Spin probe clustering in human erythrocyte ghosts

Summary A model has been developed for 5-nitroxide stearate, I(12,3), distribution in human erythrocyte ghosts which accurately predicts ESR spectral alterations observed with increased probe/total lipid (P/L) at 37°C. This spin probe occupies a class of high-affinity, noninteracting sites at low loading. Saturation occurs with increasing probe concentration, and, at higher loading, the probe inserts itself at initially dilute sites to form membranebound clusters of variable size. No low probe remains at high P/L where all I(12,3) clusters in a concentrated phase. This model allows determination of the dilute/clustered probe ratio, and shows that I(12,3) segregates in erythrocytes at what might otherwise be considered low P/L (e.g., 1/359). These findings validate the earlier use of empirical parameters to estimate probe sequestration in biological membranes.     

Fatty-acid spin probe interactions with erythrocyte ghosts and liposomes prepared from erythrocyte ghosts

Summary A model for the binding of 5-nitroxide stearate, I(12,3). to human erythrocyte ghosts was developed by comparing spin probe interactions with ghosts and liposomes prepared from ghosts. At low probe/lipid (P/L<1/2500), I(12,3) binds to a similar class of high-affinity, noninteracting sites in both ghosts and liposomes, indicating that lipid moieties are responsible for probe uptake. Saturation occurs in both systems with increasing P/L, and, at higher loading (e.g., P/L=1/360 for ghosts and liposomes), the probe inserts itself at initially dilute sites to form a class of low-affinity sites consisting of clusters of variable size. At still higher P/L ranges (>1/100), much increased probe uptake was observed in ghosts than in liposomes, which was attributed to another class of low-affinity sites, representing nonspecific interactions of I(12,3) with membrane proteins. The nature of the spectral components and ultrafiltration experiments with ghosts labeled at high P/L indicate that both dilute and clustered I(12,3) are due to membrane-incorporated probe.     

Thermotropic lipid phase separations in human platelet and rat liver plasma membranes

Electron spin resonance (ESR) studies were conducted on human platelet plasma membranes using 5-nitroxide stearate, I(12,3). The polarity-corrected order parameter S and polarity-uncorrected order parameters S(T parallel) and S(T perpendicular) were independent of probe concentration at low I(12.3)/membrane protein ratios. At higher ratios, S and S(T perpendicular) decreased with increasing probe concentration while S(T parallel) remained unchanged. This is the result of enhanced radical interactions due to probe clustering. A lipid phase separation occurs in platelet membranes that segregates I(12,3) for temperatures less than 37 degrees C. As Arrhenius plots of platelet acid phosphatase activity exhibit a break at 35 to 36 degrees C, this enzyme activity may be influenced by the above phase separation. Similar experiments were performed on native [cholesterol/phospholipid ratio (C/P) = 0.71] and cholesterol-enriched [C/P = 0.85] rat liver plasma membranes. At 36 degrees C, cholesterol loading reduces I(12,3) flexibility and decreases the probe ratio at which radical interactions are apparent. The latter effects are attributed to the formation of cholesterol-rich lipid domains, and to the inability of I(12,3) to partition into these domains because of steric hinderance. Cholesterol enrichment increases both the high temperature onset of the phase separation occurring in liver membranes from 28 degrees to 37 degrees C and the percentage of probe-excluding, cholesterol-rich lipid domains at elevated temperatures. A model is discussed attributing the lipid phase separation in native liver plasma membranes to cholesterol-rich and -poor domains. As I(12,3) behaves similarly in cholesterol-enriched liver and human platelet plasma membranes, cholesterol-rich and -poor domains probably exist in both systems at physiologic temperatures.     

Effect of calcium, insulin and growth hormone on membrane fluidity. A spin label study of rat adipocyte and human erythrocyte ghosts

