Indanedione spin labelling of Na,K-ATPase

The indanedione series of vinyl ketone spin-labelling reagents has been extended in two ways: by increasing the length of the rigid spacer between the reactive centre and the nitroxide ring, or by introducing an electrophilic substituent (that could also hinder its rotation) at the bridge head position of the nitroxide ring. Three reagents of this new series have been used to spin label the Class II thiol groups of membranous Na,K-ATPase from Squalus acanthias. With a conjugated diene spacer, the majority of spin labels are strongly held but a minor population is relatively mobile at 37 degrees C. With a conjugated triene spacer, the nitroxide is still strongly held but a portion of the label is non-covalently bound. The 4-bromo-pyrroline derivative (with short vinyl spacer) is tightly held at the attachment site, and the conventional electron paramagnetic resonance (EPR) spectra distinguish between the two enantiomeric structures which differ in their mobility at 37 degrees C. Saturation transfer EPR (ST-EPR) spectra of this label at 4 degrees C have been used to determine the dependence of the protein rotational mobility on ionic strength. Electrostatic repulsion contributes to the lateral interactions between Na,K-ATPase molecules.     

Oxygen diffusion-concentration product in rhodopsin as observed by a pulse ESR spin labeling method.

Permeation of molecular oxygen in rhodopsin, an integral membrane protein, has been investigated by monitoring the bimolecular collision rate between molecular oxygen and the nitroxide spin label using a pulse electron spin resonance (ESR) T1 method. Rhodopsin was labeled by regeneration with the spin-labeled 9-cis retinal analogue in which the beta-ionone ring of retinal is replaced by the nitroxide tetramethyl-oxypyrrolidine ring. The bimolecular collision rate was evaluated in terms of an experimental parameter W(x), defined as T1(-1)(air,x)--T1(-1)(N2,x) where T1's are the spin-lattice relaxation times of the nitroxide in samples equilibrated with atmospheric air and nitrogen respectively, which is proportional to the product of local oxygen concentration and local diffusion coefficient (transport). W-values at the beta-ionone binding site in spin-labeled rhodopsin are in the range of 0.02-0.13 microseconds-1, which are 10-60 times smaller than W's in water and 1.1-20 times smaller than in model membranes in the gel phase, indicating that membrane proteins create significant permeation resistance to transport of molecular oxygen inside and across the membrane. W(thereby the oxygen diffusion-concentration product) is larger in the meta II-enriched sample than in rhodopsin, indicating light-induced conformational changes of opsin around the beta-ionone binding site. W decreases with increase of temperature for both rhodopsin and meta II-enriched samples, suggesting that temperature-induced conformational changes take place in both samples. These changes were not observable using conventional ESR spectroscopy. It is concluded that W is a sensitive monitor of conformational changes of proteins.     

A spin label study of the thyroid hormone-binding sites in human plasma thyroxine transport proteins

The binding site topographies of the three thyroid hormone-transporting proteins in human serum--prealbumin, thyroxine binding globulin, human serum albumin--have been studied with the aid of five spin-labeled analogs of L-thyroxine in which the distance between the phenolic hydroxyl and the nitroxide nitrogen ranged from 17 to 23 A. In the presence of prealbumin, the electron spin resonance spectrum of 3-([alpha-carboxy-4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenethyl]-carbamoyl)-2,2,5,5-tetramethyl-3-pyrrolinl-yloxy-ethyl ester revealed the presence of a highly immobilized spin label. As the chain length between the thyroxyl moiety and the pyrroline ring was increased, the mobility of the nitroxide group in the prealbumin-bound labels increased. If the spin labels bind in an extended conformation, the thyroxine-binding site was estimated to be approximately 21 A in depth. This finding is consistent with the known crystal structure of prealbumin and suggests that the solution and crystal conformations of the protein are very similar. In contrast to prealbumin, the thyroxine-binding site on thyroxine-binding globulin was found to be more open and possibly deeper. Human serum albumin has two binding sites for thyroxine, one of which has a higher affinity and is deep enough to accommodate a molecule that is 23 A in length. The lower affinity site is somewhat shallower and probably wider, as thyroxine spin labels bound to this site exhibited greater mobility.     

