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

by Francis Yat Ping Kwong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1987. / Bibliography: leaves [135]-[152]

Thesis (Ph. D.)--University of Wisconsin--Madison, 1938. / Typescript. Includes abstract and vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves i-ii).

Thesis (M.S.)--University of Wisconsin--Madison, 1965. / eContent provider-neutral record in process. Description based on print version record. Bibliography: l. 82-86.

Thesis (Ph. D. in Biophysics)--University of California, Berkeley, June 1958. / Bibliography: leaves 77-80.

Thesis (Ph.D.)--University of California, Berkeley, 1961. / "UC-48 Biology and Medicine" -t.p. "TID-4500 (16th Ed.)" -t.p. Includes bibliographical references (p. 54-58).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1982. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 278-287).

Fibrinogen adsorption to human erythrocytes has been implicated as the mechanism responsible for the reversible aggregation of red cells. The object of this thesis was to measure the binding of fibrinogen to human erythrocytes and to compare the binding with aggregation behaviour. The binding of isolated ¹²⁵I-fibrinogen to red cells was estimated from the amount of radiolabel removed from solution upon addition of cells, from the amount of radiolabel retained in cell pellets produced by ultracentrifugation and corrected for intracellular radiolabel, and from radiolabel retained by cells after exhaustive washing in protein-free buffer. Binding of human albumin to red cells was determined by the same methods. The free energy of formation of red cell/red cell contacts due to isolated fibrinogen was determined by the method of Evans and Buxbaum (Biophys. J. 3̲4̲, 1-12, 1981). The solution depletion method required corrections for dilution effects and was not sensitive enough for detailed determination of binding of these proteins to red cells. Binding estimated from cell pellets and corrected for intracellular radiolabel increased linearily with protein concentration to 12 mg/mL for fibrinogen and 50 mg/mL for albumin. The binding at 1 mg/mL was between 50 and 250 molecules per cell for fibrinogen and between 1000 and 1400 molecules per cell for albumin. Binding determined from wash analyses was similar to pellet binding corrected for free radiolabel. The surface affinity of red cells for red cell beads was determined to be 1.8 ± 0.5 x 10⁻³ erg/cm² (mean ± SEM, n = 4) and 2.8 ± 0.8 x 10⁻³ erg/cm² (mean ± SEM, n = 28) at 4.4 and 8.8 mg/ml fibrinogen respectively. Adhesion between red cells and red cell beads occurred at 2.2 mg/mL fibrinogen, but was too weak to allow estimation of the surface affinity. The surface affinities and binding estimates allow calculation of the mean binding energy for fibrinogen to the red cell surface. The surface affinity per molecule, assuming 250 molecules per cell at 1 mg/mL, was 30 ± 10 and 24 ± 7 kilocalories/mole at 4.4 and 8.8 mg/mL respectively. The difference was not significant. This result poses a problem as binding energies of this order suggest that the adhesion would be irreversible. If fibrinogen binding were 1500 molecules per cell at 1 mg/mL a binding energy of 4 ± 1 kilocalories/mole would be implied. These results are interpreted as indicating that fibrinogen bound to red cells may be displaced during ultracentrifugation. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

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