Continuous wave electron paramagnetic resonance L-band spectrometer with direct digitalization using time-locked subsampling.

This article describes a novel digital L-band EPR spectrometer. The spectrometer uses direct digital detection with time-locked subsampling (TLSS). The device consists of a microwave bridge equipped with a microwave source based on direct digital synthesis (DDS) and a digital receiver. DDS technology combined with an ultra-low noise 1 GHz master clock allowed the development of a digitally controlled microwave source with exceptionally good phase noise characteristics. The obtained level of phase noise is as low as -140 dBc/Hz at 30.5 kHz from the carrier frequency of 1.15 GHz, which is important when registering the EPR dispersion signal. The receiver is equipped with a high-speed A/D converter that enables direct digitalization of the L-band microwave signal. The obtained discrete data are then buffered and averaged in a programmable logic FPGA device. Data packets from FPGA are transferred to a DSP microcontroller that correlates them with the appropriate reference signals. This detection algorithm requires time locking of the generator and the receiver, which is ensured by clocking both devices from the same reference source. This procedure allows the simultaneous detection of the absorption and dispersion signals at the magnetic field modulation frequency and at any of its harmonics. The software to control the spectrometer was designed in the LabView programming environment. The program also allows further data processing. To the best of our knowledge, the described spectrometer is one of the first full implementation of the direct digital detection technique which could replace conventional analog CW spectrometers that utilize magnetic field modulation. For an 11 µm aqueous TEMPOL solution, the new spectrometer obtained a S/N ratio greater than 160 for an EPR spectrum registered in 69 s.

An extended dynamic model of oxidative phosphorylation.

The presented model based on an earlier one (Korzeniewski, B. and Froncisz, W. (1989) Studia Biophys. 132, 173-187) simulates concentration changes in time of chemical compounds and thermodynamic forces during respiration of cell suspension in a closed chamber. A set of differential equations solved numerically describes the utilization of oxygen up to anaerobiosis and the behaviour of the system after a sudden pulse of oxygen. Flux control coefficients for most important reactions (enzymes) of oxidative phosphorylation were calculated. A good qualitative and (when a direct comparison is possible) quantitative agreement with experimental results can be observed. The following conclusions can be drawn from the simulation: (1) Wilson's steady state model is not in contradiction with sharing of the control over the respiration between some steps and displacement of the ATP/ADP carrier from equilibrium. (2) The overshoot characteristics of the delta microH+ time-course after reoxygenation can be explained without using the lag-phase kinetics of ATP-synthetase. (3) A 'hot region' (sharp changes of many parameters) can be distinguished when the oxygen concentration approaches zero; only cytochrome oxidase is clearly sensitive on oxygen concentration in all its range. (4) Control over oxidative phosphorylation is shared mainly between inputs of the system (ATP utilization and substrate dehydrogenation) and the proton leak.

Theoretical studies on the control of the oxidative phosphorylation system.

The dynamic model developed in our previous publications [1,2] was used to calculate the flux control coefficients of oxidation, phosphorylation and proton leak fluxes for isolated mitochondria and for three modes of work of intact cells (hepatocytes). The results obtained were compared with experimental data, especially those measured in the frame of the 'top-down approach' of the metabolic control theory. A good agreement for mitochondria and for intact cells was found. The control of the oxygen consumption flux is shared between the ATP utilization (main controlling factor), substrate dehydrogenation, proton leak and, in some conditions, the ATP/ADP carrier. The phosphorylation subsystem seemed to be controlled mainly by itself, while the proton leak was influenced by all three subsystems. It was also shown that the large relative change in the enzyme activity during inhibitor titration of mitochondria or cells could lead to the overestimation of some flux control coefficient values in experimental measurements. An influence of some hormones (glucagon, vasopressin, adrenaline and others) on the mitochondrial respiration was also simulated. Our results suggest that these hormones stimulate the substrate dehydrogenation as well as the phosphorylation system (ATP usage and, possibly, the ATP/ADP carrier).