Nitroxide-labeled guanine as an ESR spin probe for structural study of DNA

A guanine derivative with a covalently linked nitroxide spin label has been devised. The spin label was incorporated into oligodeoxynucleotides by post-synthetic modification. The local environment of a variety of G-rich DNA is detectable by ESR using this spin probe.     

Electron spin resonance and nuclear relaxation studies on spin-labeled glutamate dehydrogenase.

The reaction of glutamate dehydrogenase with two different stable nitroxides (spin labels) is reported. The two compounds contain a carbonyl and an iodoacetamide group as their reactive parts. The carbonyl compound inactivates the enzyme by the formation of a 1:1 covalent complex after NaBH4 reduction of an intermediate Schiff's base. Evidence indicates that the enzyme is modified at lysine-126 in the active site. The electron spin resonance (ESR) spectrum of spin-labeled enzyme indicates a high degree of immobilization of the nitroxide. The binding of reduced coenzyme NADPH is reflected by a change (immobilization) of the ESR spectrum. Nuclear relaxation of bound substrate, oxidized coenzyme, and inhibitor by the paramagnetic group is observed. This shows the existence of a binding site for these compounds close to the active site. The distances of selected protons of the binding ligands to the nitroxide are calculated. The iodoacetamide spin label reacts with several groups, one of which is not a sulfhydryl. The reaction of this particular group causes inactivation of the enzyme. Protection against this inactivation could be achieved with certain ligands. Only enzyme that was spin labeled without such protection caused paramagnetic relaxation of bound substrate and coenzyme.     

Acridine spin labels as probes for nucleic acids.

Adridine spin labels, 4-[9-(6-chloro-2-methoxy)-acridylamino]- 2,2,6,6-tetramethyl-1-piperidinyloxy (I) and 4-(9-acridylamino)- 2,2,6,6-tetramethyl-1-piperidinyloxy (II), have been synthesized and their interaction with nucleic acids studied by means of electron spin resonance (ESR). The ESR spectra of labels I and II in the presence of calf thymus DNA were characteristic of highly immobilized nitroxide radicals with maximum hyperfine splittings (2T_ˌˌ) of 58.7 and 55.5 G, respectively. The melting temperature (T_m) of DNA, determined in the presence of labels I and II by the ESR technique, were closely similar to those obtained by spectrophotometric methods. The ESR spectrum of label I bound to calf liver RNA and yeast RNA indicated that the nitroxide group of this label was highly mobile. These results suggest that spin labels I and II are suitable noncovalent probes for nucleic acids.     

Hydroxyl radical production by free and DNA-bound aminoquinone antibiotics and its role in DNA degradation. Electron spin resonance detection of hydroxyl radicals by spin trapping.

The reduced antitumor antibiotic mitomycin C in aqueous solution exposed to air gives a 36-line electron spin resonance spectrum of the semiquinone identified by computer simulation. Incubation of this radical with the spin trap N-tert-butyl-alpha-phenylnitrone (PBN) gives the PBN.OH nitroxide radical identified by independent generation. This nitroxide radical is also formed from similar treatment of a DNA to which mitomycin C is covalently attached. Incubation of the semiquinone from mitomycin C, mitomycin B, or streptonigrin (SN) with catalase or with superoxide dismutase inhibits the generation of OH, implying the intermediacy of H2O2 and O2 in its formation. The formation of the spin-trapped nitroxide radical is similarly inhibited by EDTA, suggesting the intermediacy of trace metal ions in the generation of hydroxyl radicals from SN. The results are consistent with the generation by the aminoquinone antibiotics in vivo of OH. already implicated in the degradation of DNA.     

Librational motion of an "immobilized" spin label: hemoglobin spin labeled by a maleimide derivative.

