SIMULTANEOUS SEPARATION OF NUCLEOTIDES AND NUCLEOTIDE SUGARS USING AN ION-PAIR REVERSED-PHASE HPLC - APPLICATION FOR ASSAYING GLYCOSYLTRANSFERASE ACTIVITY

摘要

Nucleotides and nucleotide sugars were simultaneously separated by high-performance liquid chromatography using an ion-pair reversed-phase method. Tetrabutylammonium hydrogen sulfate (TBAHS), which is very hydrophobic, was selected as the counterion. Using a linear elution gradient, the influence of the counterion concentration, the pH in the mobile phase, and the temperature on the solute retention times has been studied. A concentration of 2.5 mM TBAHS in a potassium phosphate buffer, pH 6.9, and ambient temperature were the optimal conditions to separate a mixture of nucleotide sugars and nucleotides. Moreover, this method has allowed the direct separation of the 4-epimeric-uridine 5'-diphosphate sugars (UDP-galactose and UDP-glucose) without prior formation of a UDP sugar-berate complex. This simple and rapid (20 min) method enabled nucleotides and nucleotide sugars to be detected down to 40 pmol. This technique was particularly attractive for assaying glycosyltransferase activity. As an example, the quantitative determination of UDP-galactose, NADH, NAD(+), and UTP during the lactose synthesis by a galactosyltransferase (EC 2.1.4.22) was successfully investigated.