Quantitative validation of different protein precipitation methods in proteome analysis of blood platelets

For the preparation of proteins for proteome analysis, precipitation is frequently used to concentrate proteins and to remove interfering compounds. Various methods for protein precipitation are applied, which rely on different chemical principles. This study compares the changes in the protein composition of human blood platelet extracts after precipitation with ethanol (EtOH) or trichloroacetic acid (TCA). Both methods yielded the same amount of proteins from the platelet preparations. However, the EtOH-precipitated samples had to be dialyzed because of the considerable salt content. To characterize single platelet proteins, samples were analyzed by two-dimensional fluorescence differential gel electrophoresis. More than 90% of all the spots were equally present in the EtOH- and TCA-precipitated samples. However, both precipitation methods showed a smaller correlation with nonprecipitated samples (EtOH 74.9%, TCA 79.2%). Several proteins were either reduced or relatively enriched in the precipitated samples. The proteins varied randomly in molecular weight and isoelectric point. This study shows that protein precipitation leads to specific changes in the protein composition of proteomics samples. This depends more on the specific structure of the protein than on the precipitating agent used in the experiment.     

Triplet state properties of the benzo[α]pyrene-DNA complex as determined by optically detected zero-field magnetic resonance

The zero-field transitions in the photoexcited triplet state of benzo[α]pyrene bound to DNA have been observed by optical detection of magnetic resonance of 2 K. The transition frequencies individual spin sublevel intersystem crossing rates were measured by monitoring the microwave-induced intensity changes of the triplettriplet absorption at 465.8 nm. The triplet state zero-field splitting and dynamics for the benzo[α]pyrene DNA complex are compared with these properties measured for benzo(α)pyrene in other solvent systems.     

Substituenteneffekte auf azido-tetrazolo-gleichgewichte elektronische und sterische effekte bei s-triazolo[4,3-c]tetrazolo[1,5-a]pyrimidinen1

The azido tetrazolo valence isomerism of twenty 9-methyl-s-triazolo[4,3-c]tetrazolo[1,5-a]pyrimidines with different substituents at position 5 has been studied by means of ¹H NMR spectroscopy. All the compounds have been found to be tetrazoles in the solid state and in (CD₃)₂SO solution; in CF₃COOH azido and tetrazolo isomers are in equilibrium. From equilibrium constants K and thermodynamic data determined it is concluded that in this series K depends on both electronic effects and steric requirements of the 5-substituents. A linear relation between K and σ was found for 5-arylderivatives because ΔS° keeps approximately constant and the substituent mainly operates on ΔH°.     

A new approach to 6-deoxy-D-allofuranose- and 6-deoxy-L-talofuranose derivatives from 1,2:5,6-di-O-isopropylidene α-D-glucofuranose

3-O-Acetyl-1,2-O-isopropylidene--D-allofuranose (2 b) was prepared from 1,2:5,6-di-O-isopropylidene--D-allofuranose (1 b). Treatment of2 b with triphenylphosphine-diethyl azodicarboxylate afforded regio- and stereospecifically the 5,6-epoxy--D-allo derivative (3). The other diastereomeric compound, 5,6-epoxy-1,2-O-isopropylidene--L-talofuranose (6) was also prepared stereoselectively from2 b via the intermediates5 a and5 b. The epoxy sugars3 and6 were converted with lithium aluminum hydride to the corresponding 6-deoxy-1,2-O-isopropylidene--D-allofuranose (4 a) and --L-talofuranose (7 a) derivatives. Hydrolysis of4 a and7 a afforded 6-deoxy-D-allose and 6-deoxy-L-talose, respectively. The corresponding 3,5-di-O-acetyl- (4 b and7 b) and the 3,5-O-(tetraisopropyldisiloxane-1,3-diyl) derivatives (4 c and7 c) are also described. Selective removal of the isopropylidene group and subsequent acetylation offers a convenient route to prepare sugar derivatives containing furanose ring, like8 b, as a suitable precursor for nucleoside analogs.Herrn Prof. Dr.K. Komarek mit den besten Wünschen zum 60. Geburtstag gewidmet.     

Synthetische und NMR-spektroskopische Untersuchungen der Benzylaminaddition an N-Maleyl-aminosäurederivate

Summary During the addition of amines to maleylamino acids in general -amino derivatives are formed. In order to change the reactivity within the vinylogous maleyl system we introduced the -benzylester group into the starting compound N-(cis--carboxyacryloyl)-phenylalanine methylester1. The obtained benzylester2 is adding benzylamine in -position yielding N-benzyl--aspartyl(-benzyl)-phenylalanine methylester4. The addition reaction was investigated by¹H-NMR-spectroscopy. The structures of the compounds have been confirmed by elemental analysis, IR,¹H-NMR and¹³C-NMR spectroscopy.

