Identification of protein surfaces by NMR measurements with a pramagnetic Gd(III) chelate.

Gd-diethylenetriamine pentaacetic acid-bismethylamide, Gd(DTPA-BMA), is shown to be a reagent suitable for the identification of protein surfaces. Compared to the conventionally used spin-label TEMPOL, Gd(DTPA-BMA) is a stronger relaxation agent, requiring lesser concentrations to achieve the same paramagnetic relaxation enhancement of solvent-exposed protein protons. It is also less hydrophobic and therefore less prone to specific binding to proteins. Relaxation enhancements predicted by a second-sphere interaction model correlated with experimental data recorded with ubiquitin, while the correlation with corresponding data recorded with TEMPOL was poor.     

PHOTOCHROMIC SYNERGISM OF BACTERIORHODOPSIN- and HALORHODOPSIN-MEDIATED PHOTOPHOSPHORYLATION IN Halobacterium halobium*

Quantitative action spectroscopy was performed in Halobacterium halobium. using four suited pigment mutants, namely the bacteriorhodopsin and halorhodopsin positive mutant strain M-l (BR⁺, HR⁺), the bacteriorhodopsin positive but halorhodopsin negative strain M-18 (BR⁺, HR^-), the bacteriorhodopsin negative but halorhodopsin positive strain L-33 (BR^-, HR⁺), and the bacteriorhodopsin and halorhodopsin negative strain L-07 (BR^-, HR⁺). The approached questions were: First, photoenergetic synergism of halorhodopsin and bacteriorhodopsin in intact cells; second, photochromism and cellular function of the blue light-absorbing intermediates, i.e. M-412 and HR-410 in bacteriorhodopsin and in halorhodopsin, respectively. Dark-adapted cells of mutant strain M-l show wavelength-dependency of quantum yield of photo-phosphorylation, φ_ATP. An 1.4-fold enhancement was found at 575 nm wavelength where the long wavelength absorbance bands of bacteriorhodopsin and halorhodopsin intersect. The enhancement vanished after a 30 min pulse of orange light (600 Wm^-2 bandpass from 495 to 750 nm), but was restored after a 30 min pulse of blue light (100 Wm^-2 bandpass from 325 to 480 nm). Photoreversibility of this enhancement probably reflects phototransformation of halorhodopsin from its ground state into its inactive intermediate, HR-410, and vice versa. The halorhodopsin-mediated enhancement with maximum quantum yield of photophosphorylation, φ_ATP= 0.06, i.e. a quantum requirement of = 17 photons/ATP, is partly substituted by a rise in phosphate potential and explained in terms of a voltage-regulated gating effect on the H⁺-driven ATP-synthase, superimposed on the chemiosmotic mechanism of energy coupling. The blue-absorbing photochromic intermediate, M-412 of bacteriorhodopsin, dissipates light energy upon photoexcitation that is reflected by a spectral decline in quantum yield of photophosphorylation to a minimum value of = 0.01 at 415 nm, i.e. a quantum requirement of = 100 photons/ATP.     

Erweiterung von Linearfunktionen in linearen Räumen

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OH-radical induced degradation of hydroxybenzoic- and hydroxycinnamic acids and formation of aromatic products—A gamma radiolysis study

The OH-radical induced degradation of hydroxybenzoic acids (HBA), hydroxycinnamic acids (HCiA) and methoxylated derivatives, as well as of chlorogenic acid and rosmarinic acid was studied by gamma radiolysis in aerated aqueous solutions. Primary aromatic products resulting from an OH-radical attachment to the ring (hydroxylation), to the position occupied by the methoxyl group (replacement –OCH₃ by ?OH) as well as to the propenoic acid side chain of the cinnamic acids (benzaldehyde formations) were analysed by HPLC–UV and LC–ESI–MS. A comparison of the extent of these processes is given for 3,4-dihydroxybenzoic acid, vanillic acid, isovanillic acid, syringic acid, cinnamic acid, 4-hydroxycinnamic acid, caffeic acid, ferulic acid, isoferulic acid, chlorogenic acid, and rosmarinic acid. For all cinnamic acids and derivatives benzaldehydes were significant oxidation products. With the release of caffeic acid from chlorogenic acid the cleavage of a phenolic glycoside could be demonstrated. Reaction mechanisms are discussed.     

Radiation damping on cryoprobes

Radiation damping on 600 and 800 MHz cryoprobes was investigated. The phase angle β between a vector 90° phase shifted to the precessing magnetization and the rf field induced in the coil was found to depend markedly on whether an FID was being acquired or not. The magnitude of the radiation damping field was sufficiently strong to restore 95% of the equilibrium water magnetization of a 90% H2O sample in a 5 mm sample tube within about 5 ms following a 165° pulse. This can be exploited in water flip-back versions of NOESY and TOCSY experiments of proteins, but care must be taken to limit the effect of the radiation damping field from the water on the Ha protons. Long water-selective pulses can be applied only following corrections. We developed a program for correcting pulse shapes if β is non-zero. The WATERGATE scheme is shown to be insensitive to imperfections introduced by radiation damping.     

Jodometrische Bestimmung des Natriums als Natriumzinkuranylacetat

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Studies on the Fast Atom Bombardment Mass Spectrometry Fragmentation of Amino Sugars Using Synthetic Derivatives of the Trisaccharide Unit β-D-Glucopyranosyl-(1-3)-O-2-deoxy-2-amino-β-d-glucopyranosyl-(1-4)-O-d-glucopyranose

Abstract

N-Phthaloyl, N-acetyl, N-benzyl, N-acetyl-N-methyl, N,N-dimethyl, N-benzoyl, and N,N-dibenzoyl derivatives of the trisaccharide β-D-glucopyranosyl-(1-3)-O-(2-deoxy-2-amino-β-D-glucopyranosyl)-(1-4)-O-β-D-glucopyranose were synthesized and analyzed by FAB MS. The intensity ratios of the peaks resulting from cleavage of the anomeric bond of the glucosamine residue and the respective molecular ion peaks turned out to be high for the N-acyl derivatives and up to two orders of magnitude lower for the N-alkyl compounds. These results show that fragmentation at the anomeric carbon of the amino sugar may be assisted by the carbonyl group and the resulting cation is stabilized by delocalization of the positive charge.     

Cadmium determination in biological samples by direct solid sampling flame atomic absorption spectrometry

A direct solid sampling flame atomic absorption spectrometric procedure for trace determination of cadmium in biological samples has been developed. Test samples (0.05–2.00 mg) were ground and weighed into small polyethylene vials, which were connected to the device for solid sample introduction into a conventional air/acetylene flame. Test samples were carried as a dry aerosol to a quartz cell, placed between the burner and the optical path, which had a perpendicular entrance and a slit in the upper part. The atomic vapor generated in the flame produced a transient signal that was totally integrated within 1 s. The effect of operating conditions and the extent of grinding on the analytical signal were evaluated. Background signals were always low and a characteristic mass of 0.29 ng Cd was obtained. Calibration was performed using different masses of solid certified reference materials. Results obtained for certified and in-house reference materials were typically within the 95% confidence interval of the certified and/or reference value, and the precision, expressed as relative standard deviation, was between 3.8 and 6.7%. The proposed system is simple and it might be adapted to conventional atomic absorption spectrometers allowing the determination of Cd in more than 80 test samples per hour, excluding weighing.