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PubMed1981

Synthesis of alpha- and beta-glycosides containing spin labels, as probes for studies of carbohydrate-protein interaction.

Nitroxide spin-labeled alpha-D-glycopyranosides were synthesized in good yield and in a highly stereoselective manner by reaction of per-O-benzyl-alpha-D-glycopyranosyl bromides with 2,2,6,6-tetramethyl-4-piperidinol under the bromide ion-catalyzed conditions devised by Lemieux et al. After hydrogenolysis, the deblocked intermediates were oxidized to give the desired, spin-labeled alpha-D-glycopyranosides. Nitroxide spin-labeled beta-D-glycopyranosides, as well as a beta-maltoside, were synthesized by standard methods. The synthesis is also described of 2-amino-2-deoxy-D-glucose and -D-galactose derivatives having a spin label at C-2, and of the spin-labeled compound 1-[4-(beta-D-galactopyranosyloxy)phenyl]-3-(2,2,6,6-tetramethylpiperidin-1-oxyl -4-yl)-2-thiourea.

Animals↗
PubMed1976

Study of the transverse diffusion of spin labeled phospholipids in biological membranes. I. Human red bloods cells.

Spin labeled analogs of phosphatidylcholine were used to study the transverse diffusion (flip-flop) of phospholipids in the erythrocyte membrane. The nitroxide spin label was placed either on the beta acyl chain or on the choline group. These labeled phosphatidylcholine molecules were incorporated into the membrane by incubation of the red cells at 22 degrees C with sonicated spin-labed phosphatidylcholine vesicles from which all traces of free fatty acids and lyso derivatives were carefully removed by bovine serum albumin treatment. This incorporation did not provide any change in the morphology of the cell as indicated by scanning electron microscopy. When spin-labeled phosphatidylcholine, having a nitroxide on the beta chain but near the polar head-group, was incorporated into the erythrocyte membrane, ascorbate treatment at 0 degrees C allows selective reduction of the signal coming from the outer layer of the membrane. When the label was on the polar head-group, the inner content of the erythrocyte rapidly reduced the label facing the cytoplasm, thus creaging a spontaneous anisotropy of the labeling. The anisotropic distribution of spin-labeled phosphatidylcholine in the erythrocyte membrane was found to be stable at 22 and 37 degrees C for more than 4 h. It is therefore concluded that the rate of outside-inside and inside-outside transition is so slow that the anisotropic distribution of the phospholipids in the erythrocyte membrane can be maintained during cell life.

Cell Membrane↗
PubMed1983

A spin label study of conformational changes in cytochrome c.

Spin-labeled pig heart cytochromes c singly modified at Met-65, Tyr-74 and at one of the lysine residues, Lys-72 or Lys-73, were investigated by the ESR method under conditions of different ligand and redox states of the heme and at various pH values. Replacement of Met-80 by the external ligand, cyanide, was shown to produce a sharp increase in the mobility of all the three bound labels while reduction of the spin-labeled ferricytochromes c did not cause any marked changes in their ESR spectra. In the pH range 6-13, two conformational transitions in ferricytochrome c were observed which preceded its alkaline denaturation: the first with pK 9.3 registered by the spin label at the Met-65 position, and the second with pK 11.1 registered by the labels bound to Tyr-74 and Lys-72(73). The conformational changes in the 'left-hand part' of ferricytochrome c are most probably induced in both cases by the exchange of internal protein ligands at the sixth coordination site of the heme.

Animals↗
PubMed1994

Determination of spin-label orientation within the myosin head.

