中文摘要：

目的建立一种测定氨甲蝶呤（MTX）两种对映体与二氢叶酸还原酶（DHFR）结合反应动力学参数的方法。方法建立DHFR反应体系，采取亲和毛细管电泳技术，以含0．2％Brij-35的pH 9．50的50mmol／L硼砂为电泳缓冲液，检测波长为254nm，在25kV的电压下，分离反应体系中各组分。观察酶反应前后的电泳图谱并根据反应生成物峰面积的变化计算相关反应动力学参数，将不同浓度的氨甲蝶呤两种对映体L-（＋）-MTX和D-（-）-MTX分别作用于所建立的DHFR反应体系，测定两种对映体的半数抑制浓度（IC50）。结果建立了亲和毛细管电泳法对氨甲蝶呤对映体与DHFR反应动力学参数的测定方法，在30min内实现DHFR反应体系中反应物和生成物的分离，并根据反应生成物峰面积的变化计算得出D-（-）-MTX的IC50为3．17×10^-7 mol／L，L-（＋）-MTX的IC50为2．48×10^-8 mol／L，两者相差13倍左右。结论亲和毛细管电泳的方法可以快速准确地用于DHFR反应动力学参数的研究，通过检测MTX对映体对DHFR的抑制能力的差异，首次发现MTX对映体对DHFR有立体选择性作用。

英文摘要：

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^-8 mol/L, respective. The IC50 value of the D-（-）-MTX was 31.67 × 10^-8 mol/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 DHFR dynamic 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.