Introduction of Deuterated Aromatic Amino Acids  [2,3,4,5,6-2H5]Phenylalanine, [3,5-2H2]Tyrosine and [2,4,5,6,7-2H5]Tryptophan Into a molecule of Photochrome Trans-Membrane Protein Bacteriorhodopsin

Oleg Mosin, Ignat Ignatov


It was carried out the introduction of functionally important deuterated aromatic amino acids - [2,3,4,5,6-2H5]phenylalanine, [3,5-2H2]tyrosine and [2,4,5,6,7-2H5]tryptophan into a molecule of photochrome trans-membrane protein bacteriorhodopsin, synthesized by a photo-organotrophic halobacterium Halobacterium halobium ET 1001. The deuterated protein (output 8-10 mg) was isolated from purple membranes by cellular autolysis by distilled water, processing of bacterial biomass by ultrasound at 22 KHz, alcohol extraction of low and high-weight molecular impurities, cellular RNA, carotenoids and lipids, with the subsequent solubilization of final product with 0,5 % (w/v) SDS-Na and fractionation by methanol, gel filtration chromatography on Sephadex G-200, reverse-phase HPLC and EI impact mass-spectrometry of methyl esters of N-Dns-[2H]derivatives of amino acids. Deuterium was detected in all residues of aromatic amino acids. However, the presence in the EI mass spectrum of the BR hydrolysate the peaks [M]+ of semi-deuterated analogues of aromatic amino acids - phenylalanine with [M]+ at m/z = 413-418, tyrosine - with [M]+ at m/z = 428-430 and tryptophan - with [M]+ at m/z = 453-457 with different levels of contributions to the deuterium enrichment of molecules testifies about the conservation of the minor pathways of biosynthesis of aromatic amino acids de novo.

Keywords: Halobacterium halobium, bacteriorhodopsin, [2,3,4,5,6-2H5]Phe, [3,5-2H2]Tyr, [2,4,5,6,7-2H5]Trp, biosynthesis, EI mass-spectrometry, RP-HPLC.

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