The text of the original in Russian 


Method of setting DEUTERIUM-tagging AROMATIC amino acid L-[2,3,4,5,6-D5]-Phenylalanine, L-[3,5-D2]-tyrosine and L-[2,4,5,6,7 -- D5]-tryptophan in the molecule MEMBRANE PROTEIN BAKTERIORODOPSINA. 


A method for obtaining membrane protein bakteriorodopsina galofilnoy bacterium Halobacterium halobium, deuterium labeled on the balance [2, 3, 4, 5, 6-2H5] phenylalanine, [3, 5-2H2] tyrosine and [2, 4, 5, 6, 7 -- 2H5] tryptophan and its subsequent allocation to the preparative yield 8-10 mg per 1 g of bacterial biomass. The method consists in growing a strain of bacteria Halobacterium halobium galofilnyh synthetic medium with deuterium-marked aromatic amino acids, protein solu 0.5% by the CSN-Na and low-temperature fractionation of methanol, followed by extraction of carotenoids, lipids, high-and low-molecular compounds, electrophoresis PAAG to 12.5% from 0.1% TAC-Na and gel-penetrating chromatography on sefadekse G-200. Inclusion of deuterium-aromatic amino acids monitored by mass spectrometry electron impact DE after hydrolysis of the protein 4-H. Ba (OH) 2 and the division turned-phase high performance liquid chromatography of methyl esters of N-CSN-amino acid derivatives. According to mass spectrometric analysis, molecular ion peaks (M) + methyl ester N-CSN-derivatives of aromatic amino acids have very low intensity and polymorphonuclear splits, so the field of molecular enrichment were strongly ushireny. In addition, mass spectra of a mixture of additive, so the mixture can be analyzed only if there are different components of the spectra recorded in the same conditions. Current computations involve solution of the system of n equations with n unknowns to a mixture of n components. For components, the concentration exceeding 10 mol.%, Accuracy and reproducibility of the analysis is +0.5 mol.% (With a confidence probability 90%). Therefore, in order to obtain reproducible results it is necessary to provide individual chromatographic derivatives 2H-tracer amino acids from protein hydrolysates. To solve this task, we used the method of request-phase HPLC on oktadetsilsilanovom selikagele silasorb A18, which confirmed the effectiveness of the separation of a mixture of methyl esters of N-CSN-derived deuterium-tracer amino acid from microbial objects metilotrofnye as bacteria and microalgae. The method was able to adapt to the conditions of chromatographic separation of a mixture of methyl esters of N-CSN-derived amino acid hydrolysates of BR, is to optimize the ratio of eluent, a gradient and the speed with elyutsii column. The best separation was achieved with a gradient elyutsii methyl esters of N-Dns-amino acid derivatives of a mixture of solvents acetonitrile: trifluoroacetic acid = 100: 0.1 - 0.5, vol.%. In doing so, managed to separate tryptophan and intractable pair of phenylalanine / tyrosine. Degrees of chromatographic purity of isolated methyl esters of N-CSN-[2, 3, 4, 5, 6-2H5] phenylalanine, N-CSN-[3, 5-2H2] tyrosine and N-CSN-[2, 4, 5, 6 7-2H5] tryptophan were 89, 91 and 90% at the outputs of 78-85%. The proposed method of biosynthesis of deuterium-labeled bakteriorodopsina shows a high efficiency for tagging protein residues deuterium-labeled aromatic amino acids. With the help of the developed method can be obtained from 8-10 mg of deuterated bakteriorodopsina from 1 g of biomass. The method is also applicable to the receivable and other deuterium-labeled membrane proteins. In future we intend to get on the developed method is fully deuterated drugs bakteriorodopsina to the reconstruction of functionally active membrane proteins in heavy water with the deuterium-labeled lipids and deuterated other biologically active compounds. These studies will help to answer the question how the deuterium-tagged bakteriorodopsin within the native membranes in conditions close to the adaptation of cells to heavy water. 

Moscow State Academy of Fine Chemical Technology. MV Lomonosov Moscow State University, 117571 Moscow, prosp. Vernadskogo, d.86. 


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