The production of 2H-labeled amino acids by a new mutant of RuMP fucultative methylotroph Вrevibacterium methylicum (стр. 2 из 2)
For evaluation of deuterium enrichment methyl esters of N-DNS-amino acids were applied because the peaks of molecular ions (M+) allow to monitor the enrichment of multicomponential mixtures of amino acids being in composition with growth media metabolites, therefore EI MS allows to detect samples with amino acids of 10-9-10-12 mol (Karnaukhova, 1994). N-DNS-amino acids were obtained through the derivatization of lyophilized M9 with DNSCl. To increase the volatality of N-DNS-amino acids, the methylation with DZM was made to prevent the possible isotopic (1H-2H) exchange in molecule of Phe. With DZM treatment it occured the derivatization on aNH2 group in the molecule, so that its N-methylated derivative was formed to the addition of methyl ester of N-DNS-Phe.
Mass spectra EI MS of methyl esters of N-DNS-amino acid mixtures, obtained from M9 where used 0; 73.5 and 98% (v/v) of 2H2O (Table, Expts. (a), (d), (e)) are shown in consecutive order in Figs. 1-3. The fragmentation pathways of methyl esters ofN-Dns-amino acids by EI MS leads to the formation of the molecular ions (M+) from whom the fragments with smaller m/z ratio further are formed. Since the value of (M+) for Leu is as the same as for Ile, these two amino acids could not be clearly estimated by EI MS. A right region of mass spectra EI MS contains four peaks of molecular ions (M+) of methyl esters of N-DNS-amino acids: Phe with m/z 412; Leu/Ile with m/z 378.5; Val with m/z 364.5; Ala with m/z 336.4 (see Fig. 1 as an example). A high continuous left background region at m/z 80 - 311 is associated with the numerious peaks of concominant metabolites and fragments of further decay of methyl esters of N-DNS-amino acids.
The results, firmly established the labeling of amino acids as heterogenious, juging by the presence of clasters of adduct peaks at their molecular ions (M+); the species of molecules with different numbers of deuterium atoms were visualised. The most aboundant peak (M+)in each claster was registered by mass spectrometer as a peak with average m/z ratio, from whom the enrichment of each individual amino acid was calculated. Thus, in experiment (d) shown in Fig. 2 where used 73.5% (v/v) 2H2O the enrichment of Phe was 4.1, calculated at (M+) with m/z 416.1 (instead of m/z 412 (M+) for non-labeled compound); Leu/Ile - 4.6 (M+) with m/z 383.1 instead of m/z with 378.5 (M+)); Val - 3.5 (M+ with m/z 368 instead of m/z (M+) with 364.5); Ala - 2.5 deuterium atoms ((M+)with m/z 338.9 instead of m/z with 336.4 (M+)).
With increasing of 2H2O content in liquid M9, the levels of amino acid enrichment varried propotionaly. As seen in Fig. 3 in experiment (e) where used 98% (v/v) 2H2O the enrichment of Phe was six ((M+) with m/z 418 instead of m/z 412 (M+)); Leu/Ile - 5.1 ((M+) with m/z 383.6 instead of m/z with 378.5 (M+)); Val - 4.7 ((M+) with m/z 369.2 instead of m/z (M+) with 364.5); Ala - 3.1 deuterium atoms (M+) with m/z 339.5 instead of m/z with 336.4 (M+)). The label was distributed uniformely among the amino acid molecules, in experiment (e) the enrichment of 2H-labeled amino acids was nevertheless less than we estimated theoretically, because Leu was added in growth medium in protonated form. This effect should be seriously scrutinised before the applying this mutant for the preparation of 2H-labeled amino acids.
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