Cytotoxicity of commonly used nitroxide radical spin probes

Since nitroxide radical spin probes are used frequently to test biophysical properties of cells, their use should be restricted to conditions that do not perturb normal cell growth and viability. Eight commonly used nitroxide radical spin probes have been tested for their effects on the survival of CHO cells. These include water-soluble spin probes Tempol, Tempamine, CTPO, CTPC and 4-maleimido-Tempo, and lipid soluble spin probes 5-Doxyl-, 12-Doxyl-, and 16-Doxylstearates. With the exception of 4-maleimido-Tempo, none of the water soluble spin labels inhibited cell survival at concentrations as high as 1 mM. At concentrations of 75 microM and higher, 4-maleimido-Tempo inhibited cell survival in a dose dependent manner. At concentrations commonly used for spin labeling of cells (30-50 microM) none of the lipid soluble spin probes tested was cytotoxic. At 100 microM only 5-Doxylstearate inhibited cell survival, whereas 12-Doxylstearate and 16-Doxylstearate had no effect.     

Structural dependence of nitroxide spin labels and nitroxide spin adducts on their reducibility by ascorbate ion

Summary

The reducibility of a series of nitroxides (aminoxyls) by ascorbate was tested by measuring the nitroxide decay rates with a stopped-flow electron paramagnetic resonance technique in aqueous phosphate buffer solution. The dependence of reactivity on the structures and pH of the medium was found for both cyclic nitroxides and nitroxide adducts of phenyl N-tert butyl nitrone (PBN). In cyclic nitroxides, the ring size is a dominant factor in determining reaction rates but substituents have additional effects on the rate depending on their electronegativity. For alkyl and hydroxyalkyl adducts of PBN, at fixed ascorbate concentration, half-lives increase with lengthening of the substituent, suggesting that a long chain in the substituent sterically protects the nitroxide group and thus prevents its reduction by ascorbate.     

Reliability of nitroxide spin probes in reporting membrane properties: a comparison of nitroxide- and deuterium-labeled steroids

The reliability for the study of membrane properties of the steroid nitroxide spin probe, 3-doxylcholestane, was tested by comparison of analogous data for the deuterated steroid, cholesterol-3 alpha-d. Good agreement between the two probes was found for the dependence of their order parameters on variation of temperature or cholesterol concentration in egg phosphatidylcholine bilayers. This finding is contrasted with the results of a previous study of fatty acid probes where poor agreement was found for the spectral responses of nitroxide- and deuterium-labeled species. The angular dependence of the ESR spectra of nitroxide-labeled probes in oriented multibilayer films was examined to determine if the probes were oriented in a tilted fashion in the bilayer. The 3-doxylcholestane probe and a doxylstearic acid labeled at position 14 orient with their long molecular axes perpendicular to the bilayer plane. In contrast, the stearic acid probe nitroxide labeled at position 5 does not appear to orient in such a fashion. We suggest that the behavior of the latter probe reflects the difficulty of inserting a bulky nitroxide group into a highly ordered region of the bilayer rather than an inherent tilting of the phospholipid acyl chains. On the basis of the comparisons between various types of probes, some suggestions are made concerning the choice of ESR spin probe to obtain reliable information in membrane studies.     

Synthesis and properties of chlorophyll-derived nitroxide spin labels

A general synthesis of chlorophyll-derived spin labels is reported. The starting point is chlorophyll a and a long chain spin-labeled alcohol. The four-step synthetic route yields new spin labels in quantities practical for membrane spin-labeling studies. Two examples prepared are 12′-proxyltridecyl pyropheophorbide a and 12′-proxylhexadecyl pyropheophorbide a. The spin labels were purified and characterized by thin-layer chromatography, high-pressure liquid chromatography, mass spectroscopy, nmr and visible spectroscopy, and the number of unpaired spins per molecule. ESR spectral parameters are reported. The chlorophyll-derived spin labels intercalate into fluid phospholipid bilayers and are observed in both the bilayer phase and bound to membrane proteins in chromatophores of the purple photosynthetic bacterium, Rhodopseudomonas sphaeroides.     

