Spin labeling and analysis of erythrocyte surfaces

• Main Author: Snoek, Robert
• Language: English
• Published: University of British Columbia 2010
• Subjects: Erythrocytes
• Online Access: http://hdl.handle.net/2429/25973

Spin labeling the oligosaccharides of the red cell membrane was achieved via selective oxidation of gal/ga1NAc (with galactose oxidase) or sialic acid residues (with mild periodic acid) followed by reductive amination of the oxidized sugars with NaBH₃CN and TEMPAMINE. Spin labeling the galactose residues resulted in low yields and specificity, hindering analysis of the spin labeled cells (SL-RBC). Higher specificity and yields were obtained by labeling sialic acids. A protocol was devised which gave maximum yields with no Heisenberg exchange or membrane alterations (as detected by gel electrophoresis). Detailed analysis of the product showed the majority of the spins to be on the PAS positive membrane proteins (glycophorin A, B and C), only 8% being associated with the lipids. Isolation of glycophorin A, the major sialoglycoprotein of the red cell membrane, revealed two modified sialic acids per molecule. Successful ESR interpretations could only be done by lysing the SL-RBC (producing SL-ghosts), eliminating spins which had become internalized (rather than covalently attached to the surface) during the reductive amination step. Assuming a random distribution of biradicals (since there were two spins per glycophorin), an average separation of 16±2 angstroms was calculated between the nitroxides. The spin labeled sialic acids exhibited relatively mobile spectra with Ʈc = 9 x l0⁻¹⁰s. Upon addition of wheat germ agglutinin (WGA), a lectin known to bind to glycophorin, the mobility of the spin label decreased. Even though WGA binding to SL-ghosts showed complex behaviour as detected by Scatchard plots (which required compensation for WGA impurities and non-specific binding), the ESR was only sensitive to the specific binding, the spin mobility decreasing with increasing WGA. The fact that the spin probe was monitoring sialic acids interactions was confirmed by addition of other lectins. Only lectins which interact with glycophorin altered the ESR signal. === Science, Faculty of === Chemistry, Department of === Graduate