English

Abstract

It was carried out the introduction of functionally important deuterated aromatic amino acids - [2,3,4,5,6- 2 H 5 ]phenylalanine, [3,5- 2 H 2 ]tyrosine and [2,4,5,6,7- 2 H 5 ]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-[ 2 H]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- 2 H 5 ]Phe, [3,5- 2 H 2 ]Tyr, [2,4,5,6,7- 2 H 5 ]Trp, biosynthesis, EI mass-spectrometry, RP-HPLC.