Intramolecular N isotope analysis of glutamine in phytoplankton

Glutamine (Gln) and glutamic acid (Glu) provide the first step of incorporating inorganic nitrogen (N) into cellular organic N in photoautotrophic primary producers. Both are present at higher concentrations than other amino acids (AAs). The N atom in cellular ammonia forms the amide group of Gln, which is subse- quently used in producing Glu. Although Glu supplies the N for most other amino acids via transamination reaction, Gln and Glu interconvert with each other via -ketoglutarate. We hypothesize that the 15N of Gln’s amino-N ( 15NGln-amino), amide N ( 15NGln-amide), and intracellular ammonium have the same values under equilibrium conditions due to fast turnover. In addition, the amide-N of Gln provides N for the nucleobases and one of the sidechain N atoms in histidine. Thus, position-specific N isotope analysis of Gln will provide key information on AA and nucleotide biosynthesis in organisms. To analyze the amino and amide N’s, intracellular Gln was extracted from lysed phytoplankton cells, separated, and collected by Ion-exchange Chromatography, then divided into two fractions. One sub-fraction was oxidized by hypochlo-rite, converting the amino-N to nitrite. All the nitrogen in the second fraction were oxidized to nitrate using persulfate with UV radiation. The nitrite or nitrate produced in the two sub-fractions will then be converted to nitrous oxide and analyzed using Purge-and-Trap Isotope Ratio Mass Spectrometry, yielding both 15NGln-amino and 15NGln-total. Using the mass balance, we then can calculate the 15NGln-amide by subtracting the 15NGln-amino from 15NGln-total. We will compare the 15NGln-amino and 15NGln-total with the 15N of intracellular ammonium in cultured phytoplankton to verify our hypothesis. This study will shed light on the 15N patterns of Gln and other amino acids in different phytoplankton phylogenetic groups under various metabolic conditions, which will further advance the use of 15N-AA patterns in trophic ecology and paleo-N cycle reconstructions.