Current methods of analyzing EPR spectra of spin-labeled muscle fibers allow the determination of spin-label orientation within the fiber, rather than the orientation of the myosin head itself. In order to describe the orientational distribution of spin labeled myosin heads within the muscle fibers, the orientation of the spin label within the myosin head must be known. The iodoacetamide label orientation in the myosin head was determined to be (16.8 degrees, 28.3 degrees, 4.2 degrees) or (16.6 degrees, 72.0 degrees, 4.3 degrees). These Eulerian angles were obtained from the analysis of EPR spectra of fibers decorated with labeled myosin heads in the absence of ATP, with the assumption that the head's tilt angle is 40 degrees, as observed in a recent EM study [Pollard, T., Bhandari, D., Maupin, P., Wachsstock, D., Weeds, A. & Zot, H. (1993) Biophys. J. 64, 454-471]. Knowledge of spin-label orientation will allow for quantitative determination of myosin head orientation in the various states of the contractile cycle.

Algorithms↗
PubMed1985

On the search for new anticancer drugs 14: the plasma pharmacokinetics and tissue distribution of spin-labeled thio-TEPA (SL-O-TT).

We defined the plasma and tissue concentrations and pharmacokinetics of SL-O-TT, a spin-labeled analog of thio-TEPA, in 35-44-g male Swiss Webster mice that had received spin-labeled thio-TEPA at a dosage of 10 mg/kg. Concentrations of spin-labeled thio-TEPA in ethyl acetate extracts of tissue and plasma were determined by gas-liquid chromatography and electron spin resonance spectroscopy. Plasma concentrations of spin-labeled thio-TEPA declined in a biexponential fashion that was well described by the equation: Ct = 21.5e-0.276t + 2.30e-0.026t indicating a half-life alpha of 2.5 min and a half-life beta of 26.6 min. After 2 h there was still spin-labeled thio-TE-PA in plasma, but not in tissues. In tissues, no spin-labeled thio-TEPA was detected with gas-liquid chromatography 15 min after injection, but with electron-spin resonance label was found in lung and skeletal muscle. The main metabolite of spin-labeled thio-TEPA is spin-labeled TEPA, where oxidative desulfurization is invoked as the main metabolic mechanism. Reduction of the spin label to the hydroxylamine was also observed with time.

Animals↗
PubMed1983

Aging of the erythrocyte. 23. Changes in the permeation of spin-labeled electrolytes.

The transport of spin-labeled anions and cations was studied in bovine erythrocyte fractions of various ages. Permeation of a spin-labeled anion, 3-carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl into the cells was found to be significantly slower in older cells, suggesting a functional impairment of the band 3 protein of the erythrocyte membrane during red cell aging in vivo.

Animals↗
PubMedDate not supplied

[Binding of reversible spin-labeled inhibitors with an butyrylcholinesterase active center].

The interaction of spin-labeled metacyn, procaine, carbolin and bivalent cations (Ca2+, Co2+, Ni2+) with butyrylcholinesterase (BChE) was studied by ESR and enzyme kinetic methods. The effect of pH, ionic strength and organic solvent was analysed. Spin-labeled metacyn binds at the anionic site of BChE active centre. This complex is stabilized both with coulombic and hydrophobic interactions, ionizing group of active centre with pK 6-7 also affects the binding. Spin-labeled procaine appeared to be enzyme competitive inhibitor (Ki = 4 X 10(-5) M) and is located, most probably, at the same site. Activating effect of Ca2+ ions on BChE was confirmed. Simultaneous application of spin labels and paramagnetic ions demonstrates that cations Co2+ and Ni2+ bind with BChE in the close vicinity of spin-labeled inhibitor site. Paramagnetic cations are located more closely to the cationic part of the inhibitor molecule than to the hydrophobic one, and can be displaced by surplus of Ca2+ ions. The experimental data testify the model of anionic centre which consists of bivalent metal ions and aminoalcyl cationic group subsites and is located in a hydrophobic pocket of the enzyme surface.

Binding Sites↗
PubMed2002

Two spin labeled triazenes: relationship between biochemical and biological activities.

