Determination of dynamic parameters of interaction between methotrexate enantiomer and dihydrofolate reductase by affinity capillary electrophoresis / 中华检验医学杂志

ABSTRACT

Objective To develop a method for the determination of the dynamic parameters of interaction between methotrexate(MTX)enantiomer and dihydrofolate reductase(DHFR).Methods An affinity capillary electrophoresis(ACE)method was adopted.Using bare fused-silica capillary,the electrophoresis buffer was 50 mmoL/L sodium tetraborate with 0.2%Brij-35,pH 9.50.The temperature of separation was controlled at 25℃ and a voltage of 25 kV was applied.The separation of the reaction mixture was performed at a wavelength of 254 nm.The difference of peak areas about the product was used to calculate the inhibitory rate and IC50 values.Results We establish the detection method for the dynamic parameters of interaction between MTX enantioner and DHFR.The separation of the reaction mixture could be achieved within 30 min.The IC50 value of D-(-)-MTX and L-(+)-MTX were 3.17×10-7 and 2.48×10-8mol/L,respective.The IC50 value of the D-(-)-MTX was 31.67×10-8mol/L,the L-(+)-MTX was2.48×10-8mol/L.The IC50 value of the D-(-)-MTX was about 13 times higher than that of the L-(+)-MTX.Conclusions ACE is a rapid.simple and accurate method that can be used to monitor DHFRdynamic reaction.The IC50 values of MTX enantiomer were quite different.The result first indicated that reaction between MTX enantiomer and DHFR had three-dimensional selection.