INTERACTION OF DIBUCAINEHCl LOCAL ANESTHETICS WITH BACTERIORHODOPSIN IN PURPLE MEMBRANE: A SPECTROSCOPIC STUDY

Several spectroscopic techniques (absorption, emission, transient absorption and differential scanning calorimetry--DSC) were used to investigate the deprotonation of dibucaine.HCl in a hydrophobic environment, and the interaction sites and mechanisms of the local anesthetic dibucaine.HCl on bacteriorhodopsin (bR) in purple membrane. The important results are summarized as follows: (1) the visible absorption features of native (lambda max = 568 nm) and deionized (lambda max = 608 nm) bR are sensitive to the amount of dibucaine.HCl added; (2) the emission spectrum of dibucaine.HCl embedded in the retinal-free mutant bR is similar to that of dibucaine free base in Triton X-100 micellar solutions; (3) the phosphorescence emission of dibucaine at 77 K is completely quenched by bR and the fluorescence quenching rate for the incorporated dibucaine.HCl in bR was determined as kq = 4.09 x 10(13) M-1 s-1; (4) the incorporation of dibucaine.HCl in bR inhibits the slow component rate of formation of M412 and decreases the amount of M412 formation in the photochemical cycle of bR; and (5) the thermal stability of native bR was measured by DSC in the presence and absence of dibucaine and yielded an endothermic transition at 95.9 +/- 1.0 degrees C with 13.6 J/g (3.25 +/- 0.12 cal/g) of enthalpy changes. All observations suggest that the action site of the local anesthetic, dibucaine.HCl, is near or at the chromophore, i.e. the retinal Schiff base of bR. The anesthetic action on bR purple membrane is probably via a specific site binding, but not a conformational mechanism.     

Enhanced signal intensities obtained by out-of-phase rapid-passage EPR for samples with long electron spin relaxation times

To understand the signals that are observed under rapid-passage conditions for samples with long electron spin relaxation times, the E' defect in irradiated vitreous SiO(2) was studied. For these samples at room temperature, T(1) is 200 mciro s and T(2) ranged from 35 to 200 micro s, depending on spin concentration. At X band with 100-kHz modulation frequency and 1-G modulation amplitude there was minimal lineshape difference between the low-power, in-phase spectra and high-power spectra detected 90 degrees out-of-phase with respect to the magnetic field modulation. Signal enhancement, defined as the ratio of the intensities of the out-of-phase to the in-phase signals when B(1) for both observation modes is adjusted to give maximum signal, was 3.4 to 9.5 at room temperature. The origin of the out-of-phase signal was modeled by numerical integration of the Bloch equations including magnetic field modulation. The waveforms for the E' signal, prior to phase sensitive detection, were simulated by summing the contributions of many individual spin packets. Good agreement was obtained between experimental and calculated waveforms. At low B(1) the experimental values of T(1) and T(2) were used in the simulations. However, at higher B(1), T(2) was adjusted to match the experimental signal intensity and increased with increasing B(1). At high B(1), T(2)=T(1), consistent with Redfield's and Hyde's models. For the spin concentrations examined, the out-of-phase signals at very high power (B(1) approximately 0.33 G) displayed a linear relationship between peak-to-peak signal amplitude and spin concentration. Under the conditions used for spin quantitation the signal-to-noise for these spectra was up to 5 times higher than for the in-phase signal, which greatly facilitates quantitation for these types of samples. For samples in which T(2) is dominated by electron spin-spin interaction, lower spin concentration results in longer T(2) and the enhancement is increased.     

Electron spinT 2 of a nitroxyl radical at 250 MHz measured by rapid-scan EPR

Rapid-scan electron paramagnetic resonance (EPR) was used to measure the room temperature spin-spin relaxation timeT ₂ for the per-deuterated nitroxyl radical, 4-oxo-2,2,6,6-tetramethylpiperidin-1-oxyl-d₁₆ in water at 250 MHz. Signals were recorded on a locally constructed system using either sinusoidal or triangular magnetic field scans. Values ofT ₂ were obtained by simulation of the rapid-scan response and were systematically shorter for the high-field line (m ₁=?1) than for them ₁=0 or +1 lines. Form ₁=+1, the value ofT ₂ increased from 0.41±0.03 μs at 0.5 mM to 0.53±0.03 μs at 0.1 or 0.2 mM. Form ₁=?1,T ₂ increased from 0.35±0.03 μs at 0.5 mM to 0.42±0.03 μs at 0.1 or 0.2 mM. These values put lower bounds on the values ofT ₁ for this nitroxyl at 250 MHz.     

