Designing new metal affinity peptides by random mutagenesis of a natural metal-binding site

The metal-binding site of a Helicobacter pylori ATPase 439 (heli(WT)-tag) was successfully used as a new fusion peptide for immobilized metal ion affinity chromatography (IMAC). It produced higher yields than the frequently used his6-tag. Due to stronger binding of the peptide to metal ions, harsher elution conditions were, however, necessary. This undesired side-effect was overcome by modifying the heli(WT)-tag by polymerase chain reaction-directed mutagenesis. The modified tags were screened by an automated high-throughput IMAC system, leading to a heliM14-tag peptide that could be eluted under conditions similar to those of the his6-tag but at the same time produced 20% higher yields of the desired protein.     

Characterization of complex assemblages of organic acids in geological samples by negative electrospray ionization mass spectrometry using a double‐focusing magnetic sector field mass spectrometer

Four different geological sample types (a crude oil, a crude oil asphaltene, a reservoir core extract and a reservoir core asphaltene) have been characterized by negative ionization electrospray mass spectrometry at low and high mass resolution using a double‐focusing magnetic sector field mass spectrometer. The mass range, shape of the spectra and the signal distribution of the acidic constituents as well as the average molecular weights, the total ion abundance and signal intensity in the spectra were compared for the different sample types. Nominal mass classes have been evaluated and Kendrick mass plots were generated in order to identify homologous series. For the crude oil sample, accurate mass assignments were made by high‐resolution double‐focusing magnetic sector field mass spectrometry (DFMSFMS) and were compared with those obtained by negative ion electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS). With both instrument types, compounds with the molecular composition C_nH_2n+zO₂, among which carboxylic acids predominated, were the main acidic compound class detectable in negative ESI mass spectra. Good agreement was achieved for the double bond class distribution and the carbon number distribution of the O₂ class. In addition, minor compound classes could be identified using FTICRMS. Copyright © 2010 John Wiley & Sons, Ltd.     

Total nucleotide analysis of Hydra DNA and RNA by MEKC with LIF detection and 32P‐postlabeling

The model organism Hydra has been used for molecular studies for more than 20 years, however, its DNA base composition has not been determined yet. We have analyzed DNA and total RNA of the freshwater polyp Hydra magnipapillata with two independent procedures of high accuracy and sensitivity – fluorescence labeling of nucleotides followed by CE‐LIF detection and ³²P‐postlabeling. DNA of Hydra was digested either to deoxyribonucleoside‐5′‐monophosphates or deoxyribonucleoside‐3′‐monophosphates selectively conjugated with the fluorescent dye 4,4‐difluoro‐5,7‐dimethyl‐4‐bora‐3a,4a‐diaza‐s‐indacene‐3‐propionyl ethylene diamine hydrochloride (BODIPY FL EDA) separated and detected using CE‐LIF. Both versions of the assay revealed a high A+T composition of 78 and 71%, whereas total DNA methylation (5‐methyldeoxycytidine) was 2.6 and 3.1%. Total Hydra RNA showed highest base levels for guanine (33%) and a level of 1.4% for pseudouracil. All values were in good agreement with those determined by the ³²P‐postlabeling method.     

Tri‐ and Tetrasubstituted N‐Phthalimidoaziridines in 1,3‐Dipolar Cycloaddition Reactions

The thermal reactions of the 2,2,3‐trisubstituted N‐phthalimidoaziridine 1a with dimethyl acetylenedicarboxylate (DMAD), thioketones 4a – 4d , and dimethyl azodicarboxylate ( 5 ) proceed even at room temperature leading to the five‐membered cycloadducts 2a, 6 – 8 , and 12 , respectively, with retention of the spatial arrangement of the aziridine substituents, in contrast to the expectation based on the conservation of orbital symmetry in concerted reactions. The analogous reactions of the tetrasubstituted phthalimidoaziridine 1b with thioketones at 40° lead to the 1,3‐thiazolidine derivatives 10 and 11 as mixtures of diastereoisomers. These unexpected results may be explained by either the isomerization of the intermediate azomethine ylides or a non‐concerted stepwise cycloaddition reaction of these ylides with the dipolarophiles. The structures of some adducts have been determined by X‐ray crystallography.     

