Theoretical study of the human DNA repair protein HOGG1 activity

We have examined the role of the catalytic lysine (Lys 249) in breaking the glycosidic bond of 8-oxoguanine in the enzyme human 8-oxoguanine DNA glycosylase. Until quite recently it has been assumed that this lysine acts as a nucleophile in an S(N)2 type of reaction after being activated through a donation of a proton to a strictly conserved aspartate, also located in the active site. However, evidence from crystallographic, as well as biochemical studies, questions this assumption mainly because the lysine is not ideally positioned for such an attack. In addition, the catalytic activity is preserved even after that aspartate is mutated to a residue not accepting protons, but still keeping the interactions in the active site. In this study, we have investigated several different reaction mechanisms to discover plausible ways where the lysine could assist in breaking the glycosidic bond. We use hybrid density functional theory to characterize both associative and dissociative pathways. We find that the smallest energetical barrier involves an S(N)1 type of mechanism where the lysine electrostatically stabilizes the dissociating base and then donates a proton with a very small barrier and then finally attacks the sugar ring to create the covalently bound protein-DNA intermediate complex. The S(N)2 mechanism also has a lower barrier than the "spontaneous" bond breaking but considerably above that of the S(N)1 reaction. However, in current conditions, the reactants placed in a conformation posed for an S(N)2 reaction is substantially more stable than if posed for the S(N)1 reaction, indicating that the active site has to bind stronger to the latter in order to achieve a full catalytic effect. An analysis of the polarization of the transition states shows that the polarization is largest for the S(N)1 reaction, indicating that this path will gain most by being placed in a prepolarized active site. These findings give further support to the hypothesis that a dissociative mechanism may be the preferred mode of action for this type of enzymes.     

Extraction rate in liquid-liquid segmented flow injection analysis

The extraction mechanism in liquid-liquid segmented flow-injection systems is investigated. A film is formed on the tubing wall by the phase with the highest affinity to the tubing material. This film surrounds the segments of the other phase. To a great extent the extraction takes place at the interface of this film. The extraction rate is influenced by the segment length, the inner diameter of the tubing and the flow velocity. Short segments, small inner diameter and high linear flow velocity lead to a high extraction rate. These findings indicate that miniaturization of the flow system will lead to faster extraction and to decreased sample-zone broadening.     

Sediment extraction and clean-up for organic phosphorus analysis by electrospray ionization tandem mass spectrometry

A method to prepare NaOH sediment extracts for organic P compound analysis with electrospray ionization tandem mass spectrometry (ESI-MS-MS) was developed on natural samples. Ion exchange, rotary evaporation and mass cut-off filtering proved to be suitable for sample preparation. Samples were analyzed with ESI-MS-MS, and reproducibility and repeatability of the method was calculated. In addition, ³¹P-nuclear magnetic resonance spectroscopy (³¹P NMR) was used to measure recovery of different P compound groups such as orthophosphate (Ortho-P), orthophosphate monoesters (Monoester-P), orthophosphate diesters (Diester-P) and pyrophosphates (Pyro-P).

The developed sample preparation method resulted in an easy-to-spray liquid for the ESI-MS-MS instrumentation. The overall P recovery was 65% and ³¹P NMR showed that Diester-P, possibly in the form of DNA, was apparently lost through the filtering step most likely due to their size. Variances in the total intensities of the MS scans (relative standard deviation (R.S.D.) 35–54%) were for about 50% due to repeated MS runs. Covariances of the peaks in the MS spectra were calculated to be for about 30% due to the sample preparation procedure. Finally, with the ESI-MS-MS approach, 11 peaks in the mass spectra were found likely to represent phosphate containing compounds.     

Using a Bootstrap Method to Choose the Sample Fraction in Tail Index Estimation

Tail index estimation depends for its accuracy on a precise choice of the sample fraction, i.e., the number of extreme order statistics on which the estimation is based. A complete solution to the sample fraction selection is given by means of a two-step subsample bootstrap method. This method adaptively determines the sample fraction that minimizes the asymptotic mean-squared error. Unlike previous methods, prior knowledge of the second-order parameter is not required. In addition, we are able to dispense with the need for a prior estimate of the tail index which already converges roughly at the optimal rate. The only arbitrary choice of parameters is the number of Monte Carlo replications.     

