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1. Incorporation of stearic acid nitroxides into erythrocyte ghosts markedly depresses the fluorescence of membrane protein tryptophan residues. 5-Nitroxide stearate quenches fluorescence more efficiently than 16-nitroxide stearate. Both compounds exhibit dynamic (diffusion-limited) quenching above 0.28 mumol/mg protein and static quenching at lower nitroxide protein ratios. Static quenching can be attributed to high affinity binding of nitroxide stearates by membrane protein. The dynamic phase represents distribution of the stearate analogues into a fluid lipid system. 2. Protein fluorophores accessible to quenching by a cholesterol analogue, androstane nitroxide, are saturated at low nitroxide/protein ratios (less than 0.14 mumol/mg protein), without resolution of a static quenching phase. This suggests that sterols are segregated away from protein, probably in CLusters". 3. Paramagnetic quenching by stearate nitroxides increases abruptly between 35 and 50 degrees C. This discontinuous enhancement of quenching by temperature is reversible up to 41 degrees C but irreversible at higher temperatures. The discontinuity is also diminished by lowering pH from 7.3 through 6.5 to 6.0. Quenching by androstane nitroxide increases linearly with temperature up to approx. 41 degrees C and then rises exponentially. We attribute the reversible quenching thermotropism detected by stearate derivatives to reversible, thermotropic unfolding and/or depolymerisation of membrane proteins. The irreversible phase, detected also by the sterol derivative can be attributed to non-reversible protein denaturation. 4. Paramagnetic quenching of membrane tryptophan fluorescence by stearate derivatives is minimal at approx. pH 7.1 (35 degrees C) and increases sharply at lower and higher pH values, suggesting that two categories of protein residues, titrating between pH 6 and 8, profoundly influence the association of fatty acyl chains and penetrating protein segments. Quenching by androstane nitroxide exhibits no significant variation between pH 6 and 8, consistent with other data indicating that erythrocyte membrane sterols are segregated from membrane proteins, probably in clusters. 5. Our new approach confirms previous suggestions of a boundary layer of lipid in close association with some proteins in erythrocyte membranes, as well as experiments indicating that the lipid status in this boundary layer depends on that state of membrane proteins. However, sterols appear to be largely excluded from this boundary domain. Our data further show that lipid-protein interactions in erythrocyte membranes can vary significantly with fluctuations of temperature and pH in the physiological range.

Three classes of lipoidal nitroxide spin probes reversibly perturb erythrocyte membranes at low concentrations (10(-10)-10(-5) M). This is manifest in (a) decreased osmotic fragility and (b) alterations of surface topology. At bulk phase nitroxide concentrations providing maximal osmotic stabilization, the erythrocytes exhibit a classic echinocyte morphology. At nitroxide concentrations very slightly higher than those yielding minimal osmotic fragility (10(-5)-10(-4) M), the cellus undergo a sphering reaction and lyse. The morphologic sequence seen in intact cells is not observed in erythrocyte ghosts. We suggest that the spin probes initially concentrate in focal domains, which expand into echinocytic protrusions primarily due to localized weakening of membrane cohesion. We propose that cell lysis involves an irreversible breakdown in membrane domain structure.

1. Perylene, whether incorporated into lecithin or lecithin/cholesterol (1:1) liposomes, exhibits identical fluorescence spectra, but fluorescence in the presence of cholesterol is enhanced by 30-50%. 2. The fluorescence of perylene in pure dipalmitoyllecithin vesicles increases sharply at the transition temperature (Tt equals 41 degrees C). No such fluorescence jump is observed in lecithin/cholesterol (1:1) micelles. 3. In lecithin liposomes maximal quenching of perylene fluorescence at 25 degrees C is effected by cholestane spin label (80%) followed by androstane spin label (70%), 5-nitroxide stearate (60%) and 16-nitroxide stearate (50%). 4. In liposomes containing 5 mol % cholesterol these differences are reduced; however, the sequence of quenching efficiencies is the same except for the nitroxide stearates, which interchange their positions. 5. 5. Paramagnetic quenching of perylene fluorescence is stable below 35 degrees C and above 45 degrees C, but decreases sharply about the phase-transition temperature of dipalmitoyllecithin. 6. In lecithin/cholesterol (1:1, molar ratio) lipsomes fluorescence quenching diminishes linearly, but only slightly, with increasing temperature. 7. Cholestane spin label and androstane spin label at concentrations of greater than 20 mol % themselves suppress the quenching discontinuity at Tt, indicating a cholesterol-like structural effect. 8. The quenching phenomena observed are attributed to a non-random accommodation of fluorophore and quencher molecules (co-clustering) below the phase transition and a statistical distribution of both impurities above Tt. 9. In the presence of cholesterol the clustering tendencies are reduced or even eliminated; this is compatible with the concept that cholesterol fluidizes the phosphatide acyl chains below the transtion temperature.

1. Paramagnetic quenching of erythrocyte membrane protein fluorescence by nitroxide-labelled lipid analogues has been studied as a function of temperature and quencher concentration, as well as after cross-linking of membrane proteins by glutaraldehyde. 2. Quenching due to nitroxide stearates reveals a static component, due to binding of quencher molecules to protein, superimposed upon a diffusion-limited component. 3. Static quenching decreases progressively above 35 degree C, a temperature region where a thermotropic discontinuity is known to occur (Bieri, V. G. and Wallach, D. F. H. (1975) Biochim. Biophys. Acta 406, 415-423). 4. Diffusion-limited quenching becomes progressively more prominent as the temperature is raised above 15 degree C. 5. Exposure of membranes to varying concentrations of glutaraldehyde indicates that membrane proteins relatively poorly accessible to cross-linking are those responsible for the membrane thermotropism above 35 degree C. 6. Protein fluorophores accessible to androstane nitroxide are saturated at a low quencher/protein ratio. This ratio is stable below 35 degree C but increases by 50% between 35 and 55 degree C.