ESR spectra were recorded from rat epididymal adipocyte ghosts labeled with the 5-nitroxide stearic acid spin probe, I(12,3). Polarity-corrected and approximate order parameters, that are sensitive to the flexibility of the incorporated label, were used to evaluate the membrane lipid fluidity. Addition of CaCl2 a 37 degrees C decreased the fluidity, as indicated by positive increases in the order parameters. The ordering effect of Ca2+ was concentration-dependent, reached saturation at approx. 3--4 mM, and was completely reversed by excess EGTA. Previous studies indicated that low- and high-affinity sites on adipocyte plasma membranes are able to bind 45Ca2+, and our results suggest that Ca2+-induced alterations in the lipid fluidity involve cation binding to low-affinity sites. The cellular movements of Ca2+ and, in particular, the binding of Ca2+ to the plasma membrane may play important roles in insulin's action on fat cell function. The possibility that insulin directly alters the membrane fluidity was tested by adding hormone to freshly-prepared I(12,3)-labeled adipocyte ghosts. Insulin, at concentrations (10(-6) M) that enhance glucose uptake into intact adipocytes, did not affect the fluidity of ghosts suspended in buffers with or without Ca2+. The fluidities of I(12,3)-labeled rat adipocyte ghosts or human erythrocyte ghosts were also unaffected by various forms of human growth hormone.     

Effects of calcium,lanthanum, and temperature on the fluidity of spin-labeled human platelets

Summary Previous platelet studies have shown that calcium plays important roles in stimulus-secretion coupling, aggregation, and other membrane-associated functions. In addition, lanthanum induces platelet aggregation and the platelet release reaction and also influences platelet responsiveness to various stimuli. The spin-label results presented here suggest that one mechanism through which calcium and lanthanum mediate their effects on platelet functions may be by decreasing the lipid fluidity of the surface membrane.The structure of platelet membrane lipids was examined with the spin-label method. Washed human platelets were labeled with the 5-, 12- and 16-nitroxide stearic acid spin probes. Order parameters which measure the fluidity of the lipid environment of the incorporated probe may be calculated from the electron spin resonance (ESR) spectra of 5-nitroxide stearate [I(12,3)]-labeled cells. Evidence is presented which indicates that these spectra principally reflect properties of the platelet surface membrane lipids. The membrane fluidity increased with temperature for the range 17 to 37 °C. Either calcium or lanthanum additions to intact cells increased the rigidity of the platelet membranes at 37 °C, although the La³⁺ effect was larger and occurred at lower concentrations than that of Ca²⁺. For example, addition of 1mm La³⁺ or 4mm Ca²⁺ increased the order parameter of I(12,3)-labeled platelets by 4.3±1.7% or 2.1±0.5%. Preliminary studies conducted on purified platelet plasma membranes labeled with I(12,3) indicated that 1mm LaCl₃ or 4mm CaCl₂ additions similarly decreased the lipid fluidity at 37 °C. The above cation-induced effects on the fluidity of whole platelets were reversed by the use of the divalent cation-chelating agent ethylene glycol-bis-(-aminoethyl ether)-N,N-tetra-acetic acid (EGTA). Lastly, lanthanum (0.2–1mm) caused rapid aggregation of platelets which were suspended in a 50-mm Tris buffer pH 7.4 that did not contain adenosine.     

Structure and thermotropic phase behaviour of detergent-resistant membrane raft fractions isolated from human and ruminant erythrocytes

Detergent-resistant membrane raft fractions have been prepared from human, goat, and sheep erythrocyte ghosts using Triton X-100. The structure and thermotropic phase behaviour of the fractions have been examined by freeze-fracture electron microscopy and synchrotron X-ray diffraction methods. The raft fractions are found to consist of vesicles and multilamellar structures indicating considerable rearrangement of the original ghost membrane. Few membrane-associated particles typical of freeze-fracture replicas of intact erythrocyte membranes are observed in the fracture planes. Synchrotron X-ray diffraction studies during heating and cooling scans showed that multilamellar structures formed by stacks of raft membranes from all three species have d-spacings of about 6.5 nm. These structures can be distinguished from peaks corresponding to d-spacings of about 5.5 nm, which were assigned to scattering from single bilayer vesicles on the basis of the temperature dependence of their d-spacings compared with the multilamellar arrangements. The spacings obtained from multilamellar stacks and vesicular suspensions of raft membranes were, on average, more than 0.5 nm greater than corresponding arrangements of erythrocyte ghost membranes from which they were derived. The trypsinization of human erythrocyte ghosts results in a small decrease in lamellar d-spacing, but rafts prepared from trypsinized ghosts exhibit an additional lamellar repeat 0.4 nm less than a lamellar repeat coinciding with rafts prepared from untreated ghosts. The trypsinization of sheep erythrocyte ghosts results in the phase separation of two lamellar repeat structures (d = 6.00; 5.77 nm), but rafts from trypsinized ghosts produce a diffraction band almost identical to rafts from untreated ghosts. An examination of the structure and thermotropic phase behaviour of the dispersions of total polar lipid extracts of sheep detergent-resistant membrane preparations showed that a reversible phase separation of an inverted hexagonal structure from coexisting lamellar phase takes place upon heating above about 30 °C. Non-lamellar phases are not observed in erythrocytes or detergent-resistant membrane preparations heated up to 55 °C, suggesting that the lamellar arrangement is imposed on these membrane lipids by interaction with non-lipid components of rafts and/or that the topology of lipids in the erythrocyte membrane survives detergent treatment.     

