Rapid molecular weight determination of recombinant hirudin by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The molecular weight of recombinant hirudin ( rHV-2) was determined rapidly by matrix-assisted laser desorption/ionization time of fight mass spectrometry (MALDI-TOF-MS). The effects of the three types of matrixes were compared and discussed, alpha-cynao-4-hydroxycinnamic acid was proved to be the best matrix. It showed that MALDI-TOF-MS was superior to the traditional method of molecular weight determination of the biological macromolecules. The mass spectrum data proved that the primary structure of rHV-2 was correct and there was no amino acid deletion, mutation and modification in its expression, refolding and purification.

Determination of purity and molecular weight of calmodulin by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The purity and molecular weight of calmodulin have been determined by means of matrix-assisted laser desorption/ionization time of flight mass spectrometry, and the results have been discussed. The experimental results demonstrate that this method is high sensitive and rapid as compared with other traditional methods.

The high strain rate response of Ultra High Molecular-weight Polyethylene: From fibre to laminate

The effect of strain rate upon the uniaxial response of Ultra High Molecular-weight Polyethylene (UHMWPE) fibres, yarns and laminates of lay-up [0/90]48 has been measured in both the 0/90 and ±45 configurations. The tensile strength of the matrix-dominated ±45 laminate is two orders of magnitude less than that of the fibre-dominated 0/90 laminate, and is more sensitive to strain rate. A piezoelectric force sensor device was developed to obtain the high strain rate data, and this achieved a rise time of less than 1 μs. It is found that the failure strength (and failure strain) of the yarn is almost insensitive to strain rate within the range (10 -1-103 s-1). At low strain rates (below 10 -1 s-1), creep of the yarn dominates and the failure strain increases with diminishing strain rate. The tensile strength of the dry yarn exceeds that of the laminate by about 20%. Tests on single fibres exceed the strength of the yarn by 20%. © 2013 Elsevier Ltd. All rights reserved.

Collapse of a composite beam made from ultra high molecular-weight polyethylene fibres

Hot-pressed laminates with a [0/90]48 lay-up, consisting of 83% by volume of ultra high molecular-weight polyethylene (UHMWPE) fibres, and 17% by volume of polyurethane (PU) matrix, were cut into cantilever beams and subjected to transverse end-loading. The collapse mechanisms were observed both visually and by X-ray scans. Short beams deform elastically and collapse plastically in longitudinal shear, with a shear strength comparable to that observed in double notch, interlaminar shear tests. In contrast, long cantilever beams deform in bending and collapse via a plastic hinge at the built-in end of the beam. The plastic hinge is formed by two wedge-shaped microbuckle zones that grow in size and in intensity with increasing hinge rotation. This new mode of microbuckling under macroscopic bending involves both elastic bending and shearing of the plies, and plastic shear of the interface between each ply. The double-wedge pattern contrasts with the more usual parallel-sided plastic microbuckle that occurs in uniaxial compression. Finite element simulations and analytical models give additional insight into the dominant material and geometric parameters that dictate the collapse response of the UHMWPE composite beam in bending. Detailed comparisons between the observed and predicted collapse responses are used in order to construct a constitutive model for laminated UHMWPE composites. © 2013 Elsevier Ltd.

Acid effects on protein molecular weight determination

The acid effects of some proteins on measuring their molecular weights were studied using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry ( MALDI-TOF-MS) and electrospray ionization mass spectrometry (ESI-MS). It was found that the signal intensity was enhanced through adjusting the acid concentration in some protein samples. In this paper, both MALDI-MS and ESI-MS was applied to examine the molecular weights of several standard proteins. And the proper acid concentration was detected in these spectra. In the meanwhile, it demonstrates that some associations of proteins in solution can be preserved into the gas phase and observed by the "soft ionization" mass spectrometry.

An evaluation of the capability of a biolayer interferometry biosensor to detect low-molecular-weight food contaminants

The safety of our food is an essential requirement of society. One well-recognised threat is that of chemical contamination of our food, where low-molecular-weight compounds such as biotoxins, drug residues and pesticides are present. Low-cost, rapid screening procedures are sought to discriminate the suspect samples from the population, thus selecting only these to be forwarded for confirmatory analysis. Many biosensor assays have been developed as screening tools in food contaminant analysis, but these tend to be electrochemical, fluorescence or surface plasmon resonance based. An alternative approach is the use of biolayer interferometry, which has become established in drug discovery and life science studies but is only now emerging as a potential tool in the analysis of food contaminants. A biolayer interferometry biosensor was assessed using domoic acid as a model compound. Instrument repeatability was tested by simultaneously producing six calibration curves showing replicate repeatability (n = 2) ranging from 0.1 to 6.5 % CV with individual concentration measurements (n = 12) ranging from 4.3 to 9.3 % CV, giving a calibration curve midpoint of 7.5 ng/ml (2.3 % CV (n = 6)). Reproducibility was assessed by producing three calibration curves on different days, giving a midpoint of 7.5 ng/ml (3.4 %CV (n = 3)). It was further shown, using assay development techniques, that the calibration curve midpoint could be adjusted from 10.4 to 1.9 ng/ml by varying assay parameters before the simultaneous construction of three calibration curves in matrix and buffer. Sensitivity of the assay compared favourably with previously published biosensor data for domoic acid. © 2013 Springer-Verlag Berlin Heidelberg.