Biochemical and biological activities of two recently synthesized spin labeled triazenes, containing the nitroxyl free radical moiety at different places of the triazene structure have been studied and compared with those of the antitumor drug Dacarbazine (DTIC). Tissue distribution of the triazenes was investigated in vitro in organ homogenates, tumor (B16 melanoma) and blood of C57BL mice using the electron paramagnetic resonance (EPR) method. The spin labeled triazenes were mainly localized in the tumor and in the brain. Normal leucocites, YAC-1 mNK target Moloney lymphoma cells and B16 melanoma cells were treated with spin labeled triazenes in vitro and the effects on cell viability were compared. Spin labeled 3,3-dimethyl triazene with nitroxyl radical as a substituent in the benzen ring was more cytotoxic to B16 melanoma cells than to YAC-1 Moloney lymphoma cells and normal leucocites in comparison to the spin labeled monomethyl triazene. The spin labeled derivatives were assessed with low toxicity for BDF1 mice hybrids in vivo. These results could be interpreted in terms of a possible correlation between tissue distribution and the selective antimelanoma activity of the spin labeled triazenes.

Animals↗
PubMed1982

Spin labeling of immunoglobulin M and E carbohydrates.

The analysis of ESR spectra of spin-labeled human myeloma immunoglobulins M and E has shown the rotation of spin labels bound to carbohydrates to be restricted most probably due to close attachment of oligosaccharide chains to protein parts of molecules. The values of rotational correlation times for IgM, IgMs and Fc5, spin-labeled at carbohydrates, were found to be equal to 7,7 and 6 ns, respectively. These data pointed to the existence of internal lability of the Fc5 fragment. ESR spectra of IgE spin-labeled at carbohydrates also reflected restricted rotation of the spin label, but the presence of a more than one wide extremum spoke in favour of differences in the degree of immobilization between different oligosaccharide chains. The value of rotational correlation times calculated for IgE, spin-labeled at protein and carbohydrate moieties, were similar (60 and 66 ns). These data confirmed the previously found rigidity of IgE molecule as compared with IgG molecule.

Carbohydrates↗
PubMed1979

Spin-label studies of membrane-associated denatured hemoglobin in normal and sickle cells.

A maleimide spin label (N-(1-oxyl-2,2,5,5-tetramethylpyrrolidinyl)-maleimide) was reacted with oxyhemoglobin-free cell stromata of normal and sickle cells. The EPR spectrum of spin-labeled red cell membranes showed that the spin labels are attached to at least two different binding sites. There was a major signal, A, which characterized a strongly immobilized environment and a minor signal, B, which characterized a weakly immobilized environment. Quantitative EPR measurements using equal amounts of Hb AA and Hb SS red blood cells demonstrated that Hb SS red cell membranes had an approximately four times higher EPR signal intensity than Hb AA red cell membranes ((7.98 +/- 1.14 . 10(5) and (2.2 +/- 1.2) . 10(5) spin labels/cell, respectively). Moreover, the ratio of signal intensities A and B are different in these cells. Comparative spectrophotometric studies of membrane-associated denatured hemoglobins of Hb AA and Hb SS red cell membranes suggested that the EPR signal A is derived from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membranes. The combination of EPR spectrum of Hb AA membranes pretreated with N-ethylmaleimide and that of spin-labeled precipitated hemoglobin further strengthened this conclusion.

Anemia, Sickle Cell↗
PubMed1989

ESR behaviour of spin-labelled oligoDNAs.

Oligodeoxyribonucleotides (oligoDNAs) were spin-labelled at the 5'-end internucleotide linkage, directly or through the spacers of different lengths, with TEMPO and the labelled oligoDNAs were explored for the ESR spectroscopical behaviour in solution. Oxidation of H-phosphonate intermediates in CCl4 solutions of TEMPO and diamines was taken for the introduction of TEMPO and spacers, respectively. ESR lines of TEMPO attached to oligoDNAs, directly or through spacers, were already broadened compared to those of free TEMPO, and the broadening decreased with increasing chain length of the spacer and increased with increase in the chain length of the oligoDNAs. The ESR lines of the oligoDNA labelled directly were further broadened in the presence of a DNA complementary to the labelled oligoDNA. In addition, the broadened lines were further broadened in the presence of a larger DNA. This observation implies that spin labelling at the internucleotide linkage is useful in the DNA probe method to avoid B/F separation.