A study of glycoprotein-lectin interactions using quartz crystal microbalance

A study of biospecific interactions between lectins and glycoproteins using a quartz crystal microbalance biosensor with dissipation monitoring (QCM-D) was reported. Four lectins were covalently immobilised on the thiol-modified gold electrode of the QCM chips in order to obtain sensing surfaces. The frequency shift served as analytical signal and the dissipation shift provided additional information about the viscoelastic properties of the glycoprotein-lectin complex formed on the surface of the QCM chip. The working conditions of the assay were optimised. The interaction between different lectins and glycoproteins was characterised by specific frequency shifts and each glycoprotein displayed its own unique lectin-binding pattern. This lectin pattern can serve as a finger print for the discrimination between various glycoproteins. The biosensor enabled quantitative determination of glycoproteins in the concentration range of 50 μg mL⁻¹ to 1 mg mL⁻¹ with good linearity and R.S.D. of less than 6.0%. An additional advantage of the proposed biosensor was the possibility to re-use the same lectin surfaces during a long period of time (2 month) without changes in analytical response. This was experimentally achieved by the application of a proper regeneration solution (10 mM glycine-HCl, pH 2.5). The lectin-based quartz crystal microbalance technique is suitable both for rapid screening and for quantitative assay of serum glycoproteins.     

Effects of temperature and flow regulated carbon dioxide cooling in longitudinally modulated cryogenic systems for comprehensive two-dimensional gas chromatography

Two different modes of temperature regulation in longitudinally modulated cryogenic systems (LMCSs) for comprehensive two-dimensional gas chromatography (GC x GC) were compared. Carbon dioxide was used as coolant. In the first mode of operation, the temperature of the trap was regulated to pre-set temperature using a digital temperature controller ("the constant temperature mode"). In the second, the temperature was regulated to a fixed negative offset to the oven temperature by using a constant flow of CO2 ("the constant flow mode"). A number of problems were occasionally observed using the constant temperature mode: (1) severe band broadening of high boiling analytes in the second dimension; (2) non-Gaussian reconstructed first-dimension peak profiles; (3) high background due to modulation of first-dimension column bleed. It was concluded that these problems were associated with inefficient solute remobilization at low LMCS trap temperatures (1 and 2) or large trap temperature fluctuations (3). These problems could be avoided or significantly reduced by using the constant flow mode. Best results were obtained as the trap temperature was kept about 70 degrees C below the oven temperature.     

Trace analysis of polychlorinated dibenzo-p-dioxins, dibenzofurans and WHO polychlorinated biphenyls in food using comprehensive two-dimensional gas chromatography with electron-capture detection

Trace analysis of 2,3,7,8-polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and the 12 WHO-PCBs (four non-ortho and eight mono-ortho congeners that have been assigned toxic equivalence factors, TEFs, by the World Health Organisation) was conducted by comprehensive two-dimensional gas chromatography with a micro electron-capture detector (GC x GC-microECD). Four food matrices (fish oil from herring, spiked cows' milk, vegetable oil and an eel extract) were analysed by two GC x GC laboratories, and four GC-HRMS laboratories generated reference values. The two GC x GC laboratories used different column combinations for separating the target analytes. For the first dimension, non-polar DB-XLB and VF-1 columns were used, and for the second dimension, an LC-50 liquid crystalline column with unique selectivity for planar compounds. The congener-specific and total toxic equivalence (TEQ) data obtained using DB-XLB x LC-50 were in good agreement with results obtained by the GC-HRMS laboratories. The WHO-PCB data obtained with the VF-1 x LC-50 combination was also good, but the PCDD/F concentrations were sometimes overestimated due to matrix interferences. GC x GC-microECD using DB-XLB x LC-50 seems to fulfil the European Community requirements of a screening method for PCDD/F and WHO-PCB TEQ in food.     