The spin label Tempo-maleimide, when "immobilized" in hemoglobin, is shown to exhibit motional fluctuation whose amplitude and/or frequency depend on temperature and solution conditions. These motional fluctuations are observable by several electron spin resonance techniques. For desalted hemoglobin the fluctuations are detectable at approximately -15 degrees C using saturation transfer techniques and at approximately +25 degrees C using line-width measurements of normal absorption spectra. In ammonium sulfate precipitated hemoglobin, however, motional fluctuations are not detectable by either technique up to at least 40 degrees C. The most probable mechanism for spin-label motion appears to be either fluctuations in protein conformation which affect the label binding site or conformational transitions of the nitroxide ring itself. These motional fluctuations are shown to introduce a librational character to the overall label motion during hemoglobin rotational diffusion, with the librational motion significantly affecting the use of spin-label spectral shapes to calculate hemoglobin rotational correlation times.     

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.     

Progesterone-binding globulin interaction with its steroid ligands: study of the protein binding site topography using spin labeled steroids and electron spin resonance spectroscopy

The binding site topography of progesterone-binding globulin (PBG) purified from pregnant guinea pig serum was examined using synthesized spin-labeled ligands and electron spin resonance (ESR) spectroscopy. A series of deoxycorticosterone-nitroxide (DOC-NO) derivatives were prepared, bearing the free radical on the side chain at increasing distance (d) from the steroid nucleus. The ability of the spin-labeled steroids to specifically bind to PBG was assessed by measurement of their relative binding affinity as compared to progesterone. ESR spectra of the bound steroid nitroxide radical were used to calculate the rotational correlation times tau c for the nitroxides as a function of their distance d to the protein-bound steroid nucleus. The data showed that the side chain nitroxide exhibited an unrestrained rotation in a water-like environment when d reached about 18 A. This would correspond to a PBG steroid binding site depth of about 28 A and suggests that the bound steroid in the PBG site is oriented with the side chain at C-17 directed toward the outside of the protein binding crevice.     

A spin label study of egg white avidin.

Avidin is a tetrametric protein (mass 68,000 daltons) that binds 4 molecules of vitamin biotin (1). The biotin binding sites, 1 per subunit, are grouped in two pairs at opposite ends of the avidin molecule (GREEN, N.M., KONIECZNY, L., TOMS, E.J., and VALENTINE, R.C. (1971) Biochem. J. 125, 781). We have studied the topography of the avidin binding sites with the aid of four spin-labeled analogs of biotin: 4-biotinamido-2,2,6,6-tetramethyl-1-piperidinyloxy (II), 3-biotinamido-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (III), 3-biotinamidomethyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (IV), 4-(biotinylglycyl)-amino-2,2,6,6-tetramethyl-1-piperidinyloxy (V). Fluorescence and optical absorption spectroscopy indicated that II to V occupied the same binding sites on avidin as did biotin. The electron spin resonance spectrum of the 4:1 complex between II and avidin contained broad line components characteristic of a highly immobilized spin label. Dipole-dipole interactions between spin labels bound to adjacent sites split each of the three major hyperfine lines into doublets with a separation of 13.8 G. The distance between adjacent bound nitroxide groups was calculated from this splitting to be 16 A. The dissociation of the 4:1 complex between II and avidin was biphasic with approximately half of the labels dissociating at a rate (kdiss equal to 2.51 times 10- minus 4 s- minus 1) that was much faster than the remainder (kdiss equal to 1.22 times 10- minus 5 s- minus 1). The electron spin resonance spectrum of the 2:1 complex between II and avidin clearly showed that, immediately after mixing, the spin labels were distributed in a random fashion among the available binding sites but that they slowly redistributed themselves so that each label bound to a site which was adjacent to an unoccupied site. The final time-independent electron spin resonance spectrum exhibited a splitting 69 G between the low and high field hyperfine lines which is characteristic of a highly immobilized, noninteracting spin label. Spin labels III and IV interacted with avidin in a similar fashion to that described for II with the exception that their dipolar splittings were 11.9 G and 14.2 G, respectively. From these splittings it was estimated that the distance between adjacent avidin-bound nitroxides was 16.7 A for labeled III and 15.7 A for label IV. The electron spin resonance spectrum of label V bound to avidin was characteristic of a noninteracting highly immobilized nitroxide with a maximum splitting of 62 G. The spectrum of V bound to avidin was independent of both time and the amount of bound label. The rate of dissociation of V from a 4:1 complex with avidin was monophasic. A model is proposed in which the recognition site for the heterocyclic ring system of biotin is represented as a cleft located within a hydrophobic depression in the surface of avidin.     

