A new approach to phosphoserine and phosphothreonine analysis in peptides and proteins: chemical modification,enrichment via solid-phase reversible binding,and analysis by mass spectrometry

beta-Elimination of the phosphate group on phosphoserine and phosphothreonine residues and addition of an alkyldithiol is a useful tool for analysis of the phosphorylation states of proteins and peptides. We have explored the influence of several conditions on the efficiency of this PO(4)(3-) elimination reaction upon addition of propanedithiol. In addition to the described influence of different bases, the solvent composition was also found to have a major effect on the yield of the reaction. In particular, an increase in the percentage of DMSO enhances the conversion rate, whereas a higher amount of protic polar solvents, such as water or isopropanol, induces the opposite effect. We have also developed a protocol for enrichment of the modified peptides, which is based on solid-phase covalent capture/release with a dithiopyridino-resin. The procedure for beta-elimination and isolation of phosphorylated peptides by solid-phase capture/release was developed with commercially available alpha-casein. Enriched peptide fragments were characterized by MALDI-TOF mass spectrometric analysis before and after alkylation with iodoacetamide, which allowed rapid confirmation of the purposely introduced thiol moiety. Sensitivity studies, carried out in order to determine the detection limit, demonstrated that samples could be detected even in the low picomolar range by mass spectrometry. The developed solid-phase enrichment procedure based on reversible covalent binding of the modified peptides is more effective and significantly simpler than methods based on the interaction between biotin and avidin, which require additional steps such as tagging the modified peptides and work-up of the samples prior to the affinity capture step.     

Quinoprotein glucose dehydrogenase modified carbon paste electrode for the detection of phenolic compounds

The development of an amperometric biosensor for the determination of phenolic compounds is described, using quinoprotein glucose dehydrogenase. The enzyme is integrated into carbon paste and its ability to donate electrons to oxidized phenolic compounds during glucose oxidation is exploited. The sensor response is based on electrochemical oxidation of the phenolic compound followed by its enzymatic regeneration when the bulk solution contains glucose and the electrode is potentiostated at +500 mV (vs. Ag/AgCl/0.1 mol/L KCl). As the result of the catalytic analyte regeneration the electrodes offer very sensitive measurements of redox species like p-aminophenol and hydroquinone and catecholamines such as epinephrine, norepinephrine, and dopamine. The sensor performance is characterized for the different substrates. Highest sensitivity is achieved for p-aminophenol which could be determined at sub-nanomolar level.     

Reaktionen von komplexliganden : XIX. Einschiebung von inaminen in chrom-alkenylcarben- und chrom-aminocarber-bindungen

1-Diethylaminopropyne reacts with pentacarbonyl[methoxy(2,2-diphenylethenyl)carbene]chromium in a stereoselective way to give pentacarbonyl-[diethylamino-E-(2-methoxy-1-methyl-4,4-diphenyl-1,3-butadienyl)carbene]-chromium via insertion of the alkyne into the metal-carbene bond. The reaction of the ynamine with pentacarbonyl[methylamino(phenyl)carbene]chromium results in insertion of the alkyne and loss of carbon monoxide to give cis-tetracarbonyl[3-aza-1-methyl-2-phenyl-2-butenyl)diethylaminocarbene]-chromium. Its structure was established by oxidative degradation in an aqueous medium to give 2-benzoyl-N,N-diethylpropanamide and finally confirmed by an X-ray analysis. The new compounds are characterized spectroscopically.     

Substituenteneffekte auf die C–C-Bindungsstärke, 8. Standardbildungsenthalpien und Spannungsenthalpien von 1,1,2,2-Tetraethylethylenglykol-dimethylether und D,L-1,2-Dimethyl-1,2-diphenylethylenglykol-dimethylether

