Spin Label Concentration Research Articles

27] Cell water viscosity

This chapter focuses on the technique used to obtain information relevant to cellular aqueous viscosity. Measuring the viscosity under conditions that perturb the cellular aqueous compartment is important in elucidating the role of viscosity in relation to a variety of physiological responses. By using the proper type of study, the compartmentalization of the cytoplasm can also be related to cytoplasmic viscosity. The electron spin resonance (ESR) technique is used to obtain information relevant to cellular aqueous viscosity. The ESR technique employing spin labels is used to measure translational motion and rotational motion. It involves the use of spin labels to measure rotational motion through the parameter known as “rotational correlation time.” The rotational motion of a spin label in a solid agar block is essentially identical to that dissolved in bulk water. The treatment indicates that the measurement of viscosity by distance-dependent, spin labe–spin label collisional events, where the spin label concentration is known, yields different and preferred information as compared to diffusional information that is inferred from rotational motion data.

Polarization of solvent nuclei by nitroxide spin labels at low magnetic fields

The application of nitroxide spin labels in low-field dynamic nuclear polarization studies of solvent-solute interactions is examined. Data obtained by saturating the nitroxide EPR line at a field-frequency combination determined after employing the Breit-Rabi formalism are most satisfactory provided low radical concentrations are used. Ratio method determinations of ultimate enhancements are appropriate in most cases, although application of an exchange-based model by using data on the broadening of spectra as the spin-label concentration is increased may provide a means of determining ultimate enhancements when the ratio method is not applicable. Studies with several nitroxide radicals show that similar species give polarization results that do not change when substituents far removed from the localized unpaired electron are changed. Perfluoropyridine is shown to exhibit increased scalar-dominated polarization with more aromatic nitroxides as has been observed for hexafluorobenzene; the dipolar-dominated enhancements for both monofluorobenzene and 2-fluoropyridine are also similar.

Electron-electron double resonance and saturation-recovery studies of nitroxide electron and nuclear spin-lattice relaxation times and Heisenberg exchange rates: lateral diffusion in dimyristoyl phosphatidylcholine.

Lateral diffusion constants of the stearic acid nitroxide radical spin label 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolindinyl oxide in dispersions of dimyristoyl phosphatidylcholine have been measured. Electron-electron double resonance methods were used to determine the product of the bimolecular collision frequency and T1e, the electron spin-lattice relaxation time. T1e in turn was measured by the technique of saturation recovery. The theoretical model of Träuble and Sackmann was then used to relate the bimolecular collision frequencies to the diffusion constants. Results are in agreement with other methods. Lower spin-label concentrations than were used in previous electron paramagnetic resonance studies are needed (label-to-lipid ratio less than 0.5 mol%). Analysis of the data also yields values of the nitrogen nuclear spin-lattice relaxation time of the nitroxide moiety. These values are indicative of membrane fluidity.

Lateral diffusion of lipids in sarcoplasmic reticulum membranes is area limited.

Scandella et al.1 used the technique of electron spin resonance to investigate lateral lipid diffusion in the membrane of sarcoplasmic reticulum from skeletal muscle. Their results suggested that lipids are free to diffuse within the plane of the membrane at rates comparable with those in isolated phospholipid bilayer systems of similar composition2–4. However, their study1 was carried out at temperatures between 50 and 70 °C. I have therefore now carried out similar studies, but at physiological temperatures and using fatty acid analogue spin labels. My results show absence of spin-exchange interaction at spin label concentrations below 2–4 mol %. This suggests the existence of diffusion barriers in the plane of the membrane which separate the lipids into clusters containing, on average, about 50 phospholipid molecules.