Fatty acid-binding to erythrocyte ghost membranes and transmembrane movement

Summary Palmitate binding to human erythrocyte ghost membranes has been investigated with ghost preparations suspended in 0.2% albumin solutions. Free unbound palmitate in the extracellular water phase was measured in equilibrium studies using albumin-filled acid loaded ghosts as small semipermeable bags. The apparent dissociation constant of binding to the membrane is 13.5 nM and the binding capacity 19 nmoles per 7.2 × 109 cells.The 0°C exchange efflux kinetics of palmitate from albumin-filled ghosts is described by a model, which provides estimates of the rate constant of membrane transfer, k₃ = 0.024 s^–1, independent of the molar ratio of palmitate to albumin () and of a mean dissociation rate constant of the palmitate-albumin complex, k₁ = 0.0015 s^–1 at 0.2, allowing for a heterogeneity of the palmitate binding to albumin.The values of a third kinetically determined dependent model constant, Q, the ratio of palmitate bound to the membrane inner surface to palmitate on intracellular albumin, are not different from the Q values obtained by equilibrium experiments.The temperature dependences of k₁ and k₃ in the interval 0°C to 15°C give activation energies of 96 and 103 kJ/mole, respectively. The 0°C exchange efflux increases about 2 fold in response to a rise of pH from 6 to 9. The results suggest a carrier mediated palmitate flux at low with a V_max about 2 pmoles min^–1 cm^–2 at 0°C pH 7.3.     

Water exchange through erythrocyte membranes: p-choloromercuribenzene sulfonate inhibition of water diffusion in ghosts studied by a nuclear magnetic resonance technique

A comparison of water diffusion in human erythrocytes and ghosts revealed a longer relaxation time in ghosts, A comparison of water diffusion in human erythrocytes and ghosts revealed a longer relaxation time in ghosts, corresponding to a decreased exchange rate. However, the diffusional permeability of ghosts was not significantly different from that of erythrocytes . The changes in water diffusion following exposure to p-chloromercuribenzene sulfonate (PCMBS) have been studied on ghosts suspended in isotonic solutions. It was found that a significant inhibitory effect of PCMBS on water diffusion occurred only after several minutes of incubation at 37°C. No inhibition was noticed after short incubation at 0°C as previously used in some labelling experiments. This indicates the location in the membrane interior of the SH groups involved in water diffusion across human erythrocyte membranes. The nuclear magnetic resonance ( n . m . r . ) method appears as a useful tool for studying changes in water diffusiofl in erythrocyte ghosts with the aim of locating the water channel.     

On the Ability of High Density Lipoproteins to Remove Phospholipid Peroxidation Products from Erythrocyte Membranes

To study the transfer of oxidized phospholipids from cell membranes to high-density lipoproteins (HDL), human Cu²⁺-oxidized erythrocyte membranes were incubated with HDL₃ subfraction for 17 h at 37°C followed by isolation of the supernatant, precipitation from it of HDL₃, and determination of lipid peroxide products (LPP) in them. The incubation increased the content of lipid hydroperoxides in HDL₃ significantly (by 32 and 40% calculated per ml of sample or mg of protein) and of malondialdehyde (by 27 and 34%, respectively) compared to control (incubation of HDL₃ alone). The content of conjugated dienes did not change significantly. Fluorescence analyses of isolated HDL₃ particles showed that the content of fluorescent products (_ex = 365 nm, _em = 430 nm) in them was 2.5 times higher than in control, and the number of binding sites for the 1-anilinonaphthalene-8-sulfonic acid probe decreased by 22%. This also confirms accumulation of LPP in the lipoprotein subfraction. It seems likely that an increase in LPP (at least hydroperoxides) in HDL₃ after their incubation with oxidized membranes occurs via transport of phospholipids containing LPP from erythrocyte membranes to lipoproteins. The data on the ability of HDL₃ to accept LPP from erythrocyte membranes in vitro suggest that HDL₃ may have a protective action on cell membranes undergoing oxidation in vivo as well.     