Interaction of nitroxide spin labels with chloroplasts

Chloroplasts isolated from oats eliminated the electron spin resonance (ESR) signals from spin labels in white light and partially restored them in far-red light. Only the white light-mediated reaction was blocked by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). In contrast, oat (Avena sativa L. cv. Garry and Park) leaf mesophyll protoplasts oxidized the spin labels in both white and far-red light, with and without DCMU. Light had no obvious effect on spin label motion within chloroplast membranes. The results suggest that, in isolated chloroplasts, nitroxide spin labels may be reduced by photosystem I within the thylakoid bilayer resulting in loss of the ESR signals. The reduced forms may be reoxidized by an element of the photosynthetic electron transport chain which operates between the DCMU block and the photosystem I reaction center. In addition, a light-mediated destruction of the spin labels occurs in both chloroplasts and protoplasts. The reduced form of the nitroxide (i.e. the hydroxylamine) may be resistant to this destruction.     

The fidelity of response by nitroxide spin probes to changes in membrane organization: The condensing effect of cholesterol

The response of doxyl fatty acid spin probes in egg lecithin bilayers to added cholesterol is compared with results from ²H-NMR. Large differences are found between the profiles of order parameter vs. label position and cholesterol concentration.At constant cholesterol content, the ESR spin probe order parameter decreases continuously as the label position is moved toward the terminal methyl region of the bilayer whereas an order parameter ‘plateau’ is observed for the upper region of the bilayer by ²H-NMR. In addition, the spin probe order parameters are smaller than those observed by ²H-NMR.Differences are also observed in the profiles of order parameter vs. cholesterol content for each label position. The spin probes detect a maximal response to added cholesterol for the central portion of the chains with much weaker responses near both ends of the chains. In contrast, the ²H-NMR results indicate a large, approximately constant response for the first ten positions in the chains with a decreasing response toward the terminal methyl group. For all the positions examined, the spin probes show a weaker response than that observed by ²H-NMR.A direct measure of the perturbing effect of a spin label is made by comparing the deuterium quadrupole splittings in egg lecithin-cholesterol bilayers for stearic acid with and without an attached doxyl moiety. The spin-labelled fatty acid has a much reduced quadrupole splitting and an opposite response to cholesterol addition.     

Exchange and shuttling of electrons by nitroxide spin labels

The ability of nitroxide spin labels to act as oxidizers of reduced nitroxides (hydroxylamines) in biological and model systems was demonstrated. All of the nitroxides tested were able to act as oxidizing agents with respect to hydroxylamine derivatives of nitroxides. The rates of these reactions were first order with respect to nitroxide concentration and with respect to hydroxylamine concentration, making the reaction second order overall. The second-order rate constants are reported for a number of these reactions. These reactions proceeded to an equilibrium state and the equilibrium constants for several combinations of reactants are presented. Both the rate constants and the equilibrium constants were found to be dependent on the ring structure of the nitroxide and hydroxylamine, with piperidines being reduced more easily and pyrrolidines and oxazolidines being oxidized more easily. All of the hydroxylamine derivatives were oxidized by air to their respective nitroxides, with the rate of this oxidation greater for pyrrolidines than for piperidines. Furthermore, hydroxylamines that are permeable to lipid bilayers were able to act as shuttles of reducing equivalents to liposome-encapsulated nitroxides that were otherwise inaccessible to reducing agents. This mechanism of shuttling of electrons was able to explain the relatively rapid reduction by cells of a nonpermeable nitroxide in the presence of a permeable nitroxide.     

Metabolism of nitroxide spin labels in subcellular fraction of rat liver: I. Reduction by microsomes

As part of an ongoing study of the role of subcellular fractions on the metabolism of nitroxides, we studied the metabolism of a set of seven nitroxides in microsomes obtained from rat liver. The nitroxides were chosen to provide information on the effects of the type of charge, lipophilicity and the ring on which the nitroxide group is locted Important variables that were studied included adding NADH, adding, induction of enzymed by intake of phenobarbital and the effects of oxygen. Reduction of nonparamagnetic derivatives and oxidation to paramagnetic derivatives were measured by electron-spin resonance spectroscopy. In general, the relative rates of reduction of nitroxides were similar to those observed with intact cells, but the effects of the various variables that were studied often differed from those observed in intact cells. The rates of reduction were very slow in the absence of added NADh or NADPH. The relative effect of these two nucleotides changed when animals were fed phenobarbital and paralleled the levels of NADPH cytochrome c reductase, cytochrome P-450, cytochrome b₅ and NADH cytochrome c reductase; results with purified NADPH-cytochrome c reductase were consistent with these results. In microsomes from uninduced animals the rate of reduction was about 10-fold higher in the absence of oxygen. The products of reduction of nitroxides by microsomes were the corresponding hydroxylmines. We conclude that there are significant NADH- and NADPH-dependent paths for reduction of nitroxides by hepatic microsomes, probably involving cytochrome c reductases and not directly involving cytochrome P-450. From this, and from parallel studies now in progress in our laboratory, it seems likely that metabolism by microsomes is an important site of reduction of nitroxides. However, mitochondrial metabolism seems to play an even more important role in intact cells.     