Spin label partitioning in lipid vesicles a model study for drug encapsulation

In this paper we attempt to outline some features which determine the encapsulation of small molecules into lipid vesicles. Spin labels derived from five carboxylic acids of different lengths were synthesized and incorporated in varying amounts into multilamellar and unilamellar vesicles made up of four different phosphatidylcholines. The influence on the release process of the bilayer rigidity and of the hydrophobicity of the entrapped molecule was systematically studied. The hydrophobicity is of critical importance and was estimated by measuring the partition coefficient ( P) between octanol and buffer. In multilamellar vesicles, molecules characterized by extreme P values (log P < −0.3 and log P > 5) can be efficiently entrapped. The rate of leakage is related to the P value according to a bell-shaped curve. Moreover, gel state of the bilayer and long acyl chains of the lipids are properties which favor a good entrapment. Small unilamellar vesicles may be formed in the presence of high concentrations of hydrophilic and lipophilic spin labels. However, the formation of unilamellar vesicles produces a significant reduction of the internal volume and of the entrapped water-soluble spin labels. High fractions of lipid-soluble spin labels can be incorporated in unilamellar vesicles but the vesicle stability is diminished.

Properties of mixed monolayers of lecithin and spin probe: II. Analysis of the molecular organization

This paper seeks to investigate the molecular organization of fatty acid spin label (4-palmitamido2,2,6,6-tetramethylpiperidine-1-oxyl) and dipalmitoylphosphatidylcholine molecules in monolayer vesicles. The molar ratio of the spin probe was varied from 1 to 22% and the corresponding electron spin resonance spectra were measured at 25 and 48°C. These spectra were analyzed in terms of the dipole-dipole and exchange interactions occurring between the radicals. From a computer simulation, a quantitative analysis of these interactions could be made. Comparison of these data with theoretical models indicate that above the lipid phase transition, i.e., at 48°C, the membrane is homogeneous and the label molecules diffuse randomly in the lecithin bulk phase ( D = 3.8 x 10 −8 cm 2/ sec). At 25°C, the membrane model can be viewed as a mosaic structure composed of spin probe clusters (7.9 x 10 11 clusters/cm 2). The size of the clusters as well as the minimal mean distance between two cluster edges could be calculated as a function of the spin label molar ratio. These results are used to interpret the observations made in the first paper of this series [Chatelain et al., J. Colloid Interface Sci. 72, 287 (1979)] concerning the surface viscosity of the mixed monolayer and the kinetics of the enzymatic activity of phospholipase A 2. It can be concluded that above a critical spin label concentration (10–15%) the properties of the entire lecithin phase are influenced by the presence of the spin probe clusters.

Free fatty acids and esters can be immobilized by receptor rich membranes from torpedo marmorata but not phospholipid acyl chains

A long chain spin labeled fatty acid and the corresponding ester have been introduced into receptor rich membranes from Torpedo Marmorata. Superimposed to a mobile component, typical of the lipid phase, a strongly immobilized component is seen on the ESR spectra, both at low temperature (−4°C) and at room temperature. An estimation of the amount of immobilized signal as a function of the concentration of spin label in the membrane shows that a saturation is reached which corresponds to approximately twice the concentration of receptor protein. In the same membranes, a spin labeled phosphatidylcholine was introduced by the release of the phosphatidylcholine analog from purified phosphatidylcholine exchange protein, preloaded with this spin label. No immobilized component is seen in this latter case even at low temperatures. Therefore the immobilized component seen with the fatty acid cannot be considered as reporting on an immobilized boundary layer of phospholipids surrounding the proteins. We attribute the immobilized signal seen with fatty acids and esters to a particular interaction of amphiphilic molecules with the cholinergic receptor protein. Very likely this effect can be associated with the local anaesthetic effect detected previously with this fatty acid.

Neisseria gonorrhoeae membrane microenvironment studied by spin-label electron spin resonance: comparison of colony types

Spin-label electron spin resonance was used to characterize the microenvironment around spin probes which localize (i) in membranes, (ii) at the membrane surface, or (iii) in the cytoplasm of living Neisseria gonorrhoeae. Four colony types (T1, T2, T3, and T4) of gonococci were compared on the basis of the electron spin resonance parameters 2T parallel to, S (order parameter), and tau c (microviscosity). The concentration of spin label used had little or no effect on viability. T1 and T2 gonococci were found to have a more restricted environment for molecular motion of a membrane surface spin label than did T3 and T4. The membrane fluidity, as measured by a membrane lipid spin label, of T4 (S = 0.571) was significantly greater than that of T1 or T3 (S = 0.580). This difference was detected at 37 degrees C, at 25 degrees C, in agar-grown bacteria, and in exponential-phase cells. Studies using spin labels which probe different levels of the membrane indicated the presence of a membrane flexibility gradient. Cytoplasmic spin-label studies indicated that the cytoplasm of all gonococcal colony types was three to five times more viscous than water.

