The new algorithm of quantitative analysis in Mössbauer spectroscopy

Abstract

In this paper was demonstrated the possibility of the use of FAB mass spectrometry on an impulse mass spectrometer VG-70 SEQ ("Fisons VG Analytical", USA) for the study of biosynthetic pathways of 2 H-labeled purine ribonucleoside inosine secreted into the liquid culture (LC) by Gram-positive chemoheterotrophic bacterium Bacillus subtilis VKPM B-3157 when grown on heavy water (HW) medium with 2 % hydrolyzate of deuterated biomass of methylotrophic bacterium Brevibacterium methylicum VKPM B-5662 as a source of growth 2 H-labeled substrates. Isolation of 2 H-labeled inosine from the LC was performed by adsorption/desorption on activated carbon with following extraction by 0.3 M ammonium-formate buffer (pH = 8.9), crystallization in 80 % ethanol and ion exchange chromatography (IEC) on a column with AG50WX 4 cation exchange resin equilibrated with 0.3 M ammonium-formate buffer and 0.045 M NH 4 Cl. The investigation of deuterium incorporation into the molecule of biosynthetic [ 2 H]inosine by FAB mass spectrometry demonstrated the incorporation of 5 deuterium atoms into the molecule (the total level of deuterium enrichment-62.5 atom. % 2 H) with incorporation of 3 deuterium atoms into the ribose and 2 deuterium atoms-into the hypoxanthine residue of the molecule. Three non-exchangeable deuterium atoms were incorporated into the ribose residue owing to reactions of enzymatic izomerization of glucose in 2 H 2 O-medium due to reactions of glycolysis, associated with the Embden-Meyerhof pathway with participation of reactions of isotope (1 Н-2 Н) exchange, while two other deuterium atoms at C2, C8-positions in the hypoxanthine residue were synthesized from [ 2 H]amino acids that originated from the deuterated hydrolysate of the methylotrophic bacterium Brevibacterium methylicum VKPM B-5662.