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Seeing the big proteins The limited numbers of bacterial proteins that have been observed by MALDI (matrix-assisted laser desorption/ionization) MS thus far have been smaller than 20 kDa. However, the bacterial genome is known to express approximately 2000 proteins, most of which are larger than 20 kDa. What about the rest of the proteins? David M. Lubman and co-workers at The University of Michigan and Michigan State University detected large E. coli proteins (>25 5ka) in whole cell lysates using MAIDI-TOF (timeof-flight) MS. To make the larger proteins more amenable to MALDI analysis, the researchers treated the cells with a combination of Triton X-100 and guanidine hydrochloride, which disrupted and solubilized the inner-
MALDI-TOFuninduced E. coli whole celllysate spectrum up to 500 kDa. (Adapted with permission. Copyright 1997 John Wiiey & Sons..
Continuous-flow polarized xenon
Their system combines a fast optical pumping system with a closed gas circulation NMR probe head. Polarized xenon is generated by first pumpA clever method for boosting NMR siging rubidium vapors with a high-power nals is to expose the sample to laserdiode laser array, followed by spin expolarized xenon. For NMR studies of change to xenon (with spin-exchange surfaces, the signal can be increased by time constants of around tens of sectransferring polarization from adsorbed onds) . The sample—in this case Aeropolarized xenon by a process dubbed sil30() with about 2.5 hydroxy protons/ SPINOE (spin polarization-induced nunm2—is packed into a 10-mxn U-shaped clear Overhauser effect). Mathias tube and placed in a double-tuned Haake and colleagues at the University probe ('H and ,2-'Xe). The entire asof California at Berkeley have taken sembly is evacuated for several hours another step toward making this a routine method by introducing a system for to remove physisorbed water and oxyand the polarized is circurapid production and continuous delivery of polarized xenon to NMR samples. lated across the sample The authors report that the experiment can be run for hours and does not quire isotopically enriched xenon The approach yields an enhanced signal for the Aerosil sample with the SPINOE pulse sequence. The new system also offers the possibility of directly polarizing dilute heteronuclei on surfaces and using polarized Schematic showing the gas circulation pump (GCP), the xenon to probe the porosU-shaped sample tube within the superconducting ity of materials. (J. Am. magnet (SC), a photodiode (PD) to detect transmitted Chem. Snc. 1197 119, light, and the optical pumping cell, which is in a fringe 11711-12) magnetic Held of 150 G.
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Analytical Chemistry News & Features, February 1, 1998
membrane proteins. The matrix was composed of a-cyano-4hydroxycinnamic acid. The presence of induced and repressed protein synthesis in response to L-arabinose catabolism in E. coli cells was monitored. The molecular weights of the observed proteins were in the range of 25120 kDa. The authors compared the results with those from 1-D and 2-D gel electrophoresis {Rabid Cotnmun MassStoectrom 1997 11 1900-08)
Detecting picomolar Pb 2 + Ion-selective polymer membrane electrodes (ISEs) typically have detection limits in the micromolar range. Sample ion activities are often adjusted by adding ion buffers to improve the detection limit; however, these buffers are not suitable for many situations. Erno Pretsch and co-workers of the Swiss Federal Institute of Technology report a new method that dramatically improves the lower detection limit of ISEs by preventing primary ions in the internal electrolyte from leaching into the sample. This is accomplished by building up a concentration gradient in the electrode's membrane using an electrolyte that has a low activity for the primary ion cincl 3. high activity for an interfering ion. Two Pb2+ ISEs with different inner reference systems were investigated, one with the conventional internal filling solution (PbCl2 and MgCl2) and one with a new internal solution [Pb(N0 3 ) 2 and EDTA-Na2], With the conventional filling solution, the detection limit for Pb 2+ was 4 x 10"6 M, and no Nernstian response toward the discriminated Na+ was observed. With the new filling solution, however, linear Nernstian response for Pb2+ was observed down to 10~ n M with a detection limit of 5 x ,0" 12 M. Also with the new internal solution Nernstian response toward Na+ was observed between 10~2 and 10""3 M making it possible to determine true selectivity coefficients
Observation of the magneto-chiral effect
Concentration profiles in an ISE. Dashed line represents conventional systems; solid line represents a concentration gradient obtained with new filling solution.