A novel reversible thiol-specific spin label: papain active site labeling and inhibition

A new, highly reactive, thiol-specific spin label, (1-oxyl-2,2,5,5-tetramethyl-Δ³-pyrroline-3-methyl)methanethiosulfonate was synthesized. Its unique specificity was demonstrated with the active thiol protease, papain, which was stoichiometrically inhibited within 5 min, resulting in a conformationally sensitive spectrum, which was identical over the pH range 4.5–7.5. The spin-label modification yielded a mixed disulfide between Cys 25 of papain and the 3-methylpyrroline nitroxide which was rapidly and completely reversed by exposing the labeled papain to mild concentrations of dithiothreitol. The concentration of released nitroxide corresponded exactly to the number of reactive thiol groups in the original enzyme. Full enzymatic activity was restored after the spin label was removed. This spin label is useful as a sensitive thiol titrating agent as well as a specific conformational probe of thiol site structure by virtue of its minimal rotational freedom and distance from the covalent disulfide linkage to the macromolecule under study.     

Stereochemical properties of the binding site of liver microsomal cytochrome P-450 as studied by substrate analogous spin labels.

For the characterization of the substrate binding site optical and EPR measurements with spin labelled substrates on solubilized and pure cytochrome P-450 were performed. Analogously to the unlabelled derivatives spin labelled n-alkylamines and isocyanides with different chain lengths are type II substrates. The Ks-values evaluated from optical (P-450 = 1.98 . 10(-6) M) and ESR (P-450 = 1.98 . 10(-4) M) measurements are very similar indicating no concentration dependences. Contrary to the unlabelled n-alkylamines the spin labelled compounds show an affinity almost independent of the chain lengths. The SL-substrates with a short distance between the functional group and the NO-group bound to P-450 induce pronounced changes of the ligand field of the heme iron and a large broadening of the signal of the immobilized nitroxide indicating intensive interactions between the unpaired electron of the nitroxide group and the paramagnetic heme iron. Elongation of the alkyl chains results in spectra of the Fe3+ complexes with only slight modification and a remained unbroadened signal of the immobilized nitroxide. The binding of the substrate through their functional groups together with a 1:1 stoichiometry of the P-450 SL-IC-complex give evidence for the same binding site in the near vicinity of the heme iron.     

Heterogeneity of thylakoid membranes studied by EPR spin probe

A lipophilic nitroxyl radical, 1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl 1-adamantylacetate, has been applied to EPR spin probe study of chloroplasts and subchloroplast fragments of different types. The latter originate from grana and the grana core regions. The binding of the spin probe to the membranes was revealed by specific changes in a shape of the EPR spectra. A share of membrane-bound spin probe was different for chloroplasts and subchloroplast fragments, as well as its rotational correlation time and apparent enthalpy and entropy activation of nitroxide rotational motion. The binding of the spin probe induced a significant decrease in the amount of the oxidized P700 and changes in the kinetics of its light oxidation and dark recovery. This suggests that one of the sites of nitroxyl radical binding is the nearest surrounding of the pigment-protein complexes of Photosystem I (PSI). Distinctions in mobility of spin probe immobilized by chloroplasts and their fragments can be caused by the different environment of the PSI complexes located in various regions of thylakoid membranes. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 5, pp. 690–698.     

Studies of Pargyline-Monoamine Oxidase Binding Using a Spin Label Probe Analog

Abstract: Analogs of the monoamine oxidase (MAO) inhibitor pargyline with a nitroxide free radical moiety attached through an ether linkage to the para position on the benzene ring have been prepared and reacted with solubilized MAO preparations from rat and beef brain and pig liver. These compounds behave as normal irreversible inhibitors of catalytic activity, with some preference for B-type enzyme. When the reaction was monitored by electron spin resonance (ESR), line broadening effects indicative of binding and with an apparent relation to substrate specificity of the preparation were observed. In addition, there was a slow decrease in intensity of the ESR spectra, which could be retarded by the addition of other MAO inhibitors or increased O₂ and enhanced by flavin reduction. It appears to be related to development of the irreversible phase of MAO inhibition. Signal recovery with added O₂ and studies of a model reaction with free flavin, suggest the signal loss to be a line broadening effect due to interaction with an enzyme-generated paramagnetic species rather than to direct reduction of the nitroxide radical.     