Antibodies directed toward human erythrocyte Ca2+-ATPase: effect on enzyme function and immunoreactivity of Ca2+-ATPases from other sources

Antibodies directed against purified human erythrocyte Ca²⁺-ATPase (purified according to a procedure modified from V. Niggli, J. T. Penniston, and E. Carafoli, 1979, J. Biol. Chem., 254, 9955–9958) were raised in rabbits. In competitive radioimmunoassay tests of immunological cross-reactivity, human erythrocyte Ca²⁺-ATPase shows a consistent pattern of immunological similarity to the Ca²⁺-ATPases derived from cell surface fractions of other species, such as rat and dog erythrocyte ghosts, rat corpus luteum plasma membranes, and rat brain synaptic plasma membranes. On the other hand, a purified Ca²⁺-ATPase preparation from rabbit skeletal muscle sarcoplasmic reticulum failed to show any immunological similarity to the human enzyme. The amount of Ca²⁺-ATPase protein in the erythrocyte ghosts was estimated to be about 0.6 μg/mg ghost protein, which was not too different from the calculated value of 1.2 ± 0.2 μg/mg ghost protein (mean ± SD, n = 6) based on the calmodulin binding studies of the erythrocyte ghosts. Anti-Ca²⁺-ATPase immunoglobulin G inhibited enzyme activity and calcium transport, showing that at least one subpopulation of antibodies can block the active site of the enzyme. The antibodies had no effect on the binding of calmodulin to erythrocyte membranes.     

Proteolytic degradation of human erythrocyte band 3 by membrane-associated protease activity

Summary Antisera directed against the cytoplasmic portion of human erythrocyte Band 3 were used to follow the degradation of the band 3 molecule. Small amounts of Band 3 were degraded when well-washed red cell membrane ghosts were incubated in the cold; this process was greatly accelerated by incubating ghosts at 37°C. Band 3 labeled with pyridoxal-phosphate was digested at comparable rates. Band 3 digestion also took place when alkali-extracted ghost membranes were incubated at 37° for prolonged periods. These results suggest that human erythrocytes contain tightly bound, membrane-associated proteolytic activity.     

Thiamin transport by human erythrocytes and ghosts

Summary Thiamin transport in human erythrocytes and resealed pink ghosts was evaluated by incubating both preparations at 37 or 20°C in the presence of [³H]-thiamin of high specific activity. The rate of uptake was consistently higher in erythrocytes than in ghosts. In both preparations, the time course of uptake was independent from the presence of Na⁺ and did not reach equilibrium after 60 min incubation. At concentrations below 0.5 m and at 37°C, thiamin was taken up predominantly by a saturable mechanism in both erythrocytes and ghosts. Apparent kinetic constants were: for erythrocytes,K _m =0.12, 0.11 and 0.10 m andJ _max=0.01, 0.02 and 0.03 pmol·l^–1 intracellular water after 3, 15, and 30 min incubation times, respectively; for ghosts,K _m =0.16 and 0.51 m andJ _max=0.01 and 0.04 pmol·l^–1 intracellular water after 15 and 30 min incubation times, respectively. At 20°C, the saturable component disappeared in both preparations. Erythrocyte thiamin transport was not influenced by the presence ofd-glucose or metabolic inhibitors. In both preparations, thiamin transport was inhibited competitively by unlabeled thiamin, pyrithiamin, amprolium and, to a lesser extent, oxythiamin, the inhibiting effect being always more marked in erythrocytes than in ghosts. Only approximately 20% of the thiamin taken up by erythrocytes was protein-(probably membrane-) bound. A similar proportion was esterified to thiamin pyrophosphate. Separate experiments using valinomycin and SCN^– showed that the transport of thiamin, which is a cation at pH 7.4, is unaffected by changes in membrane potential in both preparations.     

Release of β-galactosidase from E. coli by a plasmid containing a temperature sensitive lytic function

Summary We report the construction of a plasmid which carries the øX174 lysis gene E downstream from the lambda pL promoter. This plasmid is capable of triggering lysis inE. coli when the growth temperature is raised from 30°C to 42°C. The kinetics of release of -galactosidase and intracellular protein have been determined, as well as the decrease in cell viability upon induction of lysis. In addition, -galactosidase can be released efficiently after induction by a short sonication period. Both methods allow efficient release of -galactosidase.     

