Resonant infrared multiphoton dissociation spectroscopy of gas-phase protonated peptides. Experiments and Car-Parrinello dynamics at 300 K

The gas-phase structures of protonated peptides are studied by means of resonant infrared multiphoton dissociation spectroscopy (R-IRMPD) performed with a free electron laser. The peptide structures and protonation sites are obtained through comparison between experimental IR spectra and their prediction from quantum chemistry calculations. Two different analyses are conducted. It is first supposed that only well-defined conformations, sufficiently populated according to a Boltzmann distribution, contribute to the observed spectra. On the contrary, DFT-based Car-Parrinello molecular dynamics simulations show that at 300 K protonated peptides no longer possess well-defined structures, but rather dynamically explore the set of conformations considered in the first conventional approach.     

Experiences with an identification and quantification program for inhibitor-positive milk samples

Beta-lactam antibiotics (penicillins, cephalosporins) are still the most commonly used antibiotics for dairy cows in Germany. In routine milk testing, according to the German milk quality regulation, a positive result obtained for bulk tank milk by microbiological inhibitor tests needs no further confirmation, but results in reduced milk payment of 0.05 euros kg(-1) for one month. In some cases, however, further identification of the causative agent can be of interest, either if antimicrobial drugs have not knowingly been used recently, or if improper use of such drugs is denied. As a service for milk producers, our laboratory offers further analyses of violative milk samples, aiming at the identification and quantification of the inhibitor(s). In this program, a panel of microbiological inhibitor tests, receptor tests, and enzyme immunoassays (EIA) is used in a step-by-step analysis, which primarily focusses on beta-lactams, but also includes other compounds such as sulfonamides or tetracyclines, respectively. Here we report results for violative milk samples (n=63) analysed between 2003 and 2005. In most cases (95%), beta-lactam antibiotics could be identified, although not always at levels exceeding the respective MRL values. Penicillin G (mostly together with benzylpenicilloyl metabolites) could be identified in 74.6% of all samples. Other compounds identified were, in decreasing order, ceftiofur (11%), ampicillin/amoxicillin (6.3%), isoxazolyl penicillins (3.2%), and sulfonamides (1.6%). The results indicate that penicillin G is still the predominant antibiotic responsible for violative bulk tank milk samples as detected during regulatory control.     

Liquid-vapor transition of systems with mean field universality class

We have considered a system where the interaction, v(r)=v(IS)(r)+xi(2)v(MF)(r), is given as a linear combination of two potentials, each of which being characterized with a well-defined critical behavior: for v(IS)(r) we have chosen the potential of the restricted primitive model which is known to belong to the three-dimensional Ising universality class, while for v(MF)(r) we have considered a long-range interaction in the Kac [J. Math. Phys. 4, 216 (1963)] limit, displaying mean field (MF) behavior. We study the performance of two theoretical approaches and of computer simulations in the critical region for this particular system and give a detailed comparison between theories and simulation of the critical region and the location of the critical point. Having shown by theoretical arguments that the system belongs to the MF universality class for any positive value of xi and shows nonclassical behavior only for xi=0, we examine to which extent theoretical approximations and simulation can reproduce this behavior. While in this limiting case theoretical approaches are known to fail, we find good agreement for the critical properties between the theoretical approaches and the simulations for xi(2) larger than 0.05.     

Dynamics of Methanol in Ionic Liquids: Validity of the Stokes–Einstein and Stokes–Einstein–Debye Relations

The validity of Stokes–Einstein (SE) and Stokes–Einstein–Debye (SED) relations for methanol in the physical environment of the ionic liquid (IL) 1‐ethyl‐3‐methylimidazolium bis(trifluoromethylsulfonyl)imide is studied by means of nuclear magnetic resonance (NMR) relaxation time experiments, viscosity measurements and molecular dynamics (MD) simulations. The reorientational correlation times of the hydroxyl groups of pure methanol and of methanol in the IL/methanol mixtures were determined. For that purpose an approach for estimating NMR deuteron quadrupole coupling constants, presented by Wendt and Farrar (Mol. Phys.­ 1998 , 95, 1077–1081), was confirmed. The self‐diffusion coefficients of methanol were taken from the MD simulations. The viscosities of all systems were then measured and the SE and SED relations validated. For pure methanol both relations are valid, whereas they become increasingly invalid with increasing IL concentration, as indicated by effective volumes and radii that are too low. The deviation from the SE and SED relations could be related to dynamical heterogeneities described by the non‐Gaussian parameter α(t) obtained from MD simulations. For pure methanol, α(t) is close to zero in accord with the validity of both relations. With increasing IL concentration the dynamical heterogeneities of methanol increase strongly. The times t* at the maximum of α(t) increase linearly with the relative number of methanol monomers in the mixtures. Thus, the dynamical heterogeneities are largest for single methanol molecules fully embedded in the IL environment. In their own environment methanol molecules are highly mobile, whereas in the IL‐rich region the mobility is strongly reduced leading to the non‐validity of SE and SED relations.     