Preparation and electron paramagnetic resonance characterization of spin labeled monoderivatives of horse cytochrome c.

Horse cytochrome c was reacted with the spin label (succinimidyl-2,2, 5,5-tetra-methyl-3-pyrroline-1-oxyl-carboxylate) using optimized conditions and the reaction products were separated by a combination of cation-exchange chromatography and HPLC. The purified cytochrome c derivatives were digested with TPCK treated trypsin and the resulting peptides were separated by reverse phase HPLC. The modified Lys residues were subsequently characterized by Edman degradation and mass spectrometry. These analyses showed that five distinct cytochrome c derivatives had been produced which were modified at the specific Lys residues including Lys8, Lys25, Lys72, Lys86 or Lys87, respectively. The electron paramagnetic resonance (EPR) spectra for each cytochrome c derivative revealed that for the spin label attached to Lys8 and Lys87 only one component contributes to the spectrum whereas for Lys25, Lys72 and Lys86 the spectrum consists of two components. The highest mobility with the rotational correlation time, tauB, of 0.38 ns was observed for Lys87. The longest tauB of 1.84 ns was obtained for Lys72. An attempt to correlate the spin label mobility with the local protein structure is presented. These mono derivatized cytochrome c molecules provide a unique tool for EPR studying the interaction between cytochrome c and the lipid bilayer, as well as cytochrome c oxidase and reductase.

Spin label oximetry to assess extracellular oxygen during myocardial ischemia.

We describe real-time measurement of myocardial oxygen consumption during ischemia in the intact heart. Measurement of extracellular oxygen concentration during myocardial ischemia by spin label oximetry has been limited by ischemia-induced reduction of the neutral, water-soluble nitroxide TEMPONE. We have overcome this problem by encapsulating the nitroxides. Isolated immature (7-10 d old) rabbit hearts (n = 8) were perfused aerobically within the cavity of a loop gap resonator with bicarbonate buffer containing an oxygen-sensitive, lipid-soluble nitroxide (14N-TEMPO laurate in FC-43 perfluorocarbon micelles) and a much less oxygen-sensitive and positively charged nitroxide (15N-TEMPO choline in multilamellar vesicles) as an internal standard. The ratio of the ESR signal amplitudes of these nitroxides was used as a sensitive index of oxygen concentration. Sequestration of the nitroxides decreased their reduction rate by ascorbate in comparison with nonsequestered nitroxides. Hearts were subjected to 60 min of global no-flow ischemia at 20 degrees C. Extracellular oxygen content (mean +/- SD) during aerobic perfusion was 1195 +/- 55 mumol/liter. The electron spin resonance signal from TEMPO laurate increased with the onset and progression of ischemia, consistent with a decrease in extracellular oxygen, while the signal for TEMPO choline was relatively unchanged. Extracellular oxygen content after 40 and 60 min of ischemia was reduced to 393 +/- 27 mumol/liter (p < .05) and 61 +/- 5 mumol/liter (p < .05), respectively. We conclude that spin-label oximetry can directly and precisely measure myocardial oxygen consumption at constant temperature during ischemia in the intact heart.

Comparison of high-resolution MRI and SPECT bone scintigraphy for noninvasive imaging of the temporomandibular joint.

Prospective evaluation by magnetic resonance imaging (MRI) and both single photon emission computed tomography (SPECT) and planar bone scintigraphy was undertaken in 31 temporomandibular joints (TMJs) of 21 symptomatic patients. When compared with the results of subsequent arthrography, MRI (0.88) was more sensitive than SPECT (0.76) or planar (0.56) scintigraphy for detection of internal derangement of the TMJ. A diagnostic sensitivity of 0.96 was achieved when the results of either MRI or SPECT was considered evidence of internal joint derangement. Five symptomatic TMJs, clinically thought to be abnormal, were positive on SPECT but showed no evidence of anterior disk displacement at the time of arthrography. In such instances, SPECT may be detecting functionally significant altered joint mechanics that are not evident on anatomic imaging of the TMJ.

A rectangular loop-gap resonator for EPR studies of aqueous samples.

