Alfred Deege

Cross-linked poly(vinyl alcohol) as permanent hydrophilic column coating for capillary electrophoresis

A fast method for the generation of permanent hydrophilic capillary coatings for capillary electrophoresis (CE) is presented. Such interior coating is effected by treating the surface to be coated with a solution of glutaraldehyde as cross‐linking agent followed by a solution of poly(vinyl alcohol) (PVA), which results in an immobilization of the polymer on the capillary surface. Applied for capillary zone electrophoresis (CZE) such capillaries coated with cross‐linked PVA exhibit excellent separation performance of adsorptive analytes like basic proteins due to the reduction of analyte‐wall interactions. The long‐term stability of cross‐linked PVA coatings could be proved in very long series of CZE separations. More than 1000 repetitive CE separations of basic proteins were performed with stable absolute migration times relative standard deviation (RSD > 1.2%) and without loss of separation efficiency. Cross‐linked PVA coatings exhibit a suppressed electroosmotic flow and excellent stability over a wide pH range.

Tuning a P450 Enzyme for Methane Oxidation

Cytochrome P450 (CYP) enzymes are heme-dependent monooxygenases that catalyze the oxidation of C H bonds of endogenous and exogenous organic compounds with formation of the respective alcohols. The mechanism involves the intermediacy of a high-spin oxyferryl porphyrin radical cation which abstracts a hydrogen atom from the substrate, and the short-lived alkyl radical then undergoes C Obond formation. The binding pockets of CYPs are relatively large, therefore small compounds do not have a statistically high enough probability of being properly oriented near the oxyferryl moiety for rapid oxidation to occur; additionally there are other effects that slow down or prevent catalysis. A notorious challenge is the oxidation of methane to methanol by chemical catalysis or using enzymes of the type methane monooxygenases (MMOs). It is not only the smallest alkane, but also has the strongest C H bond (104 kcalmol ). Although CYPs represent a superfamily of monooxygenases, none have been shown to accept methane, whereas MMOs are complex enzymes (many membrane bound) that have not been expressed in heterologous hosts in any significant quantities, among other problems. Herein we show that chemical tuning of a CYP, which is based on guest/host activation using perfluoro carboxylic acids as chemically inert guests, activates the enzyme for oxidation of not only medium-sized alkanes such as n-hexane, but also of small gaseous molecules such as propane and even methane as the ultimate challenge. In the present study we chose, for practical reasons, the enzyme P450 BM3 (CYP102A1) from Bacillus megaterium, which is a self-sufficient fusion protein composed of a P450 monooxygenase and an NADPH diflavin reductase. Several crystal structures of this CYP harboring a fatty acid or fatty acid derived inhibitors, as well in the absence of such compounds have been published. To engineer mutants of P450 BM3 and of other CYPs for enhanced activity and selectivity toward a variety of different compounds, including such difficult substrates as small alkanes, rational design as well as directed evolution have proven to be successful to some extent. For example, P450 BM3 variants characterized by numerous point mutations were obtained in extensive laboratory evolution, and showed for the first time notable activity toward propane by formation of the respective alcohols (2-propanol/1-propanol= 9:1); however, the ethane to ethanol conversion remains problematic and methane oxidation has not been achieved to date. Higher activity in ethane oxidation was accomplished using mutants of P450cam, but here again methane oxidation was not reported. Our chemical approach involves a chemically inert compound that serves as a guest in the binding pocket of P450 BM3, thereby filling the space and reducing the translational freedom of small alkanes or of any other substrate. On the basis of previous reports involving CYPs harboring various substrates, such guest/host interactions can be expected to induce other modes of activation effects as well, specifically water displacement at the Fe/heme site accompanied by a change in the electronic state from the inactive low-spin state to the catalytically active high-spin states. Moreover, many studies have shown that P450 enzymes and mutants thereof can harbor two different substrates simultaneously, thus leading to cooperative effects; one example is lauric acid and palmitic acid in which cooperativity has been demonstrated by isotope labeling experiments. In yet another study regarding the metabolism of bilirubin, the addition of lauric acid or the perfluorinated analogue was reported to facilitate NADPH oxidation and substrate degradation, a finding that has implications for the treatment of jaundice, uroporphyria, and possibly cancer. It has also been shown for the case of a distantly related H2O2dependent P450 enzyme that its peroxidase activity can be influenced by the addition of fatty acids, wherein increased or decreased activity is observed depending upon their chain length. In our endeavor we were guided by the binding mode of the natural substrates, fatty acids, of P450 BM3. The binding includes H-bonds originating from their carboxy function and residues Arg 47 and Tyr 51, as well as hydrophobic interactions. The use of perfluoro carboxylic acids such as 1a–h as chemically inert, yet activating guests was therefore envisioned, because perfluoro alkyl groups are known to be resistant to oxidation while having a hydrophobic character. Moreover, it is known that a CF3 residue is sterically comparable to a CH(CH3)2 group, [11a] which means that a perfluoro fatty acid fills much more space in a P450 binding pocket than a traditional fatty acid, and can additionally induce the crucial low-spin to high-spin conversion of Fe/heme. In exploratory studies, the oxidation of n-octane and n-hexane as well as isomers thereof was studied using P450 [*] Dr. F. E. Zilly, Dr. J. P. Acevedo, Dr. W. Augustyniak, A. Deege, U. W. H usig, Prof. M. T. Reetz Max-Planck-Institut f r Kohlenforschung Kaiser-Wilhelm-Platz 1, 45470 M lheim an der Ruhr (Germany) reetz@mpiso-muelheim.mpg.de