Effects of Substituents on the Strength of C - C Bonds, 8¹. - Heats of Formation and Strain of 1,1,2,2-Tetraethylethylene Glycol Dimethyl Ether and D,L .-1,2-Dimethyl-l,2-diphenylethylene Glycol Dimethyl Ether The heats of combustion of the title compounds 1 and 2 were measured calorimetrically with the result (kcal mol ^-1, s. d. in parentheses) ΔH°_c = − 1880.1 (± 0.6) and − 2373.3 (± 1.4). The heat of vaporisation of 1 ΔH^v = 14.3 (± 0.3) and the heat of sublimation of 2 ΔH^sub = 27.2 (± 0.5) were derived from their temperature dependance of the vapor pressure. The latter were determined between 30 and 80°C using a flow method. The resulting standard heats of formation ΔH°_t(g) = −122.4 (± 0.7) and −43.8 (±1.5) for 1 and 2 correspond to a strain enthalpy (_s) of 15.9 and 8.0 kcal mol^-1, respectively. The steric strain of the dimethoxyethanes 1 and 2 is about one fourth lower than the strain of the corresponding dimethylethanes 3 and 4 bearing the same substituents. Thus, a methoxy group causes less steric stress than a methyl group.     

Isolation,identification and synthesis of the sex pheromone of the citrus mealybug,Planococcus citri (risso)

The sex attractant pheromone of the citrus mealybug, $\text{Planococcus citri}$ (Risso), has been Identified as (1$\text{R}$-$\text{cis}$)-(+)-2,2-dimethyl-3-(1-methylethenyl)cyclobutanemethanol acetate (VI).     

Influence ofpH and concentration of complexing agents on fluorescence of samarium(III), gadolinium(III), and terbium(III)—Ethylenediaminetetraacetic acid or nitrilotriacetic acid complexes in aqueous solutions

The fluorescence intensity of aqueous solutions of Sm(III), Gd(III) and Tb(III) complexes withEDTA orNTA was investigated as a function of complexone concentration over a widepH range. For Sm(III) and Tb(III) complexes the ratio of intensities of hypersensitive and allowed bands was calculated in order to obtain information about intensity borrowing of the fluorescence bands as a result of vibronic coupling.     

Capillary electrophoresis of Cr(VI) reducer Arthrobacter oxydans

Rapid and effective separation of bacteria Arthrobacter oxydans was performed using capillary electrophoresis. For optimal separation of bacteria the influence of buffer concentration, pH and applied voltage were studied. It was found that the most appropriate conditions for electrophoretic mobility measurements are as follows: applied voltage 6-14 kV; buffer concentration 5-10 mM pH 6-8. At the stationary phase of growth there are always two main heterogeneous peaks. They are connected with the morphology of bacteria as well as with cell aggregation. The heterogeneity of samples may be explained by surface modifications of bacterial cells.     

The influence of the purine 2-amino group on DNA conformation and stability-II: Synthesis and physical characterization of d[CGT(2-NH2)ACG], d[CGU(2-NH2)ACG], and d[CGT(2-NH2)AT(2-NH2)ACG]

Synthesis of an N-protected derivative of 2-amino-2'-deoxyadenosine (dA') from 2'-deoxyguanosine is reported. The syntheses of several oligodeoxynucleotides containing this modified nucleoside are described, together with physical characterization via melting studies and CD conformational analysis. As expected, the 2-amino group is seen to add to the duplex stability. Although the sequence d(TA')₃ was found to undergo a salt-induced conformational transition, mixed sequences such as d(CGTA'CG) did not display this behavior. All guanine residues present in these sequences were O⁶ protected, either with the cyanoethyl group or the 4-nitrophenylethyl group, to eliminate guanine degradation during phosphorylation and condensation reactions. Procedures for the introduction and removal of these O⁶ protecting groups are described.     

IN VIVO CHLOROPHYLL a FLUORESCENCE TRANSIENTS AND THE CIRCADIAN RHYTHM OF PHOTOSYNTHESIS IN GONYAULAX POLYEDRA

Abstract— The intensity of chlorophyll a fluorescence during the early part of fluorescence induction at O , initial fluorescence, and P, peak fluorescence, was higher during the day phase of the circadian cycle than during the night phase in continuous light (LL) conditions and was positively correlated with the rate of oxygen evolution. The circadian rhythm in fluorescence in LL persisted in the presence of 10μM 3–(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), which blocks electron flow from photo-system (PS) II in photosynthesis. The rhythmic changes in fluorescence intensity are consistent with a lower rate constant for radiationless transitions during the day phase than during the night phase of the circadian rhythmicity. The circadian changes in the intensity of fluorescence were abolished at 77K, which may indicate the importance of structural changes in membranes in circadian oscillations.