5-Nitroxide stearic acid

Ohtsuru et al. (25) have recently investigated the behavior of phosphatidylcholine in a model system that simulated soy milk. They used spin-labelled phosphatidylcholine (PC ) synthesized from egg lysolecithin and 12-nitroxide stearic acid anhydride. The ESR spectrum of a mixture of PC (250 yg) and native soy protein (20 mg) homogenized in water by sonication resembled that observed for PC alone before sonication. However, when PC (250 yg) was sonicated in the presence of heat-denatured soy protein (20 mg), splitting of the ESR signal occurred. On this basis, they postulated the existence of two phases PC making up a fluid lamella phase and PC immobilized probably due to the hydrophobic interaction with the denatured protein. In a study of a soy-milk model, Ohtsuru et al. (25) reported that a ternary protein-oil-PC complex occurred when the three materials were subjected to sonication under the proper condition. Based on data from the ESR study, a schematic model has been proposed for the reversible formation-deformation of the ternary complex in soy milk (Figure 2). 

The 12-nitroxide stearic acid and the 8-nitroxide methyl palmitate were donated by J. D. Morrisett (John Baylor College of Medicine, Houston, Tex.). All other nitroxide stearic acid and stearate probes were purchased from Syva (California). All probe molecules were subsequently purified by preparative thin layer chromatography (TLC) until a single spot was obtained under all development conditions. TLC was carried out in a dry nitrogen atmosphere to avoid possible oxidation or hydrolysis during purification. Ultrapure silica gel, free from any plasticizers, was used to avoid possible further contamination. 

Figure 4. Surface pressure (dynes/cm) vs. area/molecule (A2) for 12-nitroxide stearic acid [I2-NS] at 10°, 20°> 38% 40°y and 50°C. Broken line indicates a near liquid-like film collapse.

Isotherms for monomolecular films of a series of nitroxide stearic acid and nitroxide palmitate and stearate (methyl ester) films are reported for the temperature range 9° 50°C. 

To understand the behavior of the mixed host-probe films, it is essential to understand the behavior of the pure films of both components. We previously investigated in detail the behavior of the pure films of 12-nitrox-ide stearate [2-( 10-carboxydecyl)-2-hexyl-4,4 -dimethyl-3-oxazolidinyloxyl] including its extraordinary temperature dependence (7, 8, 16). In this paper we extend our investigations to the pure films of other nitroxide stearic acid (and methyl ester) probes where the oxazolidine ring is attached to various carbon atoms of the stearic acid (or ester) hydrocarbon chain. This series of spin-label probes is one of those most extensively used to study cell membrane structure. It was used to define, among other things, an order parameter establishing the fluidity (17) and polarity profiles (18) of lipid bilayers. 

The results are shown in Figures 1 through 7. Figures 1 through 6 show isotherms over a range of temperatures for the 5-, 12-, and 16-nitroxide stearic acids, for the 5- and 16-nitroxide methyl stearates, and for the 8-nitroxide methyl palmitate over the range 9°-50°C. [We use 5-NS, 5-NS(Me), and 8-NP(Me) for the trivial names 5-nitroxide stearic acid, 5-nitroxide methyl stearate, and 8-nitroxide methyl palmitate, with... 

Observed collisions between 14N 15N spin-label pairs are indicated. DMPC and POPC molecules are also shown. POPC represents the major component (70%) of the EYPC mixture, (b) Bimolecular collision rate for a nitroxide moiety at the C16 position of the stearic acid alkyl chain with other SASLs in the DMPC alone and the DMPC with 10mol% lutein at 27°C. (From Yin, J.J. and Subczynski, W.K., Biophys../., 71, 832,1996. With permission.)... 

Oxidation of 2,2,4,4-tetraalkyloxazolidines gives stable nitroxide radicals (265) which are used as spin labels for probing biomolecular structures (69ACR17, B-76MI41800). The stearic acid derivative 12-doxylstearic acid (266) is commercially available. 

Spin labels are stable, paramagnetic molecules that, by their structme, easily attach themselves to various biological macromolecular systems such as proteins or cell membranes. Examples of spin labels that can be covalently bonded to specific sites of biological systems include nitroxide derivatives of A-ethylmaleimide, which bind specifically to -SH groups, and nitroxide derivatives of iodoacetamide, which bind specifically to methionine, lysine, and arginine residues of amino acids. Nonco-valently bonded spin-labels that can be incorporated into biological systems include nitroxide derivatives of stearic acid, of phospholipids, and of cholesterol. 

In recent years the systems most often studied by the spin-label method have been biological membranes and various models thereof. For example, using nitroxide derivatives of stearic acid, the organization of the phospholipid phase of biological membranes has been studied [6]. One interesting practical application of this type of spin-labeling is in the study of disease-state membranes. Intact erythrocyte mem-... 

Fig. 2 Chemical structures of common nitroxide spin probes Fremy s salt (potassium nitrosodi-sulfonate) TEMPO and derivatives (2,2,6,6-tetramethylpiperidine-l-oxyl), DOXYL (4,4-dimethyl-oxazolidine-l-oxyl) PROXYL (2,2,5,5-tetramethylpyrrolidine-l-oxyl) Dehydro-PROXYL (2,2,5,5-tetramethylpyrroline-l-oxyl) 5-DSA (5-DOXYL stearic acid) 16-DSA (16-DOXYL stearic...

The sensitized cascade reported in Ref [16] consisting of triplet ds-trans photoisomerization of the excited stilbene includes the triplet sensitizer (erythrosin B), the photochrome stilbene derivative probe (4-dimethylamino-4 -aminostilbene), and nitroxide radicals (5-doxyl stearic acid) quenching the excited triplet state of the sensitizer (Figure 10.11). 

A novel analytieal tool for the selective detection of local water inside soft mol. assemblies (hydrophobic cores, vesicular bilayers, and micellar structures) suspended in bulk water has been presented. Through the use of dynamic nuclear polarization (DNP), the NMR signal of water is ampUfied, as it interacts with stable radicals that possess about 658 times higher spin polarization. Stable nitroxide radicals covalently attached along the hydrophobic tail of stearic acid molecules that incorporate themselves into surfactant-based micelle or vesicle structures have been used, allowing to study the local water content and fluid viscosity inside oleate micelles and vesicles and Triton X-100 micelles to serve as model systems for soft molecular assembhes. ... 

Nitroxide lipid spin label derivatives 14-SASL, stearic acid bearing the spin label group on the 14-C atom. 14-PCSL phosphatidylcholine bearing the spin label on the 14-C atom of the sn-2 chain. 14-CLSL cardiolipin (diphos-phatidylglycerol) bearing the spin label on the 14-C atom of a single chain. 

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