Selective fermentation of gentiobiose-derived oligosaccharides by human gut bacteria and influence of molecular weight

Gentiooligosaccharides and alternansucrase gentiobiose acceptor products were fractionated by their degree of polymerization (DP) on a Bio-Gel P2 column. Fractions were characterized by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy, and incubated with human faecal bacteria under anaerobic conditions at 37 degrees C. The growth of predominant gut bacteria on the oligosaccharides was evaluated by fluorescence in situ hybridization and a prebiotic index (PI) was calculated. Lower DP gentiooligosaccharides (DP2-3) showed the highest selectivity (PI of 4.89 and 3.40, respectively), whereas DP4-5 alternansucrase gentiobiose acceptor products generated the greatest values (PI of 5.87). The production of short-chain fatty acids was also determined during the time course of the reactions. The mixture of DP6-10 alternansucrase gentiobiose acceptor products generated the highest levels of butyric acid but the lowest levels of lactic acid. Generally, for similar molecular weights, alternansucrase gentiobiose acceptor products gave higher PI values than gentiooligosaccharides.

Influence of glycosidic linkages and molecular weight on the fermentation of maltose-based oligosaccharides by human gut bacteria

A structure-function study was carried out to increase knowledge of how glycosidic linkages and molecular weights of carbohydrates contribute toward the selectivity of fermentation by gut bacteria. Oligosaccharides with maltose as the common carbohydrate source were used. Potentially prebiotic alternansucrase and dextransucrase maltose acceptor products were synthesized and separated into different molecular weights using a Bio-gel P2 column. These fractions were characterized by matrix-assisted laser desorption/ionization time-of-flight. Nonprebiotic maltooligosaccharides with degrees of polymerization (DP) from three to seven were commercially obtained for comparison. Growth selectivity of fecal bacteria on these oligosaccharides was studied using an anaerobic in vitro fermentation method. In general, carbohydrates of DP3 showed the highest selectivity towards bifidobacteria; however, oligosaccharides with a higher molecular weight (DP6-DP7) also resulted in a selective fermentation. Oligosaccharides with DPs above seven did not promote the growth of "beneficial" bacteria. The knowledge of how specific structures modify the gut microflora could help to find new prebiotic oligosaccharides.

A new and improved strategy combining a dispersive-solid phase extraction-based multiclass method with ultra high pressure liquid chromatography for analysis of low molecular weight polyphenols in vegetables

This paper reports on the development and optimization of a modified Quick, Easy, Cheap Effective, Rugged and Safe (QuEChERS) based extraction technique coupled with a clean-up dispersive-solid phase extraction (dSPE) as a new, reliable and powerful strategy to enhance the extraction efficiency of free low molecular-weight polyphenols in selected species of dietary vegetables. The process involves two simple steps. First, the homogenized samples are extracted and partitioned using an organic solvent and salt solution. Then, the supernatant is further extracted and cleaned using a dSPE technique. Final clear extracts of vegetables were concentrated under vacuum to near dryness and taken up into initial mobile phase (0.1% formic acid and 20% methanol). The separation and quantification of free low molecular weight polyphenols from the vegetable extracts was achieved by ultrahigh pressure liquid chromatography (UHPLC) equipped with a phodiode array (PDA) detection system and a Trifunctional High Strength Silica capillary analytical column (HSS T3), specially designed for polar compounds. The performance of the method was assessed by studying the selectivity, linear dynamic range, the limit of detection (LOD) and limit of quantification (LOQ), precision, trueness, and matrix effects. The validation parameters of the method showed satisfactory figures of merit. Good linearity (View the MathML sourceRvalues2>0.954; (+)-catechin in carrot samples) was achieved at the studied concentration range. Reproducibility was better than 3%. Consistent recoveries of polyphenols ranging from 78.4 to 99.9% were observed when all target vegetable samples were spiked at two concentration levels, with relative standard deviations (RSDs, n = 5) lower than 2.9%. The LODs and the LOQs ranged from 0.005 μg mL−1 (trans-resveratrol, carrot) to 0.62 μg mL−1 (syringic acid, garlic) and from 0.016 μg mL−1 (trans-resveratrol, carrot) to 0.87 μg mL−1 ((+)-catechin, carrot) depending on the compound. The method was applied for studying the occurrence of free low molecular weight polyphenols in eight selected dietary vegetables (broccoli, tomato, carrot, garlic, onion, red pepper, green pepper and beetroot), providing a valuable and promising tool for food quality evaluation.

