Electron Spin Resonance and Fluorescence Observations on Erythrocytes, Erythrocyte Membranes, 13762 Mat-A Ascites Adenocarcinoma Cells, and Their Membranes, Effects of Membrane Perturbations

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Abstract
Membranes from erythrocytes or MAT-A 13762 tumor cells were labeled with the fatty acid spin probe I(5,10) or ANS [1-anilino-8-naphthalenesulfonate] and examined by ESR or fluorescence polarization in the presence or absence of the perturbants EDTA, trypsin, glutaraldehyde and dodecylsulfate. Extraction of cell membranes with hypotonic EDTA produced fragments in which the order parameters and fluorescence polarization values increased. Fluorescence polarization values using membranes labeled with diphenylhexatriene showed an apparent increase in membrane fluidity. A large portion of I(5,10) and fluorescence probes coextract with the peripheral membrane proteins in both membrane systems. Paramagnetic quenching of tryptophan fluorescence with I(5,10) and the spectral characteristics of ANS in these membranes indicated further that significant amounts of both probes bind either at or near the protein-lipid interface or directly to protein moieties. Trypsinization of cell membranes, which preferentially cleaves the large cytoskeletal proteins, fragmented the membranes and reduced the ESR order parameter. Glutaraldehyde immobilized I(5,10) in both types of membranes. The association of cytoskeletal proteins with the membrane apparently does not have any pronounced, consistent effect on biophysical properties of the bilayer. Attempts to apply these same probes to studies of the plasma membranes of intact cells were not successful because of the diffusion of the probes into the cells. These studies also point out difficulties in using probe-group techniques to determine the nature of changes in bilayer structural parameters and emphasize the need for a better understanding of probe-group localization and behavior in such studies.