Assay of Dihydrofolate Reductase Activity by Monitoring Tetrahydrofolate Using High-Performance Liquid Chromatography with Electrochemical Detection

✍ Scribed by Kenji Aiso; Tomoko Nozaki; Minoru Shimoda; Eiichi Kokue

• Publisher: Elsevier Science
• Year: 1999
• Tongue: English
• Weight: 70 KB
• Volume: 272
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1999.4174

✦ Synopsis

We developed a method to determine dihydrofolate reductase (DHFR) activity at pH 7.4 (37°C) by monitoring its product, tetrahydrofolate (H 4 folate), using HPLC with electrochemical detection. After the assay mixture was deproteinized by 0.5 M perchlolic acid, the H 4 folate concentration was measured. Using sodium ascorbate at 20 mM, H 4 folate was stable in our assay system. The enzyme activity was also stable. The detection limit of this method was less than 1 nM of H 4 folate in the enzyme assay system, which was 1/100 lower than those for the NADPH-spectrophotometric assay, which is commonly used for analysis of DHFR activity. This value of 1 nM allowed us to control the conversion from dihydrofolate (H 2 folate) to H 4 folate less than 10% of initial substrate concentrations during assay, when we used a concentration around K m values reported for DHFR from various sources. The rate of reduction showed a linearity at concentrations around the K m . The reduction rate must be evaluated exactly around the K m , in order to obtain an accurate profile of Michaelis-Menten kinetics. This assay method has a sensitivity high enough to determine the reduction rate at H 2 folate concentrations around K m . In addition, the assay procedure is very simple. Therefore, our method may be useful for studying DHFR.