Base Sequence↗
PubMedDate not supplied

[Study of conformational transitions in myosin using 2 types of spin label].

It is shown that three types of ESR spectra of myosin labeled with both acetate and acetamide spin labels are distinguished. These three types are the spectrum of initial preparation, myosin--ADP complex spectrum and the spectrum recorded during ATP hydrolysis. The ESR spectra of myosin labeled with acetate spin label are more sensitive to conformational changes of myosin induced by complex formation and temperature.

Cyclic N-Oxides↗
PubMedDate not supplied

[Effect of various humidity on local dynamic structure of lysozyme in a spin-labeled tetragonal crystal].

The dynamics of the side groups of amino acid residues and local conformational changes in the lysozyme molecule upon dehydration and rehydration of lysozyme crystals were studied by the methods of spin label, X-ray diffraction, and molecular dynamics. The His15 residue of lysozyme from chicken egg white was modified by spin label, and spin-labeled tetragonal crystals of the protein were grown. The spatial structure of the covalently bound spin label and its immediate surroundings in the lysozyme tetragonal crystal was determined. The conformation of a fragment of the lysozyme molecule with the spin label on His15, optimized by the method of molecular dynamics, closely agreed with X-ray data. It was found by the X-ray diffraction analysis that a decrease in relative humidity to 40% is accompanied by both a decrease in the unit cell volume by 27% and a change in the diffraction field of roentgenograms from 0.23 to 0.60 HM. The dehydration of spin-labeled lysozyme crystals leads to an anomalous widening of EPR peaks without changes in their position. The dehydration in the humidity range studied has a two-stage character. The decrease in humidity to 75% is accompanied by a sharp change in the parameters measured, and on further decrease in humidity to 40% they change insignificantly. The first stage is caused by the removal of the greater part of molecules of bulk water, and the second stage is due to the removal of the remaining bulk water and possible changes in the dynamics of weakly bound water molecules and their position. The simulation of experimental EPR spectra showed that the anomalous broadening of the spectrum upon dehydration is related to an increase in the dispersion of spin label orientations induced by changes in the network of hydrogen bonds generated by water molecules in the vicinity of the spin label and a possible turn (by no more than 5 degrees) of the entire protein molecule. After rehydration, the physical state of the lysozyme crystal did not return to the starting point.

Crystallization↗
PubMed1977

Preparation of spin-labeled opiates: morphine and codeine.

The preparation of 6-spin-labeled codeine and morphine is described. Treatment of either 6-chlorocodide or 8-bromocodide with 4-amino-2,2,6,6-tetramethylpiperidino-1-oxyl free radical in dimethylformamide afforded 6-spin-labeled codeine. Similar treatment of 6-chloromorphide afforded 6-spin-labeled morphine. Exclusive formation of the 6-isomer in these reactions is explained by halide-ion-catalyzed isomerization of the 6-halo opiate to the 8-halo isomer followed by a normal SN2' displacement of the halogen. Both spin-labeled compounds displayed weak in vivo analgesic activity and did not bind appreciably to receptors in brain homogenate.

Codeine↗
PubMed2003

Quantification of blood flow in brain tumors: comparison of arterial spin labeling and dynamic susceptibility-weighted contrast-enhanced MR imaging.