A functional central limit theorem for a generalized occupancy problem

A functional central limit theorem is proved for a class of finitely exchangeable random variables which are based on an occupancy scheme.     

An L-band crossed-loop (Bimodal) EPR resonator

Our crossed-loop resonator design has been enhanced to increase the filling factor and has been extended from S-band to L-band. High isolation between the two modes results in shorter dead time in pulsed EPR experiments than would occur with a reflection resonator of the same Q.     

Electron spin relaxation of triarylmethyl radicals in fluid solution

Electron spin relaxation times of a Nycomed triarylmethyl radical (sym-trityl) in water, 1:1 water:glycerol, and 1:9 water:glycerol were measured at L-band, S-band, and X-band by pulsed EPR methods. In H(2)O solution, T(1) is 17+/-1 micros at X-band at ambient temperature, is nearly independent of microwave frequency, and exhibits little dependence on viscosity. The temperature dependence of T(1) in 1:1 water:glycerol is characteristic of domination by a Raman process between 20 and 80 K. The increased spin-lattice relaxation rates at higher temperatures, including room temperature, are attributed to a local vibrational mode that modulates spin-orbit coupling. In H(2)O solution, T(2) is 11+/-1 micros at X-band, increasing to 13+/-1 micros at L-band. For more viscous solvent mixtures, T(2) is much shorter than T(1) and weakly frequency dependent, which indicates that incomplete motional averaging of hyperfine anisotropy makes a significant contribution to T(2). In water and 1:1 water:glycerol solutions continuous wave EPR linewidths are not relaxation determined, but become relaxation determined in the higher viscosity 1:9 water:glycerol solutions. The Lorentzian component of the 250-MHz linewidths as a function of viscosity is in good agreement with T(2)-determined contributions to the linewidths at higher frequencies.     

PISEMA powder patterns and PISA wheels

Resonance patterns observed in 2D PISEMA (polarization inversion spin exchange at magic angle) spectra from a transmembrane alpha-helix have been demonstrated to yield structural details of the protein. This paper presents a mathematical discussion of the PISEMA powder spectrum as the image in the frequency plane of a quadratic function from the sphere of unit vectors. The simplicity of this function allows easy calculation of the powder spectrum. Based on this analysis of powder patterns, four degeneracies are discussed which arise in determining possible orientations associated with PISA spectra. This paper also gives parametric equations for PISA wheels, which are specific patterns observed in PISEMA spectra of oriented peptides. These wheels are useful both in assigning the resonances and in determining the orientation of the helix with respect to the magnetic field. The union of these PISA wheels gives the entire powder spectrum.     

Modelling,simulation, animation,and real-time control (MoSART) for a class of electromechanical systems: a system-theoretic approach

This paper describes an interactive modelling, simulation, animation, and real-time control (MoSART) environment for a class of ‘cart-pendulum’ electromechanical systems that may be used to enhance learning within differential equations and linear algebra classes. The environment is useful for conveying fundamental mathematical/systems concepts through computer-aided analysis, design, graphical visualization, and 3D animation. Referred to as Cart-Pendulum Control3D-Lab1Control3D-Lab-short for control 3D animation laboratory. The author to whom correspondence should be addressed., the environment is based on Microsoft Windows/Visual C++/Direct-3D and MATLAB/Simulink2MATLAB and Simulant are trademarks of The Masterworks, Inc.. Pull-down menus provide access to systems/models/control laws, exogenous signals, parameters, animation models and visual indicators, a suite of (easy-to-modify) Simulant diagrams containing models and control laws, MATLAB m-files for detailed analysis and design, and detailed documentation for each of the above. Three blocks enable animation, joystick inputs, and real-time animation within Simulant. Examples are presented to illustrate the utility of the environment as a valuable tool for analysing/visualizing the above class of electromechanical systems and for enhancing mathematics instruction.