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

Nucleotides and nucleotide sugars were simultaneously separated by hig h-performance liquid chromatography using an ion-pair reversed-phase m ethod. Tetrabutylammonium hydrogen sulfate (TBAHS), which is very hydr ophobic, was selected as the counterion. Using a linear elution gradie nt, the influence of the counterion concentration, the pH in the mobil e phase, and the temperature on the solute retention times has been st udied. A concentration of 2.5 mM TBAHS in a potassium phosphate buffer , pH 6.9, and ambient temperature were the optimal conditions to separ ate a mixture of nucleotide sugars and nucleotides. Moreover, this met hod has allowed the direct separation of the 4-epimeric-uridine 5'-dip hosphate sugars (UDP-galactose and UDP-glucose) without prior formatio n of a UDP sugar-berate complex. This simple and rapid (20 min) method enabled nucleotides and nucleotide sugars to be detected down to 40 p mol. This technique was particularly attractive for assaying glycosylt ransferase 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.