Upgrading of wood pre-hydrolysis liquor for renewable barrier design: a techno-economic consideration

A techno-economic assessment of an upgrading procedure and outtake of a pre-hydrolysate in a presumed dissolving pulp mill was performed. Pre-hydrolysis of spruce wood chips in pilot scale produced input data for energy and mass balances and was performed with and without subsequent membrane filtration to produce hydrolysate fractions rich in galactoglucomannan and with some lignin. The hydrolysate is a viable raw material for the production of renewable thin oxygen barrier films as demonstrated herein in the formulation of free standing films with very low oxygen permeability at both moderate and high relative humidities. Approximately 50,000 ton dry solid upgraded pre-hydrolysate suitable for production of oxygen barriers could be produced according to the presumed dissolving pulp mill producing about 500,000 air dry ton dissolving pulp per year and applying a liquor to wood ratio of 4:1. Utilization of the pre-hydrolysis liquor hence adds value to and realizes the dissolving plant as a biorefinery. A sensitivity analysis indicates that the market price of the upgraded pre-hydrolysate has the largest positive effect on the return on investment for separation and upgrading of a pre-hydrolysate. Increased investment cost and increased annuity factor show negative effects.     

No-alignment-strategies for exploring a set of two-way data tables obtained from capillary electrophoresis-mass spectrometry

Hyphenated techniques such as capillary electrophoresis-mass spectrometry (CE-MS) or high-performance liquid chromatography with diode array detection (HPLC-DAD), etc., are known to produce a huge amount of data since each sample is characterized by a two-way data table. In this paper different ways of obtaining sample-related information from a set of such tables are discussed. Working with original data requires alignment techniques due to time shifts caused by unavoidable variations in separation conditions. Other pre-processing techniques have been suggested to facilitate comparison among samples without prior peak alignment, for example, 'binning' and/or 'blurring' the data along the time dimension. All these techniques, however, require optimization of some parameters, and in this paper an alternative parameter-free method is proposed. The individual data tables (X) are represented as Gram matrices (XXT), where the summation is taken over the time dimension. Hence the possible variations in time scale are eliminated, while the time information is at least partly preserved by the correlation structure between the detection channels. For comparison among samples, a similarity matrix is constructed and explored by principal component analysis and hierarchical clustering. The Gram matrix approach was tested and compared to some other methods using 'binned' and 'blurred' data for a data set with CE-MS runs on urine samples. In addition to data exploration by principal component analysis and hierarchical clustering, a discriminant partial least squares model was constructed to discriminate between the samples that were taken with and without the prior intake of a drug. The result showed that the proposed method is at least as good as the others with respect to cluster identification and class prediction. A distinct advantage is that there is no need for parameter optimization, while a potential drawback is the large size of the Gram matrices for data with high mass resolution.     

Evaluation of modulators and electron-capture detectors for comprehensive two-dimensional GC of halogenated organic compounds

Different cryogenic and a heated GC x GC modulator(s) were evaluated and compared for the analysis of high-boiling halogenated compounds. The cryogenic modulators investigated were: (i) the longitudinally modulated cryogenic system; (ii) the liquid-nitrogen-cooled jet modulator (KT2001); (iii) a dual-jet CO2 modulator (made in-house); (iv) a semi-rotating cryogenic modulator (made in-house) and (v) a CO2 loop modulator (KT2003); the heated modulator was the slotted heater system (sweeper). Each modulator was optimised with respect to analyte peak widths at half height in the second-dimension. n-Alkanes, chlorinated alkanes, polychlorinated biphenyls (PCBs) and fluorinated polycyclic aromatic hydrocarbons (F-PAHs) were used as test analytes. The flow rate of the coolant was found to be an important parameter, i.e. the flow rate of the gaseous nitrogen in the KT2001, and of the liquid CO2 in the other cryogenic modulators. For the slotted heater the stroke velocity and pause time were important parameters. This modulator had a limited application range in terms of temperature due to a necessary 100 degrees C difference between sweeper and oven temperature. All cryogenic modulators were found to be suitable for the GC x GC analysis of high-boiling compounds, but the CO2 modulators are to be preferred to the KT2001 due to a wider application range and slightly narrower peaks. As regards the performance of three commercially available electron-capture detectors (ECDs), the aim was to obtain narrow peak widths in GC x GC, i.e. to avoid band broadening caused by the cell volume. The most important parameters were the flow rate of the make-up gas and the detector temperature which both should be as high as possible. Comparison of analyte peak widths obtained with ECD mode and flame ionisation detection (FID) showed that all ECDs exhibited band broadening compared to the FID. The narrowest peaks were obtained with the Agilent micro-ECD, which has a cell volume of only 150 microl.