Ibaraki 305-8687, Japan. tishii@ffpri.affrc.go.jp
Ichita
Junji
J
Matsue
Hajime
H
Ono
Hiroshi
H
Maeda
Ikuko
I
D002240:Carbohydrate Sequence; D002247:Carbon Isotopes; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D010368:Pectins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D062367:ortho-Aminobenzoates / Q000378:metabolism*
D002247:Carbon Isotopes; D005456:Fluorescent Dyes; D009844:Oligosaccharides; D062367:ortho-Aminobenzoates; C042491:rhamnogalacturonan I; D010368:Pectins; C000219:anthranilamide; C003181:polygalacturonic acid