Erythrocyte plasma membrane fluidity in avian muscular dystrophy

Erythrocytes from closely related dystrophic and normal adult chickens showed no differences in either membrane fluidity or polarity at 25°C, measured by electron paramagnetic resonance spectroscopy of fatty acid spin labels. The effects of temperature and spin label concentration on order parameters were identical for erythrocytes from dystrophic and normal birds. Similarly, erythrocyte ghosts from dystrophic and normal chickens showed identical spectral properties.

Erythrocyte membrane abnormalities in Duchenne muscular dystrophy monitored by saturation transfer electron paramagnetic resonance spectroscopy.

Saturation transfer electron paramagnetic resonance and the spin label 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl were used to study erythrocytes from patients with Duchenne muscular dystrophy or Becker syndrome and from age-matched normal boys. There were significant differences in the spectral intensities of erythrocytes from Duchenne patients when compared to controls. Spectral intensities increased with time in the former; no such change was observed in the latter. Saturation transfer electron paramagnetic resonance spectra of erythrocytes from patients with Becker syndrome were significantly different from those from Duchenne patients but were not significantly different from normals. These observations suggest the possible usefulness of these techniques in the differential diagnosis of Duchenne muscular dystrophy. Spin label concentration spectral studies suggest that the observed spectral differences between Duchenne patients and controls were due to differential spin exchange phenomena.

Fatty acid enhancement of human serum albumin binding properties. A spin label study.

The introduction of a new spin-labeled anionic ligand, 1-gamma-aminobutyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene, is reported. Under the experimental conditions, the first molar equivalent of this ligand is 93% bound to human serum albumin. With the addition of palmitate, the free spin label concentration decreases greatly, by almost 80%, in the presence of a fatty acid:albumin ratio of 3:1 to 4:1. The spectral characteristics of the bound spin label are also affected. The changes seen in the intensity of and the splitting between the high and low field extrema are indicative of perturbations of the protein molecule. It is seen then that the binding of each molar equivalent of fatty acid effects the conformation state of albumin and allosterically affects albumin binding properties. Computer spectral subtractions, furthermore, suggest that the binding of the first molar equivalent of palmitate specifically increases the affinity of the first two 1-gamma-amino-butyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene binding sites. The present results indicate that fluctuations in serum free fatty acid levels within the physiological range may have a major modulatory effect on the free serum levels of certain drugs and/or physiological substances that bind to albumin.

Spectral characterization of 15N spin labels

Nitrocide spin labels containing 15N have hyperfine lines at magnetic field positions different from those for 14N spin labels. This allows the use of an 14N and an 15N spin label for specific experiments where two events can be monitored at the same time. Before this technique can be used to full advantage, the spectral characteristics of 15N spin labels under various conditions must be known. This report describes, in a comparative way, the effects of restricted rotational motion and nitroxide—nitroxide interactions on 15N-Temponë and the corresponding 14N spin label. An interesting feature is that 15N extends the useful range of motion of spin labels to slower tumbling rates than those conveniently handled with 14N. A general description of line shape changes due to spin label concentration effects is made without resorting to formalism.

Spin Label Translational Diffusion in Solid Tristearin

Translational diffusion of the intermediate chain length spin label 7N14 has been detected and studied in a lipid environment which is in the bulk solid state. Under favorable circumstances this can occur at temperatures as much as 50 degrees C below the optical melting point. Translational diffusion allows 7N14 molecules to coalesce into impurity pools of high spin label concentration. Two other spin labels, 2N3 and 14N27, do not show a tendency to form such impurity pools. While 2N3 undergoes rapid tumbling at temperatures far below the melting point of the tristearin matrix, the molecules remain in an isolated state with no evidence of spin exchange. 14N27 is restricted in rotational motion in the solid matrix and also does not form impurity pools.