Metabolism of nitroxide spin labels in subcellular fraction of rat liver. I. Reduction by microsomes

As part of an ongoing study of the role of subcellular fractions on the metabolism of nitroxides, we studied the metabolism of a set of seven nitroxides in microsomes obtained from rat liver. The nitroxides were chosen to provide information on the effects of the type of charge, lipophilicity and the ring on which the nitroxide group is located. Important variables that were studied included adding NADH, adding NADPH, induction of enzymes by intake of phenobarbital and the effects of oxygen. Reduction to nonparamagnetic derivatives and oxidation back to paramagnetic derivatives were measured by electron-spin resonance spectroscopy. In general, the relative rates of reduction of nitroxides were similar to those observed with intact cells, but the effects of the various variables that were studied often differed from those observed in intact cells. The rates of reduction were very slow in the absence of added NADH or NADPH. The relative effect of these two nucleotides changed when animals were fed phenobarbital, and paralleled the levels of NADPH cytochrome c reductase, cytochrome P-450, cytochrome b5 and NADH cytochrome c reductase; results with purified NADPH-cytochrome c reductase were consistent with these results. In microsomes from uninduced animals the rate of reduction was about 10-fold higher in the absence of oxygen. The products of reduction of nitroxides by microsomes were the corresponding hydroxylamines. We conclude that there are significant NADH- and NADPH-dependent paths for reduction of nitroxides by hepatic microsomes, probably involving cytochrome c reductases and not directly involving cytochrome P-450. From this, and from parallel studies now in progress in our laboratory, it seems likely that metabolism by microsomes is an important site of reduction of nitroxides. However, mitochondrial metabolism seems to play an even more important role in intact cells.     

Computer modeling of nitroxide spin labels on proteins

Electron paramagnetic resonance using site‐directed spin labeling can be used as an approach for determination of protein structures that are difficult to solve by other methods. One important aspect of this approach is the measurement of interlabel distances using the double electron–electron resonance (DEER) method. Interpretation of experimental data could be facilitated by a computational approach to calculation of interlabel distances. We describe an algorithm, PRONOX, for rapid computation of interlabel distances based on calculation of spin label conformer distributions at any site of a protein. The program incorporates features of the label distribution established experimentally, including weighting of favorable conformers of the label. Distances calculated by PRONOX were compared with new DEER distances for amphiphysin and annexin B12 and with published data for FCHo2 (F‐BAR), endophilin, and α‐synuclein, a total of 44 interlabel distances. The program reproduced these distances accurately (r² = 0.94, slope = 0.98). For 9 of the 11 distances for amphiphysin, PRONOX reproduced the experimental data to within 2.5 Å. The speed and accuracy of PRONOX suggest that the algorithm can be used for fitting to DEER data for determination of protein tertiary structure. © 2011 Wiley Periodicals, Inc. Biopolymers 97: 35–44, 2012.     

Metabolism of nitroxide spin labels in subcellular fractions of rat liver. II. Reduction in the cytosol

As part of an ongoing study of the role of subcellular fractions on the metabolism of nitroxides, we studied the metabolism of a set of five nitroxides in cytosol derived from rat hepatocytes. The nitroxides were chosen to provide information on the effects of the type of charge and the ring on which the nitroxyl group is located. The rates of reduction were fastest for a six-membered positively charged nitroxide ('CAT-1') and slowest for an anionic five-membered ring nitroxide ('PCA'). Changing levels of glutathione, sulphydryl groups in general, NADPH or NADH had little or no effect on the rates of reduction, while the addition of ascorbate oxidase essentially abolished reduction of the nitroxides. The products of reduction by the cytosol were the corresponding hydroxylamines. The overall rates of reduction of neutral or anionic nitroxides were much slower than those observed with intact cells. We conclude that the primary source of metabolism of nitroxides by cytosol is reduction by ascorbate and that under most conditions reduction of nitroxides in the cytosol is not a major factor in the metabolism of nitroxides by cells.     