Poly(vinyl alcohol)-coated microfluidic devices for high- performance microchip electrophoresis

The channels of microfluidic glass chips have been coated with poly(vinyl alcohol) (PVA). Applied for microchip electrophoresis, the coated devices exhibited a suppressed electroosmotic flow and improved separation performance. The superior performance of PVA‐coated channels could be demonstrated by electrophoretic separations of labeled amines and by video microscopy. While a distorted sample zone is injected using uncoated channels the application of PVA‐coated channels results in an improved shape of the sample zone with less band broadening. Applying PVA‐coated microchips for the separation of amines labeled with Alexa Fluor 350™ even sub‐second separations, utilizing a separation length of only 650 νm, could be obtained, while this was not possible using uncoated devices. By using PVA‐coated devices rather than an uncoated chip a threefold increase in separation efficiencies could be observed. As the electroosmotic flow (EOF) was suppressed, the anionic compounds were detected at the anode whereas the dominant EOF in uncoated devices resulted in an effective mobility to the cathode. Besides improved separation performance another important feature of the PVA‐coated channels was the suppressed adsorption of fluorescent compounds in repetitive runs which results in an improved robustness and detection sensitivity. Applying PVA‐coated channels, rinsing or etching steps could be omitted while this was necessary for a reliable operation of uncoated devices.

Design and performance of a microchip electrophoresis instrument with sensitive variable-wavelength fluorescence detection.

A modular instrument for high‐speed microchip electrophoresis (MCE) equipped with a sensitive variable‐wavelength fluorescence detection system was developed and evaluated. The experimental setup consists mainly of a lamp‐based epifluorescence microscope for variable‐wavelength fluorescence detection and imaging and a programmable four‐channel bipolar high‐voltage source capable of delivering up to +/– 10 kV per channel. The optical unit was equipped with a high‐sensitivity photomultiplier tube and an adjustable aperture. The system was applied to MCE separations of flurescein isothiocyanate (FITC)‐labelled amines utilizing blue light (450–480 nm) for excitation as well as for the separation of rhodamines utilizing excitation light in the green spectral region (531–560 nm). At optimized conditions baseline separation of four FITC‐labelled amines could be obtained in less than 50 s at a detection limit of 460 ppt (1 nM) with a signal‐to‐noise ratio of 3:1. Three rhodamines could be baseline‐separated in less than 6 s at a detection limit of 240 ppt (500 pM). The relative standard deviations of absolute migration times determined in repetitive MCE separations of FITC‐labelled amines were below 2.5% (n= 25). By the application of cyclodextrin‐modified electrolytes, chiral separation of FITC‐labelled amines could be performed in seconds demonstrating the potential of microchip electrophoresis for chiral high‐throughput screening.