PURPOSE: To implement an arterial spin labeling technique that is feasible in routine examinations and to test the method and compare it with dynamic susceptibility-weighted contrast material-enhanced magnetic resonance (MR) imaging for evaluation of tumor blood flow (TBF) in patients with brain tumors. MATERIALS AND METHODS: Thirty-six patients with histologically proven brain tumors were examined at 1.5 T. A second version of quantitative imaging of perfusion by using a single subtraction with addition of thin-section periodic saturation after inversion and a time delay (Q2TIPS) technique of pulsed arterial spin labeling in the multisection mode was implemented. After arterial spin labeling, a combined T2- and T2*-weighted first-pass bolus perfusion study (gadopentetate dimeglumine, 0.2 mmol/kg) was performed by using a double-echo echo-planar imaging sequence. In regions of interest, maps of absolute and relative cerebral blood flow were computed and analyzed with arterial spin labeling and dynamic susceptibility-weighted contrast-enhanced MR imaging, respectively. RESULTS: Both techniques yielded the highest perfusion values in imaging of glioblastomas and the lowest values in imaging of two low-grade gliomas that both showed strong gadopentetate dimeglumine enhancement. There was a close linear correlation between dynamic susceptibility-weighted contrast-enhanced MR imaging and arterial spin labeling in the tumor region of interest (linear regression coefficient, R = 0.83; P <.005). Blood flow is underestimated with arterial spin labeling at low flow rates. High- and low-grade gliomas can be distinguished at the same level of significance with both methods. Absolute TBF is less important for tumor grading than is the ratio of TBF to age-dependent mean brain perfusion. CONCLUSION: Arterial spin labeling is a suitable method for assessment of microvascular perfusion and allows distinction between high- and low-grade gliomas.

Adolescent↗
PubMed1977

Complement-induced decrease in membrane mobility: introducing a more sensitive index of spin-label motion.

We have used spin-labeling to investigate complement-induced changes in lipid organization of antibody-sensitized sheep erythrocyte membranes. The spectrum of methyl 5-doxylstearate incorporated into the lipid component of sheep erythrocyte membranes is typical of a membrane bilayer. The membranes from complement-lysed erythrocytes have a small, but statistically significant, reduction in fluidity when compared to membranes from osmotically-lysed erythrocytes, as indicated by a small increase in T'. In theory, measurements of the widths of the outer hyperfine extrema should be more sensitive to motion than the separation of the outer hyperfine extrema (2T'). Our results indicate that the half-width at half-height of the outer hyperfine extrema show a severalfold greater percentage change than T'. The sign and magnitude of these changes are in general agreement with previous predictions. Our results imply that motional corrections to the S formalism of Hubbell, Gaffney, and McConnell are necessary because spin-label motion appears to be explicitly represented in this type of electron spin resonance spectra.

Animals↗
PubMed1979

Detection of conformational changes in complex III of the respiratory chain by a maleimido spin label.

Changes in the conformation of Complex III (CoQH2-cytochrome c reductase) of the mitochondrial respiratory chain were detected upon oxidoreduction using the nitroxide spin label, 3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl. EPR spectra of the spin label show a transition from a greater to a lesser degree of immobilization when the labeled enzyme, reduced either with ascorbate or sodium dithionite, is oxidized with potassium ferricyanide or ferricytochrome c. These observations are interpreted to indicate that Complex III is more compact in the reduced state at least in the locality of the spin label. An apparent increase in the concentration of total spins during oxidation of the complex suggests change in the interaction between the spin label and other paramagnetic centers and not an oxidation of spin label, itself, since reduced free spin label could not be reoxidized. Addition of antimycin A had no effect on the EPR spectrum of the spin-labeled enzyme, indicating that this inhibitor does not initiate a conformational change in the region of the spin label. Experiments in which N-ethyl-[2-3H] maleimide was bound to Complex III show that binding occurs primarily to a subunit with a molecular weight of 45,000. Although no qualitative differences were observed, it was found that less radioactivity appears in samples reduced with dithionite than in those reduced with ascorbate. This difference appears to be caused by decomposition products of dithionite.

Ascorbic Acid↗
PubMed1980

Aging of the erythrocyte. I. Increase in the microviscosity of cell interior as determined by the spin label method.

By means of the spin labelling technique changes in the microviscosity of the cell interior of density-seperated bovine red blood cells were estimated. An increase in the rotational correlation time of the Tempamine spin label indicated an increase in the microviscosity of the red blood cell interior during in vivo aging. This increase is apparently due to alterations in the electrolyte content and may influence the course of diffusion-limited reactions in the cell.

Animals↗