Assay of dihydrofolate reductase activity by monitoring tetrahydrofolate using high-performance liquid chromatography with electrochemical detection.
We developed a method to determine dihydrofolate reductase (DHFR) activity at pH 7.4 (37 degrees C) by monitoring its product, tetrahydrofolate (H(4)folate), using HPLC with electrochemical detection. After the assay mixture was deproteinized by 0.5 M perchloric 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.
Aiso
K
K
Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, Tokyo, 183-0057, Japan.
Nozaki
T
T
Shimoda
M
M
Kokue
E
E
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004563:Electrochemistry / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009249:NADP / Q000032:analysis; D051381:Rats; D013762:Tetrahydrofolate Dehydrogenase / Q000032:analysis* / Q000378:metabolism*; D013763:Tetrahydrofolates / Q000032:analysis*; D013816:Thermodynamics
D007202:Indicators and Reagents; D013763:Tetrahydrofolates; C030371:5,6,7,8-tetrahydrofolic acid; D009249:NADP; D013762:Tetrahydrofolate Dehydrogenase