Labeling of Aldehyde Fragments for PAGE & MS/HPLC

In this study, laminaran was oxidized with H2O2 and Fe+2 ions in 1 mM NaHPO4 pH 4.0 buffer. It was suspected that the oxidation would hydrolyze the glucan into monomers or scission glucose to aldehyde fragments. Following the method of Klock & Starr (1988) aldehydes were labeled with a fluorescent amine (ANTS) for PAGE for visualization or a lipophilic amine (TBT) for HPLC/MS for molecular weight determination. After determining visually by PAGE assay that ring scission was the principal method of glucan oxidation, oxidized carbohydrate were labeled with with a heavy lipophillic group and fragments were counted to determine the oxidation frequency carbons of glucose in the laminaran molecule. The oxidations were carried out in gross excess of glucose monomers relative to iron ions and hydrogen peroxide to minimize the likelihood of re-oxidation of glucan fragments.