Fluorescent labeling of pectic oligosaccharides with 2-aminobenzamide and enzyme assay for pectin

An Arabidopsis Cell Wall Proteoglycan Consists of Pectin and Arabinoxylan Covalently Linked to an Arabinogalactan Protein

“…If free pectin and/or xylan polysaccharides were present in YS, they would be detectable by reducing end assays or reducing end labeling due to the presence of terminal anomeric carbons not bound in a glycosidic bond. The reducing end of polysaccharides can be labeled with the hydrophobic fluor 2-amino-benzoamide (2-AB) and detected by a gain in absorbance at UV-254 nm (Ishii et al, 2002). Following incubation of YS2 with 2-AB and purification of the treated YS2 by SEC and reversephase chromatography, the resulting 2-AB-treated YS2 showed no gain in absorbance at 254 nm and had the same retention time as nontreated YS2.…”

In VitroStabilization and Minimum Active Component of Polygalacturonic Acid Synthase Involved in Pectin Biosynthesis

“…16) The enzyme has been characterized in a detergent-solubilized form of suspension cultures of tobacco cells 17) and petunia pollen tubes. 18) The activity was also detected in azuki bean 19) and pumpkin 20) in a detergent-permeabilized form. The solubilized enzyme y To whom correspondence should be addressed.…”

“…Fax: +81-6-6850-5382; E-mail: txi@chem.sci.osaka-u.ac.jp Abbreviations: BES, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid; Bis-Tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid; DP, degree of polymerization; GalUA, galacturonic acid; HG, homogalacturonan; MES, 2-morpholinoethanesulfonic acid; MOPS, 3-morpholinopropanesulfonic acid; OGA, oligogalacturonic acid; PGA, polygalacturonic acid; PIPES, piperazine-1,4 0 -bis (2-ethanesulfonic acid) has been shown to add GalUA from UDP-GalUA onto the nonreducing end of oligogalacturonic acid (OGA). 18,20,21) It was found to be located in the lumen of the Golgi using sucrose density gradient centrifugation coupled with proteinase treatment of the Golgi-rich membrane fraction. 22) Recently, a new assay method for polygalacturonate 4--galacturonosyltransferase was developed using fluorescence-labeled OGAs as acceptor substrates.…”

“…22) Recently, a new assay method for polygalacturonate 4--galacturonosyltransferase was developed using fluorescence-labeled OGAs as acceptor substrates. [18][19][20] When the enzyme was incubated with fluorescencelabeled OGA and UDP-GalUA, it transferred more than 10 GalUA units onto the fluorogenic OGA.…”

Derivation of Hydroxamic Acid from Pectin and Its Applications in Colorimetric Determination

“…2,3) Pectin and the oligosaccharides have also been noted as dietary supplements because of their functions in the intestinal tract, and as a result, a sensitive assay for these compounds was developed. 4,5) Hydroxamic acid (HX) is a compound formed by condensation between carboxylic acid and hydroxylamine (HA). HX is known to develop color in the presence of Fe 3ϩ ions in HCl solution and this reaction has been employed determining carboxylic acids.…”