The cell membrane is mainly composed of lipid bilayers with inserted proteins and carbohydrates. Lipid bilayers made of purified or synthetic lipids are widely used for estimating the effect of target compounds on cell membranes. However, the composition of such biomimetic membranes is much simpler than the composition of biological membranes. Interactions between compounds and simple composition biomimetic membranes might not demonstrate the effect of target compounds as precisely as membranes with compositions close to real organisms. Therefore, the aim of our study is to construct biomimetic membrane closely mimicking the state of natural membranes. Liposomes were prepared from lipids extracted from L-α-phosphatidylcholine, Escherichia coli, yeast (Saccharomyces cerevisiae) and bovine liver cells through agitation and sonication. They were immobilized onto silicon dioxide (SiO2) sensor surfaces using N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid buffer with calcium chloride. The biomimetic membranes were successfully immobilized onto the SiO2 sensor surface and detected by nanoplasmonic sensing. The immobilized membranes were exposed to choline carboxylates. The membrane disruption effect was, as expected, more pronounced with increasing carbohydrate chain length of the carboxylates. The results correlated with the toxicity values determined using Vibrio fischeri bacteria. The yeast extracted lipid membranes had the strongest response to introduction of choline laurate while the bovine liver lipid extracted liposomes were the most sensitive towards the shorter choline carboxylates. This implies that the composition of the cell membrane plays a crucial role upon interaction with choline carboxylates, and underlines the necessity of testing membrane systems of different origin to obtain an overall image of such interactions.
- MeSH
- biomimetické materiály chemie MeSH
- buněčná membrána chemie MeSH
- cholin analogy a deriváty MeSH
- liposomy chemie MeSH
- membránové lipidy chemie MeSH
- Saccharomyces cerevisiae MeSH
- skot MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The power supply for IEF based on features of the Cockcroft-Walton voltage multiplier (CW VM) is described in this work. The article describes a design of the IEF power supply, its electric characteristics, and testing by IEF analysis. A circuit diagram of the power supply included two opposite charged branches (each consisting of four voltage doublers). The designed CW VM was powered by 230 V/50 Hz alternate current and it generated up to 5 kV and 90 mW at the output. Voltage and current characteristics of the power supply were measured by known load resistances in the range from 10 kΩ to 1 GΩ, which is a common resistance range for IEF strip geometry. Further, the power supply was tested by a separation of a model mixture of colored pI markers using a 175 × 3 × 0.5 mm focusing bed. Automatically limited power load enabled analysis of samples without previous optimization of the focusing voltage or electric current time courses according to sample composition. Moreover, the developed power supply did not produce any intrinsic heat and was easy to set up with cheap and commonly available parts.
The article presents a new concept of preparative solution IEF where time requirements and efficiency are similar to gel-based IEF whereas simple fraction handling as well as quick and complete protein recovery typical for solution-based IEF methods are maintained. The presented method is based on the IEF in separation medium soaked in a segmented strip of nonwoven fabric. The strip is positioned in an open horizontal V-shaped trough. Suggested focusing method combines free solution IEF under continuous evaporation and whole channel dispensing. Separation medium based on ethylene glycol/water mixture enhances viscosity enough to reduce electroosmosis and prevents the medium from completely drying out. Generation of pH gradient and final local pH is visually traced by colored low-molecular pI markers added to input mixture, which enables an optimization of focusing process and collection of individual fractions at desired pH range. The proposed method was tested by fractionation of the proteins and bioactive peptides originating from raw whey. Moreover, subsequent HPLC analysis of the individually collected solution IEF fractions was used for identification of whey components. We confirmed that the method is capable to process directly few tenths of milliliters of raw samples including the salty ones.
A divergent-flow isoelectric focusing (DF IEF) technique has been applied for the separation and preparative analysis of peptides. The parameters of the developed DF IEF device such as dimension and shape of the separation bed, selection of nonwoven material of the channel, and separation conditions were optimized. The DF IEF device was tested by the separation of a peptide mixture originating from the tryptic digestion of BSA, cytochrome c, and myoglobin. The pH gradient of DF IEF was created by the autofocusing of tryptic peptides themselves without any addition of carrier ampholytes. The focusing process was monitored visually using colored pI markers, and the obtained fractions were analyzed by RP-HPLC and ESI/TOF-MS. DF IEF operating in the autofocusing mode provides an efficient preseparation of peptides, which is comparable with a commercially available MicroRotofor multicompartment electrolyzer and significantly improves sequence coverage of analyzed proteins. The potential of the DF IEF device as an efficient tool for the preparative scale separations was demonstrated by the isolation of caseinomacropeptide (CMP) from a crude whey solution.
- MeSH
- design vybavení MeSH
- isoelektrická fokusace přístrojové vybavení metody MeSH
- kaseiny chemie MeSH
- peptidové fragmenty analýza chemie izolace a purifikace MeSH
- peptidy analýza izolace a purifikace MeSH
- proteiny analýza izolace a purifikace MeSH
- skot MeSH
- trypsin chemie MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
