title: Size exclusion chromatography with multi detection in combination with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry as a tool for unraveling the mechanism of the enzymatic polymerization of polysaccharides.
abstract: Determination of the size distributions of natural polysaccharides is a challenging task. More advantageous for characterization are well-defined synthetic (hyper)-branched polymers. In this study we concentrated on synthetic amylopectin analogues in order to obtain and compare all available data for different distributions and size dependence of molecular weights. Two groups of well-defined synthetic branched polysaccharides were synthesized via an in vitro enzyme-catalyzed reaction using the enzyme phosphorylase b from rabbit muscle and Deinococcus geothermalis glycogen branching enzyme. Synthetic polymers had a tunable degree of branching (2%-13% determined via (1)H NMR) and a tunable degree of polymerization (30-350 determined indirectly via UV spectrometry). The systems used for separation and characterization of branched polysaccharides were SEC-DMSO/LiBr and multi detection (refractive index detector, viscosity detector, and multi angle light scattering detector) and SEC-water/0.02% NaN(3); and SEC-50 mM NaNO(3)/0.02% NaN(3) and multi detection. Additionally the side chain length distribution of enzymatically debranched polysaccharides was investigated by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis. With this combination of characterization techniques, we were able not only to characterize the amylopectin analogues but also to solve parts of the molecular mechanism of their enzymatic polymerization. Moreover our materials showed potential to be standards in the field of natural polysaccharides characterization.
authors.affiliation: Department of Polymer Chemistry, Zernike Institute for Advanced Materials, University of Groningen, The Netherlands.
authors.forename: Jelena
authors.initials: J
authors.lastname: Ciric
authors.forename: Jorrit
authors.initials: J
authors.lastname: Oostland
authors.forename: Jan Willem
authors.initials: JW
authors.lastname: de Vries
authors.forename: Albert J J
authors.initials: AJ
authors.lastname: Woortman
authors.forename: Katja
authors.initials: K
authors.lastname: Loos
mesh_terms: D015061:1,4-alpha-Glucan Branching Enzyme / Q000378:metabolism*; D000687:Amylopectin / Q000737:chemistry; D000818:Animals; D002850:Chromatography, Gel / Q000379:methods*; D034301:Deinococcus / Q000201:enzymology; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000166:cytology / Q000201:enzymology; D010763:Phosphorylase b / Q000378:metabolism*; D058105:Polymerization; D011108:Polymers / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry*; D011817:Rabbits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D011108:Polymers; D011134:Polysaccharides; D000687:Amylopectin; D010763:Phosphorylase b; D015061:1,4-alpha-Glucan Branching Enzyme