The conformations of nitroxide-labelled fatty acid probes of membrane structure as studied by 2H-NMR

The electron spin resonance (ESR) spectrum of a nitroxide spin probe intercalated in a membrane is influenced by the amplitude of anisotropic motion of the nitroxide group and by the geometry of the oxazolidine ring of the nitroxide. In the analysis of the ESR spectra of nitroxide-labelled fatty acid probes, it is generally assumed that the five-membered oxazolidine ring system is oriented rigidly perpendicular to the long molecular axis of the probe. This assumption is tested in the present study, using 2H-NMR of specifically deuterium-labelled nitroxide spin probes. Evidence is presented that the nitroxide does not display the assumed geometry in membranes. The departure from this geometry depends on the position of the nitroxide label on the acyl chain, with a more pronounced departure for position 5 relative to position 12. These and previous data provide an explanation for the discrepancies between spin-probe ESR and 2H-NMR order parameters in membranes.     

Adamantyl nitroxide: A spin label for probing membrane surfaces

In an effort to understand more about the perturbing properties of adamantane-like molecules on biological membranes, the spin probe adamantyl nitroxide (2,2′-dimethyl-5-adamantyl oxazolidine-N-oxyl) was synthesized, purified and characterized. Electron paramagnetic resonance (EPR) spectra were then obtained from 1:50 and 1:200 mixtures of adamantyl nitroxide with dipalmitoyl and dipalmityl phosphatidylcholine multibilayers. Above the phase transition temperature of these lipids (41°C for dipalmitoyl phosphatidylcholine and 43°C for dipalmityl pholphatidylcholine) the spectra of adamantyl nitroxide are similar to control spectra obtained in liquid oleic acid. Below the phase transition temperatures, however, spectral differences were observed depending on: (1) the concentration of the spin probe in the lipid; (2) the linkage between the polar head group and the hydrocarbon tails of the phospholipid; (3) the temperature of the sample. Partitioning of adamantyl nitroxide between the aqueous and hydrocarbon phases of the sample is most prominent at probe-to-lipid ratios of 1:200 and at temperatures below the pre-transition temperature of the lipid (around 33°C). Computer simulations of the above results, as well as additional experiments performed at 35 GHz, show that the results arise from true partitioning and not from asymmetric probe motion.Two conclusive results of these experiments are that spectra of adamantyl nitroxide in phospholipid multibilayers are sensitive to probe concentration and to the physical characteristics of the phospholipid which they probe. The spectral differences which arise when adamantyl nitroxide is used with ether- and ester-linked phospholipids indicate that it is a sensitive probe of membrane surfaces. Employment of this molecule in membrane research should prove to be useful in obtaining additional information about membrane surface events.     

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.     

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.     

The conformations of nitroxide-labelled fatty acid probes of membrane structure as studied by 2H-NMR

The electron spin resonance (ESR) spectrum of a nitroxide spin probe intercalated in a membrane is influenced by the amplitude of anisotropic motion of the nitroxide group and by the geometry of the oxazolidine ring of the nitroxide. In the analysis of the ESR spectra of nitroxide-labelled fatty acid probes, it is generally assumed that the five-membered oxazolidine ring system is oriented rigidly perpendicular to the long molecular axis of the probe. This assumption is tested in the present study, using ²H-NMR of specifically deuterium-labelled nitroxide spin probes. Evidence is presented that the nitroxide does not display the assumed geometry in membranes. The departure from this geometry depends on the position of the nitroxide label on the acyl chain, with a more pronounced departure for position 5 relative to position 12. These and previous data provide an explanation for the discrepancies between spin-probe ESR and ²H-NMR order parameters in membranes.