Surfactant-assisted intercalation of high molecular weight poly(ethylene oxide) into vanadyl phosphate di-hydrate

A high molecular weight poly(ethylene oxide)/layered vanadyl phosphate di-hydrate intercalation compound was synthesized via the surfactant-assisted approach. Results confirmed that surfactant molecules were replaced with the polymer, while the lamellar structure of the matrix was retained, and that the material presents high specific surface area. In addition, intercalation produced a more thermally stable polymer as evidenced by thermal analysis. (C) 2011 Elsevier Ltd. All rights reserved.

Purification and Characterization of a Low-Molecular-Weight Phospholipase A2 from Developing Seeds of Elm1

Phospholipase A2 (PLA2) was purified about 180,000 times compared with the starting soluble-protein extract from developing elm (Ulmus glabra) seeds. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified fraction showed a single protein band with a mobility that corresponded to 15 kD, from which activity could be recovered. When analyzed by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry, the enzyme had a deduced mass of 13,900 D. A 53-amino acid-long N-terminal sequence was determined and aligned with other sequences, giving 62% identity to the deduced amino acid sequence of some rice (Oryza sativa) expressed sequence tag clones. The purified enzyme had an alkaline pH optimum and required Ca2+ for activity. It was unusually stable with regard to heat, acidity, and organic solvents but was sensitive to disulfide bond-reducing agents. The enzyme is a true PLA2, neither hydrolyzing the sn-1 position of phosphatidylcholine nor having any activity toward lysophosphatidylcholine or diacylglycerol. The biochemical data and amino acid sequence alignments indicate that the enzyme is related to the well-characterized family of animal secretory PLA2s and, to our knowledge, is the first plant enzyme of this type to be described.

Electrospinning and crosslinking of low-molecular-weight poly(trimethylene carbonate-co-L-lactide) as an elastomeric scaffold for vascular engineering

The growth of suitable tissue to replace natural blood vessels requires a degradable scaffold material that is processable into porous structures with appropriate mechanical and cell growth properties. This study investigates the fabrication of degradable, crosslinkable prepolymers of l-lactide-co-trimethylene carbonate into porous scaffolds by electrospinning. After crosslinking by γ-radiation, dimensionally stable scaffolds were obtained with up to 56% trimethylene carbonate incorporation. The fibrous mats showed Young’s moduli closely matching human arteries (0.4–0.8 MPa). Repeated cyclic extension yielded negligible change in mechanical properties, demonstrating the potential for use under dynamic physiological conditions. The scaffolds remained elastic and resilient at 30% strain after 84 days of degradation in phosphate buffer, while the modulus and ultimate stress and strain progressively decreased. The electrospun mats are mechanically superior to solid films of the same materials. In vitro, human mesenchymal stem cells adhered to and readily proliferated on the three-dimensional fiber network, demonstrating that these polymers may find use in growing artificial blood vessels in vivo.

Buckling in polymer monolayers: Molecular-weight dependence

We present systematic investigations of buckling in Langmuir monolayers of polyvinyl acetate formed at the air-water interface. On compression the polymer monolayers are converted to a continuous membrane with a thickness of ~2–3 nm of well-defined periodicity, lambdab. Above a certain surface concentration the membrane undergoes a morphological transition buckling, leading to the formation of striped patterns. The periodicity seems to depend on molecular weight as per the predictions of the gravity-bending buckling formalism of Milner et al. for fluidlike films on water. However anomalously low values of bending rigidity and Young's modulus are obtained using this formalism. Hence we have considered an alternative model of buckling-based solidlike films on viscoelastic substrates. The values of bending rigidity and Young's modulus obtained by this method, although lower than expected, are closer to the bulk values. Remarkably, no buckling is found to occur above a certain molecular weight. We have tried to explain the observed molecular-weight dependence in terms of the variation in isothermal compressive modulus of the monolayers with surface concentration as well as provided possible explanations for the obtained low values of mechanical properties similar to that observed for ultrathin polymer films.

Effect of Molecular Weight on the Thermal and Impact Sensitivity of Polymers in Propellants

Abstract is not available.