Spin label studies on the human erythrocyte membrane. Two sites and two phases for fatty acid spin labels

Human erythrocytes, untreated and glutaraldehyde-treated, were spin labeled with three kinds of fatty acid labels, and their electron spin resonance (ESR) spectra were studied in detail at various temperatures. 1. The better spectral resolution could be obtained by packing the erythrocytes in a hematocrit capillary tube, because of the preferential parallel orientation of the cylindrical axes of erythrocyte-disc to the centrifugal axis. 2. It was demonstrated by the incorporation and the release of the labels that the membrane possessed two kinds of the fatty acid "sites": the tightly and weakly binding "sites" at the approximate molar ratio of 1:1. The rough estimates of the binding constants were obtained, which reproducibly varied with the blood donors over a period of a year. 3. The temperature dependency of the ESR spectra revealed the presence of two distinct phases, perhaps the solid and fluid phases. With lowering of the temperature, the fluid phase became more solid but the solid phase unchanged. The pretreatment of the erythrocytes with glutaraldehyde increased the amount of the frozen phase, corresponding to the decrease of the membrane flexibility.