We report for the first time the efficient use of accelerated solvent extraction (ASE) for extraction of ricin to analytical purposes, followed by the combined use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and MALDI-TOF MS/MS method. That has provided a fast and unambiguous method of ricin identification for in real cases of forensic investigation of suspected samples. Additionally, MALDI-TOF MS was applied to characterize the presence and the toxic activity of ricin in irradiated samples. Samples containing ricin were subjected to ASE, irradiated with different dosages of gamma radiation, and analyzed by MALDI-TOF MS/MS for verification of the intact protein signal. For identification purposes, samples were previously subjected to SDS-PAGE, for purification and separation of the chains, followed by digestion with trypsin, and analysis by MALDI-TOF MS/MS. The results were confirmed by verification of the amino acid sequences of some selected peptides by MALDI-TOF MS/MS. The samples residual toxic activity was evaluated through incubation with a DNA substrate, to simulate the attack by ricin, followed by MALDI-TOF MS/MS analyses.
- MeSH
- aceton chemie MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- hexany chemie MeSH
- peptidy analýza chemie MeSH
- ricin analýza chemie MeSH
- rozpouštědla chemie MeSH
- sekvence aminokyselin MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- tandemová hmotnostní spektrometrie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The enzymatic regioselective monopalmitoylation of racemic 9-(2,3-dihydroxypropyl)- adenine (DHPA), an approved antiviral agent, has been performed by an immobilized form of Candida antarctica B lipase (CAL-B) using a 4:1 DMF/hexane mixture as the reaction medium. To improve the chemical yield of the desired monopalmitoylation reaction, solid-phase chemical modifications of the lipase were evaluated. The reaction yield was successfully increased obtaining 100% product after a second treatment of the product solution with fresh immobilised chemically glycosylated-CAL-B.
- MeSH
- adenin analogy a deriváty chemie MeSH
- Candida enzymologie MeSH
- enzymy imobilizované chemie MeSH
- fungální proteiny chemie MeSH
- glykosylace MeSH
- hexany chemie MeSH
- katalýza * MeSH
- lipasa chemie MeSH
- lipoylace MeSH
- polymery chemie MeSH
- rozpouštědla chemie MeSH
- stereoizomerie MeSH
- Publikační typ
- časopisecké články MeSH
High performance countercurrent chromatography (HPCCC) was successfully applied for the separation of nostotrebin 6 from cultivated soil cyanobacteria in a two-step operation. A two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (4:5:4:5, v/v/v/v) was employed for the HPCCC separation. In the first-step operation, its neutral upper phase was used as stationary phase and its basic lower phase (1% NH3 in lower phase) was employed as mobile phase at a flow rate of 1 mL/min. In the second operation step, its neutral upper phase was used as stationary phase, whereas both its neutral lower phase and basic lower phase were employed as mobile phase with a linear gradient elution at a flow rate of 0.8 mL/min. The revolution speed and temperature of the separation column were 1,000 rpm and 30 °C, respectively. Using HPCCC followed by clean-up on Sephadex LH-20 gel, 4 mg of nostotrebin 6 with a purity of 99% as determined by HPLC/DAD-ESI-HRMS was obtained from 100 mg of crude extract. The chemical identity of the isolated compound was confirmed by comparing its spectroscopic data (UV, ESI-HRMS, ESI-HRMS2) with those of an authentic standard and data available in the literature.
- MeSH
- acetáty chemie MeSH
- cholinesterasové inhibitory chemie izolace a purifikace MeSH
- cyklopentany chemie izolace a purifikace MeSH
- hexany chemie MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- methanol chemie MeSH
- Nostoc chemie MeSH
- protiproudá chromatografie MeSH
- půdní mikrobiologie MeSH
- rozpouštědla chemie MeSH
- voda chemie MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Chlorosomes from green photosynthetic bacteria are large photosynthetic antennae containing self-assembling aggregates of bacteriochlorophyll c, d, or e. The pigments within chlorosomes are organized in curved lamellar structures. Aggregates with similar optical properties can be prepared in vitro, both in polar as well as non-polar solvents. In order to gain insight into their structure we examined hexane-induced aggregates of purified bacteriochlorophyll c by X-ray scattering. The bacteriochlorophyll c aggregates exhibit scattering features that are virtually identical to those of native chlorosomes demonstrating that the self-assembly of these pigments is fully encoded in their chemical structure. Thus, the hexane-induced aggregates constitute an excellent model to study the effects of chemical structure on assembly. Using bacteriochlorophyllides transesterified with different alcohols we have established a linear relationship between the esterifying alcohol length and the lamellar spacing. The results provide a structural basis for lamellar spacing variability observed for native chlorosomes from different species. A plausible physiological role of this variability is discussed. The X-ray scattering also confirmed the assignments of peaks, which arise from the crystalline baseplate in the native chlorosomes.
- MeSH
- alkoholy chemie MeSH
- anizotropie MeSH
- bakteriochlorofyly chemie metabolismus MeSH
- buněčné struktury metabolismus MeSH
- Chlorobium metabolismus MeSH
- esterifikace MeSH
- hexany chemie MeSH
- kvarterní struktura proteinů MeSH
- radiační rozptyl MeSH
- rentgenové záření MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