The method can be applied to other ions in addition to Pb2+, as long as the activity of the ion can be buffered at low levels in the internal filling solution of the ISE. The authors have observed similar results with Ca2+ and Ag+ ISEs. In all cases, improved lower detection limits and selectivities were observed. The robustness of the system is currently being evaluated. (J. Am. Chem. Soc. 1997,119,11347-48)
Measuring oxidative damage Free radicals are known to cause aging and age-related diseases. A marker is needed to measure the amount of oxidative damage caused by free radicals. Stefan A. J. Coolen and colleagues at the University of Technology Eindhoven and the State Institute for Quality Control of Agricultural Products (both in The Netherlands) use the antipyretic drug antipyrine, which is used to measure total hepatic oxidase activity, as a marker for oxidative stress because its metabolic pathway is well-known. In oxidative damage, excess free radicals will react with antipyrine as well as with biomolecules. To determine which free-radical products of antipyrine might be found in the human body during oxidative stress, antipyrine in water was exposed to a 60 Co y-source to generate free hydroxyl radicals. The hydroxylated derivatives that formed were separated by HPLC and micellar electrokinetic capillary chromatography (MEKC). The authors found that the reaction between hydroxyl radicals and antipyrine occurs according to pseudo-first-order reac-
Optical activity, whether it be natural or magnetically induced, causes the polarization of transmitted light to be rotated. Natural and magnetically induced optical activity closely resemble one another—the difference being that the former is an intrinsic property of a substance that lacks mirror symmetry and the latter is induced in any substance by applying a magnetic field that breaks she molecule's symmetry. Although the two effects have vastly different origins, a situation can arise in which symmetries Etre broken tit the same time, resulting in an additional optical effect, referred to as the magneto-chiral effect. G.LJ.A Rikken and E. Raupach of Max Planck Institut fur Festkorperforschung/CNRS (France) report for the first time en the direct tobervatton oo the magneto-chiral optical effect. This link between natural and magnetically induced optical activity, albeit a weak one, has long been predicted; however, until now there has been no hard evidence for the existence of such a relationship. In their experiment, differences in luminescence intensity of tris(3-trifluoroacetyl-±-camphorato) europiuni(III) complexes, which show significant magnetic circular dichroism in aqueous solution, are measured in directions parallel and antiparallel to an externally applied magnetic field
tion kinetics and that the correlation coefficient between HPLC and MEKC was 0.9984. The reaction products (ortho-, meta-, and/>ara-hydroxylated antipyrine) were characterized by NMR and MS, and the values of the dissociation constants (9, 8.6, and 9, respectively) were determined. The ortho- -nd mee ta-hydroxylated isomers differed from the enzymatic metabolites formed in humans. The effects of different doses of radiation were also discussed. (J. Chromatoer A 1 9 9 7 788 95-103)
Schematic diagram of apparatus for detecting magneto-chiral luminescence. (Adapted with permission. Copyright 1997 Macmillan Magazines.)
As shown in the schematic diagram, the sample (S) is subjected to filtered (F) unpolarized llght trom a mercury discharge lamp. The sample is excited, and its luminescence is collected with optical fibers and detected by silicon photodiodes. Specific emission wavelengths can be chosen with a grating monochromator (M). Differences in the intensity between parallel and antiparallel directions are detected by a lock-in amplifier (LA). The authors suggest that, because magneto-chiral anisotropy has now been shown to exist in emission, the relations between Einstein's coefficients in radiative processes imply its existence in absorption, and the Kramers-Kronig relations imply its existence in the real part of the refractive index. (Nature 1997, 390,493-94)
Formation of radiation products of a 1 -mM antipyrine solution versus time during exposure to radiation (dose rate = 1.5 kGy/h). (1) 0 Gy, (2) )065 Gy, and (3) 2650 Gy. (a) Para-, (b) meta-, and (c) ortho-hydroxylated antipyrine; (d) antipyrine. (Adapted with permission. Copyright 1997 Elsevier Science.)
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