Electron spin echo studies of hemoglobin cyanide and nitroxide derivatives

Electron spin echo envelope modulation studies are performed on human hemoglobin cyanide, hemoglobin nitroxide and hemoglobin nitroxide + inositol hexaphosphate at neutral pH. The modulation data are Fourier transformed and are analyzed in the frequency domain. The frequency components observed from hemoglobin cyanide indicate modulation from the coordinated nitrogen N-1 of the proximal imidazole (His-F8). In the case of hemoglobin nitroxide, the binding of inositol hexaphosphate causes the nitrogen N-3 of the proximal imidazole to be protonated in some of the subunits. From a comparison with other studies on these derivatives of hemoglobin, these subunits are identified as the alpha-subunits.     

Oxidation of hydroxylamines to nitroxide spin labels in living cells

In the presence of oxygen, cells can oxidize hydroxylamines, which are the products of the reduction of nitroxides in cells, back to nitroxides. Lipid-soluble hydroxylamines are oxidized much more rapidly than water-soluble ones, and most of this oxidation is inactivated by heat or trichloroacetic acid, indicating that the principal mechanism is enzyme-linked. The rates of oxidation of some lipophilic hydroxylamines are comparable to the rates of reduction of the corresponding nitroxides. Hydroxylamines formed by reduction of aqueous soluble nitroxides are not oxidized by cells, except for slight oxidation of some pyrrolidine derivatives. The latter is due to autoxidation. The kinetics of oxidation of reduced lipid-soluble nitroxides are all first-order with respect to hydroxylamines, regardless of the position of the nitroxide group along the carbon backbone, indicating that the oxidation occurs within the membrane. The oxidation of hydroxylamines in cells in inhibited by cyanide but not by antimycin A or SKF-525A. We also describe an effective method to oxidize hydroxylamines and follow this reaction; the method is based on the use of perdeuterated [15N]Tempone.     

The fluidity and organization of mitochondrial membrane lipids of the brown adipose tissue of cold-adapted rats and hamsters as determined by nitroxide spin probes.

A detailed study of lipid fluidity and organization in the mitochondria of the brown adipose tissue from warm- and cold-adapted rats (nonhibernators) and hamsters (hibernators) is made in order to delineate any relationship between lipid properties and the ability to lower body temperature after cold-adaptation. Complete phospholipid analyses are presented; the data are very similar for cold- and warm-adapted rats, and for cold- and warm-adapted hamsters, but the rat lipids have a higher degree of unsaturation than those of the hamsters. Spin probe analogs of stearic acid and cholestane were used to investigate at the molecular level the fluidity and order of the mitochondrial lipids. Studies were made on intact mitochondria, and in liposomes and oriented multibilayers of extracted lipids. In no case was evidence found for a phase transition in the lipids, or for a relationship between the lipid fluidity in brown adipose tissue mitochondria and the ability to survive at lowered body temperatures. The spin probes generally had a decreased mobility in mitochondria relative to extracted lipids. The electron spin resonance spectra were analyzed to include order- and time-dependent phenomena by a recent stochastic method. The results show that more approximate analyses for order parameters and correlation times can yield incorrect conclusions. As segmental motion decreases in rate, order parameters will be overestimated. Decreasing rates of pseudoisotropic motion lead to incorrect estimates of rotational correlation times. Either of the above can result in the inference of an artifactual phase transition in the lipids.     

Saturated and unsaturated lipid spin labels with terminally located nitroxide groups

Synthetic routes are described to a new series of nitroxide lipid spin labels useful for studying the effects of unsaturation and chain length on motion experienced by nitroxide spin labels in biological membrane systems. The labels incorporate a terminally-located proxyl nitroxide group on a saturated or unsaturated fatty acid chain. Syntheses utilize as the key step either an alkylation of an acetylide anion with a nitroxide iodide or else a Wittig coupling between a nitroxide ylid and an aldehyde. Spin labels described include 17-proxylstearolic acid, 17-peroxyl-stearic acid, 17-proxyloleic acid, 16-proxylheptadecanoic acid, 9-proxyldecanoic acid and two phosphatidyl choline derivatives.