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.     

A Mini‐Twister Variant and Impact of Residues/Cations on the Phosphodiester Cleavage of this Ribozyme Class

Nucleolytic ribozymes catalyze site‐specific cleavage of their phosphodiester backbones. A minimal version of the twister ribozyme is reported that lacks the phylogenetically conserved stem P1 while retaining wild‐type activity. Atomic mutagenesis revealed that nitrogen atoms N1 and N3 of the adenine‐6 at the cleavage site are indispensable for cleavage. By NMR spectroscopy, a pK_a value of 5.1 was determined for a ¹³C2‐labeled adenine at this position in the twister ribozyme, which is significantly shifted compared to the pK_a of the same adenine in the substrate alone. This finding pinpoints at a potential role for adenine‐6 in the catalytic mechanism besides the previously identified invariant guanine‐48 and a Mg²⁺ ion, both of which are directly coordinated to the non‐bridging oxygen atoms of the scissile phosphate; for the latter, additional evidence stems from the observation that Mn²⁺ or Cd²⁺ accelerated cleavage of phosphorothioate substrates. The relevance of this metal ion binding site is further emphasized by a new 2.6 Å X‐ray structure of a 2′‐OCH₃‐U5 modified twister ribozyme.     

Hastings’s Additivity Counterexample via Dvoretzky’s Theorem

The goal of this note is to show that Hastings’s counterexample to the additivity of minimal output von Neumann entropy can be readily deduced from a sharp version of Dvoretzky’s theorem.     

Zur Methode der Bleisuperoxydbestimmung

Ohne Zusammenfassung