Characterization of the Band 3 substrate site in human red cell ghosts by NDS-TEMPO,a disulfonatostilbene spin probe: The function of protons in NDS-TEMPO and substrate-anion binding in relation to anion transport

Summary NDS-TEMPO is a specific disulfonatostilbene spin label for the Band 3 substrate site (K. F. Schnell, W. Elbe, J. Käsbauer & E. Kaufmann,Biochim. Biophys. Acta 732:266–275, 1983). The pH dependence of NDS-TEMPO binding and of chloride and sulfate binding was studied in resealed human erythrocyte ghosts. pH was varied from 6.0 to 9.0. The ESR spectra from NDS-TEMPO-labeled red cell ghosts exhibited a strong immobilization of membrane-bound NDS-TEMPO. Changes of pH had no effect upon the mobility of membrane-bound NDS-TEMPO. A mutual competition between NDS-TEMPO binding and the binding of the substrate-anions, chloride and sulfate, was observed throughout the entire pH range. The maximal number of NDS-TEMPO binding sites per cell was in the range of 9.0×10⁵ to 1.10×10⁶ and was found to be insusceptible to changes of pH. The NDS-TEMPO/substrate-site and the chloride/substratesite dissociation constants amounted to 1.25 m and to 17mm and were independent of pH from pH 6.0 to 8.0, while the sulfate/substrate-site dissociation constant displayed a strong pH dependency with a maximum of 50mm at about pH 7.0. The NDS-TEMPO inhibition constants from the chloride and the sulfate flux experiments were 0.5 m (0°C) and 1.8 m (25°C), respectively, and are in close accordance with the NDS-TEMPO/substrate-site dissociation constants. Our studies provide strong evidence for the assumption that NDS-TEMPO binds in fact to the substrate site of Band 3. They show that the strong pH dependence of the chloride and of the sulfate transport cannot result from the pH dependency of substrate-anion binding, but point to the participation of ionizable regulator sites in transport catalysis. These regulator sites seem to be positioned outside the substrate site of the Band 3 transport domain.     

Inhibition of the phosphate self-exchange flux in human erythrocytes and erythrocyte ghosts

Summary The phosphate self-exchange flux in resealed erythrocyte ghosts and in amphotericin B (5.5 m) permeabilized erythrocytes has been studied. The phosphate self-exchange flux exhibits an S-shaped concentration dependence and a self-inhibition in permeabilized red cells while in erythrocyte ghosts no self-inhibition of the phosphate flux has been observed. The apparent halfsaturation constants and the apparent Hill coefficients were assessed by the double reciprocal Hill plots of versus 1/[P] ⁿ . The phosphate half-saturation constants amount to approx. 125mm in ghosts and to about 75mm in permeabilized cells while the apparent Hill coefficients amount to 1.15 and to 1.65 (pH 7.2, 25°C), respectively. Both chloride and sulfate elicit a mixed-type inhibition of the phosphate self-exchange flux. In permeabilized cells, chloride and sulfate shift the flux optimum towards higher phosphate concentrations and reduce the apparent Hill coefficients. In erythrocyte ghosts, the apparent Hill coefficients are insensitive to these anions. The double reciprocal Hill plots indicate a mixed-type inhibition of the phosphate self-exchange flux by DNDS, salicylate and dipyridamole and a noncompetitive inhibition of the phosphate self-exchange flux by phlorhizin. By contrast, the Hill-Dixon plots for chloride and sulfate indicate a competitive inhibition of the phosphate self-exchange flux in erythrocyte ghosts and a mixed-type inhibition in permeabilized cells and provide Hill coefficients of greater than unity for chloride and sulfate. The Dixon plots for DNDS, salicylate, phlorhizin and dipyridamole show a noncompetitive inhibition of the phosphate flux and provide apparent Hill coefficients of 0.95–1.0 for inhibitor binding. Using the Debye-Hückel theory, the effects of ionic strength upon phosphate transport and inhibitor binding can be eliminated. The results of our studies provide strong evidence for the assumption that electrostatic forces are involved in phosphate transport and in inhibitor binding.     

