A reversed phase HPLC method for the analysis of nucleotides to determine 5’-PDE enzyme activity
5’-Phosphodiesterase (5'-PDE) can be extracted from barley roots and used as a catalyst in the hydrolysis of RNA to produce 5'-nucleotides. The assay of enzyme activity is essential for the production of 5'-PDE. To improve the conventional assays, we developed and validated a new method for the analysis of 5’-PDE enzyme activity using reversed phased high performance liquid chromatography (RP-HPLC). The method is based on the quantification of the four 5’-nucleotides namely cytidine 5’-monophosphate (5’-CMP), uridine 5’-monophosphate (5’-UMP), guanosine 5’-mono-phosphate (5’-GMP) and adenosine 5’-mono-phosphate (5’-AMP), produced in the enzymatic hydrolysis of yeast RNA. The optimal condition for the enzymatic hydrolysis of RNA to detect the enzyme activity was investigated. The results show that when the hydrolysis takes place at 70 oC for 30 min at pH 5.0, the hydrolysis reaction has highest yield for the four of the 5’-nucleotides. 5’-PDE demonstrated highest catalytic capability. These four 5’-nucleotides were utilized for the analysis of enzyme activity of 5’-PDE with our newly developed HPLC method. Excellent reproducibility, precision, and linearity were obtained for this HPLC method.
KEY WORDS: Enzyme activity, 5’-PDE, Barley roots, Nucleotides, RNA