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Selected AbstractsCharacterization of carbonic anhydrase from Neisseria gonorrhoeaeFEBS JOURNAL, Issue 6 2001Björn Elleby We have investigated the steady state and equilibrium kinetic properties of carbonic anhydrase from Neisseria gonorrhoeae (NGCA). Qualitatively, the enzyme shows the same kinetic behaviour as the well studied human carbonic anhydrase II (HCA II). This is reflected in the similar pH dependencies of the kinetic parameters for CO2 hydration and the similar behaviour of the kinetics of 18O exchange between CO2 and water at chemical equilibrium. The pH profile of the turnover number, kcat, can be described as a titration curve with an exceptionally high maximal value of 1.7 × 106 s,1 at alkaline pH and a pKa of 7.2. At pH 9, kcat is buffer dependent in a saturable manner, suggesting a ping-pong mechanism with buffer as the second substrate. The ratio kcat/Km is dependent on two ionizations with pKa values of 6.4 and 8.2. However, an 18O-exchange assay identified only one ionizable group in the pH profile of kcat/Km with an apparent pKa of 6.5. The results of a kinetic analysis of a His66,Ala variant of the bacterial enzyme suggest that His66 in NGCA has the same function as a proton shuttle as His64 in HCA II. The kinetic defect in the mutant can partially be overcome by certain buffers, such as imidazole and 1,2-dimethylimidazole. The bacterial enzyme shows similar Ki values for the inhibitors NCO,, SCN, and N3, as HCA II, while CN, and the sulfonamide ethoxzolamide are considerably weaker inhibitors of the bacterial enzyme than of HCA II. The absorption spectra of the adducts of Co(II)-substituted NGCA with acetazolamide, NCO,, SCN,, CN, and N3, resemble the corresponding spectra obtained with human Co(II)-isozymes I and II. Measurements of guanidine hydrochloride (GdnHCl)-induced denaturation reveal a sensitivity of the CO2 hydration activity to the reducing agent tris(2-carboxyethyl)phosphine (TCEP). However, the A292/A260 ratio was not affected by the presence of TCEP, and a structural transition at 2.8,2.9 m GdnHCl was observed. [source] Prediction of the association state of insulin using spectral parametersJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 4 2003Vladimir N. Uversky Abstract Human insulin exists in different association states, from monomer to hexamer, depending on the conditions. In the presence of zinc the "normal" state is a hexamer. The structural properties of 20 variants of human insulin were studied by near-UV circular dichroism, fluorescence spectroscopy, and small-angle X-ray scattering (SAXS). The mutants showed different degrees of association (monomer, dimers, tetramers, and hexamers) at neutral pH. A correlation was shown between the accessibility of tyrosines to acrylamide quenching and the degree of association of the insulin mutants. The near-UV CD spectra of the insulins were affected by protein association and by mutation-induced structural perturbations. However, the shape and intensity of difference CD spectra, obtained by subtraction of the spectra measured in 20% acetic acid (where all insulin species were monomeric) from the corresponding spectra measured at neutral pH, correlate well with the degree of insulin association. In fact, the near-UV CD difference spectra for monomeric, dimeric, tetrameric, and hexameric insulin are very distinctive, both in terms of intensity and shape. The results show that the spectral properties of the insulins reflect their state of association, and can be used to predict their oligomeric state. © 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 92:847,858, 2003 [source] Optimum acquisition of Raman spectra in pigment analysis with IR laser diode and pulsed UV irradiationJOURNAL OF RAMAN SPECTROSCOPY, Issue 10 2006Alejandro López-Gil Abstract Fluorescence, due to the binding media, is the main problem that one encounters in the molecular analysis of artistic pigments using Raman spectroscopy. With the object of minimizing this problem, we propose the use of a semiconductor IR laser in Raman spectral acquisition and the application of local irradiation with a pulsed UV laser on the analysis zone. The Raman analysis with an IR source shows advantages compared to that with visible lasers, such as the reduction of fluorescence and its shot noise, although a price has to be paid in the form of the small intensity of the obtained spectra with the same acquisition time. Also, in this paper we demonstrate that controlled levels of pulsed UV radiation over the analyzed painting (pigment + binding media) can improve even more the Raman spectral quality obtained with the IR laser, which leads us to conclude that the local use of a pulsed UV laser, prior to Raman analysis with the IR source, can bring optimum quality results in pigment identification. The spectral quality of these experimental results for different pigments has been measured by calculating in each case the signal-to-noise ratio (SNR) of the corresponding spectra. From a quantitative point of view, in some practical cases (chromium yellow and ultramarine blue) an SNR improvement of 16 dB is achieved when pulsed UV irradiation and IR laser Raman analysis are used instead of a simple Raman analysis with a visible laser. Copyright © 2006 John Wiley & Sons, Ltd. [source] The local isotropy hypothesis and the turbulent kinetic energy dissipation rate in the atmospheric surface layerTHE QUARTERLY JOURNAL OF THE ROYAL METEOROLOGICAL SOCIETY, Issue 603 2004M. Chamecki Abstract We test the applicability of the local isotropy hypothesis to surface-layer turbulent flow; turbulent velocities measured with a three-dimensional sonic anemometer are used for this purpose, and the predictions of local isotropy for the spectra, second- and third-order structure functions are assessed against measured data. Also investigated are scale interactions via the correlation between velocities and velocity increments, and the ability of isotropic spectral models to reproduce measured spectra. In general, second-order structure functions display a narrower inertial range than the corresponding spectra; both the known effects of path-averaging and the predictions of the spectral models show that the sonic anemometer is unable to resolve the whole inertial range, even at a measurement frequency of 60 Hz. We confirm previous results that unstable runs tend to be more isotropic, but find that, for third-order statistics, isotropy does not hold well for the data analysed. Turbulence intensity, and not atmospheric stability, plays a determining role on the correlation coefficient between velocities and velocity increments. The observed anisotropic behaviour has important implications for the calculation of the turbulent kinetic energy dissipation rate from Kolmogorov's four-fifths law, whose estimates are consistently smaller than those from the inertial range of the spectrum or the structure functions. Copyright © 2004 Royal Meteorological Society [source] Phosphorescence of SiO2 optical fibres doped with Ce3+ ionsPHYSICA STATUS SOLIDI (C) - CURRENT TOPICS IN SOLID STATE PHYSICS, Issue 3 2007I. Veronese Abstract Phosphorescence emitted by a new dosimetric system based on a SiO2 optical fibre doped with Ce3+ ions was investigated. The defects in the matrix that, acting as electron traps, originate the phosphorescence signal were studied by means of thermally stimulated luminescence in the temperature interval 313-533 K. A continuous trap distribution with activation energies extending from 0.8 to 1.5 eV was observed. On the basis of these findings, the temperature dependence of the shape of isothermal phosphorescence decay was analysed and the corresponding spectrum was compared with that of radioluminescence. (© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] |