Fast atom bombardment mass spectrometry in evaluation of biosynthetic pathways of purine ribonucleoside [2h] inosine

In this paper was demonstrated the possibility of the use of fast atom bombardment (FAB) mass spectrometry on an impulse mass spectrometer VG-70 SEQ ("Fisons VG Analytical", USA) for the study of biosynthetic pathways of 2H-labeled purine ribonucleoside inosine secreted into the culture liquid (CL) 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 2H-labeled substrates. Isolation of 2H-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 % (v/v) 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 NH4Cl. The investigation of deuterium incorporation into the molecule of biosynthetic [2H]inosine by FAB mass spectrometry demonstrated the incorporation of 5 deuterium atoms into the molecule (the total level of deuterium enrichment - 62.5 atom% 2H) 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 2H2O-medium due to reactions of glycolysis, associated with the Embden-Meyerhof pathway with participation of reactions of isotope (1Н-2Н) exchange, while two other deuterium atoms at C2, C8-positions in the hypoxanthine residue were synthesized from [2H]amino acids that originated from the deuterated hydrolysate of the methylotrophic bacterium Brevibacterium methylicum VKPM B-5662.