     

Zur Aktivierung partiell silylierter Kohlenhydrate mittels Triphenylphosphan/Azodicarbonsäureester

On the Activation of Partially Silylated Carbohydrates Using Triphenylphosphane/Diethylazodicarboxylate Reaction of methyl α-D-glucopyranoside ( 1 ) with two equivalents of t-butyldimethylchlorosilane yields methyl 2,6-bis[O-(t-butyldimethylsilyl)]-α-D-glucopyranoside ( 1a ) and methyl 3,6-bis[O(t-butyldimethylsilyl)]-α-D-glucopyranoside ( 1b ) in a ratio of 4:1. The anomeric β-pyranoside 2 affords methyl 2,6-bis[O(t-butyldimethylsilyl)]-β-D-glucopyranoside ( 2a ) and methyl 3,6-bis[O(t-butyldimethylsilyl)]-β-D-glucopyranoside ( 2b ) in nearly equal amounts. 2b is isomerized to methyl 4,6-bis[O(t-butyldimethylsilyl)]-β;-D-glucopyranoside ( 2c ) (83%) and 2a (10%) with triphenylphosphane/diethylazodicarboxylate. Structures were assigned by NMR.-analysis and CD.-analysis of the corresponding benzoates 1c , 1d and 2d and of the acetates 2e and 2f . 1a is transformed into methyl 4-azido-2, 6-bis[O(t-butyldimethylsilyl)]-4-deoxy-α-D-galactopyranoside ( 3 ) with triphenylphosphane/diethylazodicarboxylate/HN₃. 2a and 2c yield the 3-azido-allosides 5 and 7 respectively under similar conditions. The activation by triphenylphosphane/diethylazodicarboxylate is high enough to introduce also p-nitrobenzoate groups with inversion of configuration at the reaction center. By this way 1a and 2a give methyl 2, 6-bis[O(t-butyldimethylsilyl)]-4-O-p-nitrobenzoyl-α-D-galactopyranoside ( 4 ) and methyl 2, 6-bis[O-(t-butyldimethylsilyl)]-3-O?ptrobenzoyl-β-D-allopyranoside ( 6 ) respectively. For elucidation of structures the acetate derivatives 3a-7a were prepared.     

Strukturelle Abwandlungen anN-Acetylneuraminsäure, 3

From methyl-5-acetylamino-7,8-anhydro-4,9-O-bis-(t-butyldimethylsilyl)-3,5-dideoxy--D-glycero-D-galacto-2-nonulopyranosidonic acid methylester (1) the derivatives1 a and1 b were obtained by removing the 9-O-(t-butyldimethylsilyl)group withBu ₄NF, followed by acetylation. Treatment of1 b with 80% acetic acid and acetanhydride/pyridine yields the 8-epi-N-acetylneuraminic acid derivative2 a and the 7-epi-N-acetylneuraminic acid derivative3 a in a ratio of 3:1 (Scheme 1). The structure elucidation of2 b was achieved by converting2 b via the 4,9-bis-O-(tBDMSi)-8-O-tosyl-derivative2 d into the epoxide1 (Scheme 2). Using the same sequence the epoxides4 and5 were transformed into theN-acetylneuraminic acid derivative6 a and the 7,8-bis-epi-N-acetylneuraminic acid derivative7 a (Scheme 3). After treatment with sodium hydroxide and 0.025m HCl and Dowex 50 H⁺ the 8-epi-, 7-epi- and 7,8-bis-epi-N-acetylneuraminic acids2,3, and7 were obtained. These three compounds were tested withCMP-N-acetylneuraminic acid synthetase.

Herrn KollegenK. Schlögl mit den besten Wünschen zum 60. Geburtstag.     

An integral representation for decomposable measures of measurable functions

Summary We start with a measurem on a measurable space (,A), decomposable with respect to an Archimedeant-conorm on a real interval [0,M], which generalizes an additive measure. Using the integral introduced by the second author, a Radon-Nikodym type theorem, needed in what follows, is given.The integral naturally leads to a -decomposable measurem on the space of all measurable functions from to [0, 1]. The main result of the present paper is the converse of this, namely that, under natural conditions, any -decomposable measurem on can be represented as an integral of a certain Markov-kernelK. We extend this representation to measures on which have values in a set of distribution functions.These results generalize the work done by the first author in the case of additive measures.     

A characterization of subgroups of the orthogonal group

LetG be a subgroup of the general linear group GL_n(K), where charK 2. Put Kⁿ =V. AssumeG is generated by the setS of all elements inG for which dimV( – 1) = 1, and suppose ²=1_V for each inS. If {V(–1)¦S} contains a simplex, if – 1_V G, and if inG is a product of dim v(–1) elements inS wheneverV(–1) is not contained in the kernel of–1, thenG is a subgroup of an orthogonal group.This research was supported in part by NSERC Canada grant A7251.To Helmut Mäurer on his 60th birthday     

Zdanovskii’s Rule and Isopiestic Measurements Applied to Synthetic Bayer Liquors

Isopiestic measurements of mixed electrolyte solutions involving sodium hydroxide and other components of Bayer liquors were performed at 50?°C and 100?°C. Most of the systems studied obey Zdanovskii’s rule exactly or very closely. However, those with sodium chloride as one of their components show deviations from Zdanovskii’s rule, which are well predicted by our thermodynamic modelling software based on Pitzer’s equations.