Spin labelling studies of carbohydrate polymers
Creators: name: Aplin, John Dalzell
Publisher: name: University of British Columbia
Year: 2010

description: Nitroxide reporter groups were used in two different capacities for esr studies of naturally occurring carbohydrates. As 'probes'—small molecules in solution—in aqueous polysaccharide sols and gels, their rotational and translational diffusion was found to be independent of the presence of polymer despite the pronounced effect of the latter on viscosity and other macroscopic properties. As 'labels'—covalently attached to the macromolecule—they were more useful, esr lineshape analysis yielding information about rotational reorientation, accessibility from solution and proximity to neighbouring unpaired spins. Cyanogen bromide activation, carbodiimide and chloroacetamide coupling and other methods were used to affix spin labels to agarose, Sepharoses, alginate, cellulose, wood, and the terminal non-reducing sialic acids of fetuin, bovine submaxillary mucin, the outer surface of the human erythrocyte, and tissue sections from human colon. In the solution glycoproteins and at the cell surface, rotational reorientation of the labels was found to be largely independent of the overall rotational diffusion of the cell or macromolecule. The gel-sol-gel transition of labelled agarose was examined, and it was found that the two forms, as defined macroscopically, are not clearly distinguishable at the microscopic level by the mobility of the polymer backbone. The Sepharose 4B gel bead was studied using nitroxide as a model ligand. Dipolar interactions between immobilised labels were observed, suggesting the possibility of multiple site binding of a protein in solution to the stationary phase in chromatographic separations. The randomness of the distribution of attachment sites was tested using spin dilution, solvent-mediated shrinkage of the beads and electron exchange with paramagnetic ions in the solution phase. Only the last revealed any inhomogeneity. The dependence of rotational correlation time (T) on the length of the 'spacer arm' connecting the nitroxide to the matrix was studied for alkylamine and oligoglycine linking groups, each of which was found to behave in similar fashion, T decreasing towards an asymptote with increasing spacer length. The data indicated that units containing ~ 12 bonds should be optimal for affinity chromatography, and literature reports provided ample corroboration. The dependence on concentration of electron exchange between nitroxides affixed to the surface of cellulose, and paramagnetic ions in solution, provoked the definition and derivation of accessibility parameters (z[sub i]) which increased, as expected, with spacer length. Negatively charged reducing agents (ascorbate, dithionite) gained access to nitroxides even at ~ 0.35 nm from the surface, while ferrous ions achieved only partial reduction; at ~ 1.8 nm the three agents were equally effective. These results were discussed in terms of proposed models for the cellulose-water interface. Measurement of electron-electron dipolar interactions enabled a new calculation of surface area to be made and the effect of drying to be studied.