Synthesis and characterization of an aliphatic monoimide-bridged polysilsesquioxane by the sol–gel route

Based on differential scanning calorimetry data, it was shown that the reaction of (3-triethoxysilylpropyl)succinic anhydride and (3-amino)propyltriethoxysilane at 110 °C resulted in the formation of polyamic acid, whereas the thermal treatment at 220 °C led to the generation of an aliphatic monoimide-bridged polysilsesquioxane as proved by FT-IR. X-ray powder diffraction studies showed a prominent reflection at 2θ = 6.66° (d = 1.32 nm) revealing that a crystalline area is formed. ²⁹Si CP-MAS-NMR and ¹³C CP-TOSS-MAS-NMR measurements proved that no cleavage of the Si–C bond occurred, and a highly condensed material was obtained.     

Notched Ring Test for Measuring Slow Cracking Resistance in Plastics Pipes and Fittings

It is known, that the lifetime of polyethylene pipes is essentially limited by slow crack growth (SCG). For state of the art PE materials common SCG testing methods have reached their limits with respect to extension of testing times. A comparatively new method is the Notched Ring Test (NRT) as developed by Choi et al.^[1] Pipe rings notched at the inner wall are used. The test is carried out in 80 °C water under constant bending load. The arrangement of the notch at the inner wall reduces testing times using the residual stress of extruded pipes. A disadvantage of this method is that there is no clearly defined failure time because SCG takes place between two phases of creeping. The output of this test is an “on-set slow cracking time” (crack initiation), obtained by analysis of the displacement curve. In this work it has been shown that the NRT method yields to brittle fracture within acceptable time frames.^[2] Methods for data analysis are presented. This test could be very useful applied in research and development for resin evaluation and as a tool in quality control in pipe production for evaluating the process conditions.     

Presmectic wetting and supercritical-like phase behavior of octylcyanobiphenyl liquid crystal confined to controlled-pore glass matrices

The influence of controlled-pore glass (CPG) confinement on the phase behavior of octylcyanobiphenyl liquid crystal (LC) is studied by means of x-ray scattering and high precision calorimetry. For CPG samples with pore diameter 2R>24 nm, the smectic order parameter temperature dependence eta(T) reveals apparent presmectic ordering far above the bulk smectic A-nematic (SmA-N) phase transition for both nontreated and silane-treated CPG matrices. The behavior of eta(T) is qualitatively similar in all samples, well obeying the mean field approach (MFA) in which the surface wetting tendency plays the dominant role. In contrast, the critical fluctuations remain important in the specific heat data, which cannot be described within the MFA. We show experimentally that randomness and surface wetting become dominant over finite-size effects for 2R approximately<10 nm, in agreement with theoretical analysis. In nontreated samples, the noncritical character of the static disorder and the interfacial LC-CPG coupling almost completely suppress the quasi-SmA-N and nematic-isotropic phase transitions at 2R approximately 15.1 and approximately 7.5 nm, respectively.     

Superparamagnetic gamma-Fe(2)O(3) nanoparticles with tailored functionality for protein separation

Polymer coated superparamagnetic gamma-Fe(2)O(3) nanoparticles were derivatized with a synthetic double-stranded RNA [poly(IC)], a known allosteric activator of the latent (2-5)A synthetase, to separate a single 35 kDa protein from a crude extract which cross reacted with antibodies raised against the sponge enzyme.     

CO2 Activation and Hydrogenation by PtHn− Cluster Anions

Gas phase reactions between PtH_n^? cluster anions and CO₂ were investigated by mass spectrometry, anion photoelectron spectroscopy, and computations. Two major products, PtCO₂H^? and PtCO₂H₃^?, were observed. The atomic connectivity in PtCO₂H^? can be depicted as HPtCO₂^?, where the platinum atom is bonded to a bent CO₂ moiety on one side and a hydrogen atom on the other. The atomic connectivity of PtCO₂H₃^? can be described as H₂Pt(HCO₂)^?, where the platinum atom is bound to a formate moiety on one side and two hydrogen atoms on the other. Computational studies of the reaction pathway revealed that the hydrogenation of CO₂ by PtH₃^? is highly energetically favorable.