Portal de Periódicos da CAPES

Rapid analysis of nucleotide-activated sugars by high-performance liquid chromatography coupled with diode-array detection, electrospray ionization mass spectrometry and nuclear magnetic resonance

2004; Elsevier BV; Volume: 1034; Issue: 1-2 Linguagem: Inglês

10.1016/j.chroma.2004.02.023

1873-3778

Michael Ramm, Jean‐Luc Wolfender, Emerson Ferreira Queiroz, Kurt Hostettmann, Matthias Hamburger,

Infant Nutrition and Health

A generally applicable method for HPLC analysis of sugar nucleotides was established. Separation was achieved using ion-pair chromatography on a reversed-phase column. Ion-pair reagents were selected and various parameters optimized with respect to separation of 11 of the most important sugar nucleotides and compatibility with on-line detection by electrospray ionization MS and NMR. The method was applied to the on-line analysis of the GDP-D-mannose-4,6-dehydratase (Gmd) and GDP-4-keto-6-deoxy-D-mannose reductase (Rmd) catalyzed conversion of GDP-D-mannose to GDP-D-rhamnose. By LC-NMR, the intermediate product of the reaction was shown to be a mixture of GDP-4-keto-6-deoxy-D-mannose and GDP-3-keto-6-deoxy-D-mannose. Nucleotide co-factors of enzymatic reactions such as ATP and NADH did not interfere with the analysis of nucleotide-activated sugars.