A new rectangular geometry of the loop-gap resonator for the use with a flat cell has been developed. Maxwell's equations for the resonators with two, four, six, and eight gaps have been solved assuming the existence of only the magnetic z-component. The formulas obtained were numerically solved for the electric and magnetic field distributions over the cross-sections of the resonators. The presence of a nodal plane for the electric field in the center of the resonator allows the use of a flat cell instead of a capillary for EPR measurements. Using the field distributions obtained, the quality factor and EPR signal amplitude for various shapes and gap numbers for the resonators containing a flat cell filled with water were examined numerically. This allowed finding the geometry that yields the maximum EPR signal intensity. Several X-band resonators were built in order to verify the results obtained theoretically. The experiments confirmed the ability of a novel resonant structure to accommodate a flat cell filled with an aqueous sample. It has been found that the optimum aqueous sample volume for the X-band rectangular loop-gap resonator equals 16 mm3. For a saturable aqueous sample this gives a fourfold improvement in the S/N ratio over the circular 1 mm i.d. loop-gap resonator equipped with 0.6 mm i.d. capillary.

The cervical nerves and foramina: local-coil MR imaging.

A detailed study of the magnetic resonance (MR) appearance of the cervical nerves and neural foramina is presented. Using a "butterfly" local coil and a 1.5-T GE Signa MR system, the authors obtained 3-mm-thick axial, parasagittal, and 45 degrees oblique MR images of the cervical neural foramina in four normal volunteers. For the oblique images, subjects were rotated 45 degrees toward the right posterior oblique. Neural and vascular structures in the foramina were identified by correlation of the MR images with corresponding cryomicrotomic sections from four cadavers. The 45 degrees oblique images were more useful than axial or parasagittal images for demonstrating anatomic relations in the neural foramina. MR imaging with local coils demonstrates the complex anatomy of cervical neural foramina in various planes.

Probing iso-1-cytochrome c structure by site-directed spin labeling and electron paramagnetic resonance techniques.

A cysteine-specific methanethiosulfonate spin label was introduced into yeast iso-1-cytochrome c at three different positions. The modified forms of cytochrome c included: the wild-type protein labeled at naturally occurring C102, and two mutated proteins, S47C and L85C, labeled at positions 47 and 85, respectively (both S47C and L85C derived from the protein in which C102 had been replaced by threonine). All three spin-labeled protein derivatives were characterized using electron paramagnetic resonance (EPR) techniques. The continuous wave (CW) EPR spectrum of spin label attached to L85C differed from those recorded for spin label attached to C102 or S47C, indicating that spin label at position 85 was more immobilized and exhibited more complex tumbling than spin label at two other positions. The temperature dependence of the CW EPR spectra and CW EPR power saturation revealed further differences of spin-labeled L85C. The results were discussed in terms of application of the site-directed spin labeling technique in probing the local dynamic structure of iso-1-cytochrome c.

Planar-pair local coils for high-resolution magnetic resonance imaging, particularly of the temporomandibular joint.

The two-loop--one-gap loop--gap resonator (planar pair) has been investigated as a local receiving coil for high-resolution proton imaging at 1.5 T (63.8 MHz). Sensitivity contours were determined on the bench by measuring the voltage induced in the coil when it is placed on the surface of a tank containing 0.1-M NaCl, as a function of the coordinates and orientation of a radiating dipole inside the tank. Contours were obtained as a function of the diameter D of the two loops, and their separation d. For comparison, considerable data also were obtained for single-turn circular loops as a function of loop diameter. Coil quality factors, Q's, are reported in free space and the saline tank as a function of geometric parameters. Contours also were obtained using phantoms in a whole-body imager. Planar-pair coils couple to slabs of approximately (2D + d) X D cross section, and are superior to circular coils for imaging anatomic structures that are somewhat elongated. A particularly important feature of planar-pair local coils is that they are intrinsically isolated from the whole-body coil of the imager, and no special passive or active circuits are required when the whole-body coil is used for excitation. Planar-pair local coils have been optimized for imaging of the temporomandibular joint, and an image is shown.