Gating Movements of Colicin A and Colicin Ia Are Different

The effect of temperature on fusion of Sendai virus with target membranes and mobility of the viral glycoproteins was studied with fluorescence methods. When intact virus was used, the fusion threshold temperature (20–22°C) was not altered regardless of the different types of target membranes. Viral glycoprotein mobility in the intact virus increased with temperature, particularly sharply at the fusion threshold temperature. This effect was suppressed by the presence of erythrocyte ghosts and/or dextran sulfate in the virus suspension. In these cases also, no change in the fusion threshold temperature was observed. On the other hand, reconstituted viral envelopes (virosomes) bearing viral glycoproteins but lacking matrix proteins were capable of fusing with erythrocyte ghosts even at temperatures lower than the fusion threshold temperature and no fusion threshold temperature was observed over the range of 10–40°C. The mobility of viral glycoproteins on virosomes was much greater and virtually temperature-independent. The intact virus treated with an actin-affector, jasplakinolide, reduced the extent of fusion with erythrocyte ghosts and the mobility of viral glycoproteins, while the treatment of virosomes with the same drug did not affect the extent of fusion of virosomes with erythrocyte ghosts and the mobility of the glycoproteins. These results suggest that viral matrix proteins including actins affect viral glycoprotein mobility and may be responsible for the temperature threshold phenomenon observed in Sendai virus fusion.This revised version was published online in August 2005 with a corrected cover date.     

Plant catechols prevent lipid peroxidation in human plasma and erythrocytes

The antioxidant activity of several plant catechol derivatives was tested in buffer, plasma, and human erythrocytes. In buffer, chlorogenic acid (CGA), caffeic acid (CA), and dihydrocaffeic acid (DCA) reduced ferric iron equally well in the ferric reducing antioxidant power (FRAP) assay. Low concentrations of the polyphenols enhanced the ability of plasma to reduce ferric iron by about 10%. In plasma, lipid hydroperoxide and F₂-isoprostane formation induced by a water-soluble free radical initiator were reduced by CGA at concentrations as low as 20 M. During incubation at 37°C, human erythrocytes took up DCA, but not CGA, and intracellular DCA enhanced the ability of erythrocytes to reduce extracellular ferricyanide. When intact erythrocytes were exposed to oxidant stress generated by liposomes containing small amounts of lipid hydroperoxides, extracellular CGA at a concentration of 5 M decreased both lipid peroxidation in the liposomes, and spared -tocopherol in erythrocyte membranes. These results suggest that the catechol structure of these compounds convey the antioxidant effect in plasma and in erythrocytes.     

Direct Measurement of Nitrite Transport Across Erythrocyte Membrane Vesicles Using the Fluorescent Probe, 6-Methoxy-N-(3-sulfopropyl) quinolinium

Nitrite was shown to quench the fluorescence of 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) almost twofold more than chloride. SPQ loaded inside vesicles prepared from asolectin and isolated erythrocyte ghosts allowed for the direct measurement of nitrite movement across these membranes. Movement of nitrite across asolectin occurred by diffusion as HNO₂ in a pH-dependent manner. By contrast, erythrocyte ghosts had very low diffusion rates for nitrous acid. Erythrocyte ghosts preloaded with 50 mM nitrite to quench SPQ fluorescence were utilized to study heteroexchange with externally added anions. SPQ fluorescence increases (becomes unquenched) with added bicarbonate and nitrate, indicating that nitrite is moving out of the preloaded vesicles. The pH optimum for this exchange was approximately 7.6 and exchange was inhibited by N-ethylmaleimide (NEM) and dihydro-4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS). These data indicate that nitrite moves across erythrocyte plasma membranes as NO₂- by a heteroexchange mechanism with other monovalent anions.     

Chemical synthesis of dinitrodiphenylsulfone derivatives of ethanolamine and serine and its application to the study of neighbor analysis of amino-phospholipids in the erythrocyte membrane

Summary The dinitrodiphenylsulfone derivatives of serine and ethanolamine have been prepared and their chromatographic and spectral properties are described. This cross-linking agent was used to determine the neighbor analysis of amino-phospholipids in the erythrocyte membrane. The results with erythrocyte ghosts show that at 50 m probe 31–50% of the total phosphatidylethanolamine is cross-linked to itself and 10–12% of the phosphatidylethanolamine is cross-linked to phosphatidylserine. Approximately 10–12% of the phosphatidylserine is cross-linked to itself and 16–20% of phosphatidylserine is cross-linked to phosphatidylethanolamine. The cross-linking of amino-phospholipids of ghosts with difluorodinitrodiphenylsulfone (9 Å span) is compared with cross-linking of these phospholipids by difluorodinitrobenzene (5 Å span). It is important to use the same sample of ghosts for this type of study since biological variability was seen in ghosts prepared from different batches of stored blood.