Two types of kinetics of membrane potential of water plant leaves illuminated by ultraviolet light

The effects of ultraviolet-B (UV-B) radiation on plasma membrane of water plant (Elodea canadensis, Vallisneria spiralis) cells were investigated by using microelectrode methods. A fast and reversible depolarization of membrane potential occurs initially during exposure of leaf cells to UV on a white light background, after which a slow phase of depolarization sets in. On action series, UV is pulsed for 15 s, with dark interval of 3 min, no monotonous response of systems on the UV excitation is observed. The action spectrum of the fast UV response lies in the interval of 300-330 nm and that of the slow phase-in the interval of 280-300 nm. The input impedance of membranes remains unchanged during the period of exposure. It is concluded that the H(+)-extruding complex of plant cell plasma membranes really consists of two types of interrelated electronic H(+)-pumps: an H(+)-pump of redox-active nature and the H(+)-pump of the H(+)-ATPase enzyme complex. Clearly, during the exposure of leaf cells to UV light, initially, the H(+)-pump of redox-active nature and then H(+)-ATPase are inhibited. It is proposed that the initial chromophore of UV-B light on plasma membrane can be one of the components of H(+)-pump of redox-active nature. It is probably the molecular of quinone.     

Effect of pressure on coupled electronic ground and excited states determined from luminescence spectra of trans-dioxorhenium(V) complexes

Pressure-dependent luminescence spectra of trans-dioxo complexes of rhenium(V) with ancillary ethylenediamine ligands exhibit resolved vibronic structure in the O=Re=O symmetric stretching mode at room temperature. The intensity distribution within the vibronic progression changes with pressure, leading to band shapes that are also pressure-dependent. These spectroscopic features arise from coupled electronic states and depend on the energy differences between ground and excited states, which vary by 2500 cm(-1) for the three complexes with ethylenediamine, tetramethylethylenediamine, and tetraethylethylenediamine ancillary ligands. We describe the pressure-dependent vibronic structure and band shapes with anharmonic adiabatic potential energy surfaces for the ground states of all complexes. The calculated spectra reveal the pressure dependence of the energies of electronic origins, luminescence band maximums, offsets between ground- and emitting-state potential minimums, and vibrational frequencies. The largest pressure effects are observed where the coupled electronic states are close in energy.     

Rapid quantitative capillary zone electrophoresis method for monitoring the micro-heterogeneity of an intact recombinant glycoprotein

A simple high-resolution capillary zone electrophoresis (CZE) method capable of rapidly assessing the micro-heterogeneity of a 24 kDa molecular weight glycoprotein, has been developed. Separation is carried out using a bare silica capillary at a pH of 2.5 in a commercially available electrophoresis buffer system composed of triethanolamine and phosphoric acid. Over 30 peaks were detected within a run time of 15 min using a 27 cm capillary and approximately 60 peaks were detected using a 77 cm capillary. Although most of the peaks arise from differences in the oligosaccharide structures present on the one glycosylation site on this molecule, other forms of micro-heterogeneity due to the presence of the nonglycosylated form of this glycoprotein and various types of chemical degradation, e.g., deamidation, are also responsible for the multitude of peaks observed. Although the exact chemical identity of each peak in the resulting electropherogram of this glycoprotein is not known, useful information can be obtained for assessing comparability, stability, and batch consistency. Factors impacting the resolution, precision, accuracy, and robustness of the assay are also discussed along with inherent advantages and limitations associated with measuring the micro-heterogeneity of intact glycoproteins.     

Evaluation of multidimensional (ion-exchange/reversed-phase) protein separations using linear and step gradients in the first dimension

The performance characteristics of multidimensional liquid chromatographic protein separations were evaluated using on-line electrospray mass detection, and a novel workflow for automated LC/MS data processing. Two-dimensional ion exchange/reversed-phase LC separations of Escherichia coli cytosol were conducted using either a continuous linear or discontinuous step gradient in the first dimension. Chromatographic profiles of the top 100 most abundant components were characterized to assess overall separation reproducibility within each mode, and to characterize differences in component distribution between the two modes of operation. Analysis of the resulting data indicates that multidimensional separations of complex protein mixtures can be done reproducibly. Furthermore, under the conditions employed within this study, a linear first dimension gradient was more effective at fractionating the protein mixture, distributing fewer major components to multiple second dimension cycles than an equivalent step gradient. The application of on line mass spectrometry, and automated processing of the resulting data, proved valuable for producing component level analysis of multidimensional protein separations.     

Profiling of impurities in illicit methamphetamine by high-performance liquid chromatography and capillary electrochromatography

High performance liquid chromatography (HPLC) with photodiode array (PDA) UV and fluorescence (FL) detection, and capillary electrochromatography (CEC) with laser-induced fluorescence (LIF) detection were investigated for the analysis of acidic extracts derived from illicit methamphetamine. These compounds include major impurities from the hydriodic acid/red phosphorous reduction method, i.e., 1,3-dimethyl-2-phenylnaphthalene and 1-benzyl-3-methylnaphthalene, and other trace-level, structurally related impurities. For certain of these solutes, HPLC with conventional FL detection gave at least a 60× increase in sensitivity over UV detection. In addition, other highly fluorescent impurities were detected in methamphetamine produced via four other synthetic routes. The use of a rapid scanning FL detector (with acquisition of “on the fly” excitation or emission) provided structural information and gave “optimum” excitation and emission detection wavelengths. CEC with LIF detection using UV laser excitation provided greatly improved chromatography over HPLC, with good detection limits in the low ng/ml range. Both methodologies provide good run-to-run repeatability, and have the capability to distinguish between samples.