Direct evidence of nitrogen coupling in the copper(II) complex of bovine serum albumin by S-band electron spin resonance technique.

ESR spectra of the tight binding Cu(II) complex of bovine serum albumin (BSA) has been studied using S-band. At physiological pH, only one form of copper binding to BSA was detected from the ESR spectra. From previous X-band ESR spectra, nitrogen superhyperfine splittings were observable in the g perpendicular region; however, the resolution of the g parallel region was not sufficient to confirm the exact donor atoms of the complex. Using low-frequency ESR (2-4 GHz) at 77 K, we have resolved the nitrogen superhyperfine structure in the g parallel region. A computer simulation method has been developed for distinguishing between three and four nitrogen donor atoms. The Hyde-Froncisz theory of g and A strain broadening has been modified to use a field-swept calculation for the line shape. The observed intensity pattern and the computer simulation of such spectra positively confirm the structure of Cu(II) ion coordinated to four in-plane nitrogen atoms in frozen aqueous solutions of Cu(II)-BSA complexes at physiological pH. This is the first time that this binding site has been confirmed on the protein instead of a protein fragment or model compound. This work is another example of the usefulness of the S-band ESR technique for characterizing the metal-protein interactions when random variation in g factors cause line broadening in conventional X-band ESR spectra.

Spin-label oximetry at Q- and W-band.

Spin-lattice relaxation times (T1s) of small water-soluble spin-labels in the aqueous phase as well as lipid-type spin-labels in membranes increase when the microwave frequency increases from 2 to 35 GHz (Hyde, et al., J. Phys. Chem. B 108 (2004) 9524-9529). The T1s measured at W-band (94 GHz) for the water-soluble spin-labels CTPO and TEMPONE (Froncisz, et al., J. Magn. Reson. 193 (2008) 297-304) are, however, shorter than when measured at Q-band (35 GHz). In this paper, the decreasing trends at W-band have been confirmed for commonly used lipid-type spin-labels in model membranes. It is concluded that the longest values of T1 will generally be found at Q-band, noting that long values are advantageous for measurement of bimolecular collisions with oxygen. The contribution of dissolved molecular oxygen to the relaxation rate was found to be independent of microwave frequency up to 94 GHz for lipid-type spin-labels in membranes. This contribution is expressed in terms of the oxygen transport parameter W=T1⁻¹(Air)-T1⁻¹(N2), which is a function of both concentration and translational diffusion of oxygen in the local environment of a spin-label. The new capabilities in measurement of the oxygen transport parameter using saturation-recovery (SR) EPR at Q- and W-band have been demonstrated in saturated (DMPC) and unsaturated (POPC) lipid bilayer membranes with the use of stearic acid (n-SASL) and phosphatidylcholine (n-PC) spin-labels, and compared with results obtained earlier at X-band. SR EPR spin-label oximetry at Q- and W-band has the potential to be a powerful tool for studying samples of small volume, ~30 nL. These benefits, together with other factors such as a higher resonator efficiency parameter and a new technique for canceling free induction decay signals, are discussed.

New model of charged molecule redistribution induced in spherical vesicles by direct current electric field.

A new electrophoresis model of charged components in a spherical phospholipid vesicle is proposed. In the new model the effective local tangential electric field is a result of the uniform external electric field modified by the electric field of redistributed charges. The modification is calculated on the basis of the Gouy-Chapman surface potential theory. Numerical calculations of steady-state distribution of charged molecules and the transmembrane potential are performed. The results show significant difference from the old, simplified model that neglects modification of the external electric field caused by redistributed charges.

Spin-label oximetry: kinetic study of cell respiration using a rapid-passage T1-sensitive electron spin resonance display.

