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
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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 <i>b</i> from rabbit
muscle and <i>Deinococcus geothermalis</i> glycogen branching
enzyme. Synthetic polymers had a tunable degree of branching (2%β13%
determined via <sup>1</sup>H NMR) and a tunable degree of polymerization
(30β350 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<sub>3</sub>; and SEC-50 mM NaNO<sub>3</sub>/0.02%
NaN<sub>3</sub> 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