Redox‐Controlled Hydrogen Bonding: Turning a Superbase into a Strong Hydrogen‐Bond Donor

Herein the synthesis, structures and properties of hydrogen‐bonded aggregates involving redox‐active guanidine superbases are reported. Reversible hydrogen bonding is switched on by oxidation of the hydrogen‐donor unit, and leads to formation of aggregates in which the hydrogen‐bond donor unit is sandwiched by two hydrogen‐bond acceptor units. Further oxidation (of the acceptor units) leads again to deaggregation. Aggregate formation is associated with a distinct color change, and the electronic situation could be described as a frozen stage on the way to hydrogen transfer. A further increase in the basicity of the hydrogen‐bond acceptor leads to deprotonation reactions.     

A new ELISA for the quantification of equine procalcitonin in plasma as potential inflammation biomarker in horses

In human medicine, procalcitonin (PCT) is a very common and well-established biomarker for sepsis. Even though sepsis is also a leading cause of death in foals and adult horses, up to now, no data about the role of equine PCT in septic horses has been available. Based on monoclonal antibodies targeted against human PCT, we report here the development of a sandwich ELISA for the quantification of equine PCT in equine plasma samples. The ELISA was characterized for intra- and interassay variance and a working range from 25 to 1,000 ng mL^?1 was defined as within this range; both intra- and interassay variances were below 15 %. The target recovery ranged between 73 and 106 %. The ELISA was used to determine the equine PCT concentration in 24 healthy and 5 septic horses to show the potential for clinical evaluation of equine PCT. Significantly different (P?=?0.0006) mean equine PCT concentrations were found for the healthy control group and the sepsis group (47 and 8,450 ng mL^?1).     

Parallel electromembrane extraction in the 96-well format

The repeatability and extraction recoveries of parallel electromembrane extraction (Pa-EME) was thoroughly investigated in the present project. Amitriptyline, fluoxetine, and haloperidol were isolated from eight samples of pure water, undiluted human plasma, and undiluted human urine, respectively; in total 24 samples were processed in parallel. The repeatability was found to be independent of the different sample matrices (pure water samples, human plasma, and water) processed in parallel, although the respective samples contained different matrix components. In another experiment seven of the 24 wells were perforated. Even though the perforation caused the total current level in the Pa-EME setup to increase, the intact circuits were unaffected by the collapse in seven of the circuits. In another approach, exhaustive extraction of amitriptyline, fluoxetine, and haloperidol was demonstrated from pure water samples. Amitriptyline and haloperidol were also isolated exhaustively from undiluted human plasma samples; the extraction recovery of fluoxetine from undiluted human plasma was 81%. Finally, the sample throughput was increased with the Pa-EME configuration. The extraction recoveries were investigated by processing 1, 8, 68, or 96 samples in parallel in 10 min; neither the extraction recoveries nor the repeatability was affected by the total numbers of samples. Eventually, the Pa-EME was combined with ultra performance liquid chromatography (UPLC) to combine high-throughput sample preparation with high-throughput analytical instrumentation. The aim of the present investigation was to demonstrate the potential of electromembrane extraction as a high throughput sample preparation platform; and hopefully to increase the interest for EME in the bioanalytical field to solve exisiting and novel analytical challenges.     

Purification of Plasma-Derived Coagulation Factor VIII by Immobilized-Zn<Superscript>2+</Superscript> and -Co<Superscript>2+</Superscript> Affinity Chromatography

Coagulation factor VIII (FVIII) is a glycoprotein that plays a crucial role in the clotting cascade. Replacement therapies with recombinant and plasma-derived concentrates of FVIII are used for treatment of hemophilia A. We have previously purified the human plasma FVIII by immobilized metal affinity chromatography (IMAC) using Cu²⁺ as the metal ligand. In this work we report the purification of FVIII using Zn²⁺ and Co²⁺, two metal ions that bind proteins more weakly. Human plasma was directly applied to the anion-exchange ANX Sepharose FF column and the eluate was used as starting material for the studies in IMAC columns. Using imidazole as desorbing agent, FVIII was recovered with 65% activity in the IMAC-Zn²⁺ column and with 74% activity in the IMAC-Co²⁺ column. Purification factors were 4 and 9, respectively. Using a pH gradient, FVIII was eluted at pH 5.0 with 17% activity in the IMAC-Zn²⁺ and 77% activity in the IMAC-Co²⁺. Vitamin K-dependent proteins, a family of proteins that includes Prothrombin and coagulation factor IX, coeluted with FVIII in the ANX Sepharose FF column and were recovered with the unbound proteins on both IMAC columns. Therefore, Co²⁺ and Zn²⁺ columns were as effective as the Cu²⁺ column in separating FVIII from vitamin K-dependent proteins. Finally, we have shown that FVIII remained complexed with the von Willebrand factor.