An unusual ESR display has been developed that exhibits sensitivity to bimolecular collisions of dissolved oxygen in water with nitroxide radical spin probes at oxygen concentrations as low as 0.1 microM, requiring only 1 microliter of sample. The method involves observation of the ESR rapid-passage signal when tuned to the dispersion using a loop-gap resonator. The bimolecular collision rate determines the phase of the signal. The method has been used in a closed-chamber geometry to study respiration of asynchronous populations of Chinese hamster ovary (CHO) cells. An integral of the Michaelis-Menten equation permits direct comparison with experiment and is shown to be incompatible with the data. The theory of diffusion limitation also is developed and shown to be inconsistent with experiment. The average oxygen concentration is found to decrease as Vmaxt, where t is the time after sealing the chamber, to a critical oxygen concentration of 5.2 microM. Below 5.2 microM, the concentration can be fitted to an exponential form, exp(-t/tau), where tau = 15 sec for 4000 cells per microliter. It is believed that this experimental behavior is determined by complex enzyme kinetics.

A complication in prescan strategy when using surface coils.

The "prescan" process for setting the gain of an MRI scanner can be confounded by an effect arising from the spatial variation of the angle between the vector reception field of a local or surface coil and the excitation field of a whole-body coil. It is most pronounced when the phase-encoding direction is parallel to the plane of the coil but not along the magnetic field. The largest signal is shown to reside in a data row that is shifted from the one that corresponds to zero phase-encoding gradient. The effect depends on the field-of-view and the dimensions of the receiving coil. Strategies are suggested by which manufacturers of scanners can cope with this intrinsic problem. In the meantime, users of surface coils should be aware of the difficulty.

An evaluation of paramagnetic broadening agents for spin probe studies of intact mammalian cells.

Six transition metal ion complexes have been examined for their effects on the cell survival as well as their effectiveness in inducing the broadening of the electron spin resonance (ESR) spectra of nitroxide spin probes. These paramagnetic species are Ni(EDTA), Ni(DTPA), potassium tris(oxalato) chromate (chromium oxalate), K3Fe(CN)6, Cu(DTPA), and NiCl2. At 100 mM concentration, the typical concentration used in cell studies to broaden the extracellular nitroxide ESR signal, only Ni(EDTA) and Ni(DTPA) are found to be non-toxic to Chinese hamster ovary cells. The relative cytotoxicities of the six metal ion complexes are Cu(DTPA) greater than K3Fe(CN)6 greater than NiCl2 greater than chromium oxalate greater than Ni(DTPA) greater than Ni(EDTA). Thus, potassium ferricyanide and NiCl2, two most commonly used paramagnetic broadening agents, are relatively toxic to the cell. In contrast, among the six paramagnetic species tested here, chromium oxalate appears to be the most effective agent at non-toxic concentrations in inducing the broadening of the ESR spectra of both cationic and neutral nitroxide spin probes. By considering both their cytotoxicity and their effectiveness in causing line broadening of the nitroxide ESR spectra, chromium oxalate is a good paramagnetic broadening agent for spin probe studies of intact mammalian cells.

Analysis of powder and single crystal electron paramagnetic resonance spectra of deoxy myoglobins containing cobalt (II) proto-and mesoporphyrins IX at various microwave frequencies.

Artificial myoglobins (Mbs) substituted for protoheme with Co(II) proto-and mesoporphyrins IX (proto-and meso-CoMbs, respectively) were prepared. The principal values and eigenvectors of g tensors and the hyperfine coupling tensors of the paramagnetic Co(II) centers of their deoxy forms have been determined by single crystal EPR spectroscopy at 77 K in order to elucidate orientation and electronic structure of the prosthetic group in myoglobin. The orientation of the porphyrin plane of deoxy meso-CoMb were found to be identical to that of deoxy proto-CoMb. However, the in-plane hyperfine coupling constants of deoxy meso-CoMb were more anisotropic and larger than those of deoxy proto-CoMb, suggesting an increase in the electron spin density on the Co(II) ion upon the exchange of protoporphyrin IX with mesoprophyrin IX. Powder EPR spectra of these CoMbs, which were measured at S- and L-band microwave frequencies, exhibited well resolved 59Co hyperfine splittings and can be clearly interpreted by the use of the EPR parameters obtained from single crystal EPR measurements.