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核心技术专利：CN118964589B侵权必究

核心技术专利：CN118964589B侵权必究

Degan P, Carpano P, Cercignani G, Montagnoli G

I.S.T., Istituto Nazionale per la Ricerca sul Cancro, Laboratorio di Mutagenesi, Genova, Italia.

Int J Biochem. 1989;21(3):291-5. doi: 10.1016/0020-711x(89)90186-9.

Covalent coupling of fluorescein to methotrexate (MTX) by a 5-carbon spacer yields a dihydrofolate reductase (DHFR) inhibitor (FMTX) with Ki = 11 nM. 2. FMTX shows a fluorescence quenching with respect to fluorescein which is relieved by binding to the enzyme. 3. The dissociation constants (Kd) of MTX, FMTX, NADPH and 7,8-dihydrofolate (DHF) from bovine liver DHFR have been determined by fluorometric titrations. 4. The Kd values for NADPH, MTX and FMTX from the complementary binary complexes (MTX.DHFR, FMTX.DHFR and NADPH.DHFR) were also obtained; these show a 2- to 4-fold decrease with respect to those obtained by titration of the free enzyme. 5. A competitive assay for MTX has been developed by exploiting the fluorescence enhancement of DHFR-bound FMTX. This assay may be useful for the routine determination of MTX in the concentration range from 10(-9) to 10(-7) M.

通过一个含5个碳原子的间隔臂将荧光素与甲氨蝶呤（MTX）共价偶联，得到一种二氢叶酸还原酶（DHFR）抑制剂（FMTX），其抑制常数（Ki）为11 nM。2. FMTX相对于荧光素表现出荧光猝灭，而与酶结合后荧光猝灭得以解除。3. 通过荧光滴定法测定了MTX、FMTX、NADPH和7,8 - 二氢叶酸（DHF）与牛肝DHFR的解离常数（Kd）。4. 还获得了来自互补二元复合物（MTX·DHFR、FMTX·DHFR和NADPH·DHFR）的NADPH、MTX和FMTX的Kd值；相对于通过滴定游离酶获得的值，这些值显示出2至4倍的降低。5. 通过利用与DHFR结合的FMTX的荧光增强，开发了一种MTX的竞争性检测方法。该检测方法可用于常规测定浓度范围为10⁻⁹至10⁻⁷ M的MTX。

Degan P, Carpano P, Cercignani G, Montagnoli G

I.S.T., Istituto Nazionale per la Ricerca sul Cancro, Laboratorio di Mutagenesi, Genova, Italia.

Int J Biochem. 1989;21(3):291-5. doi: 10.1016/0020-711x(89)90186-9.

Covalent coupling of fluorescein to methotrexate (MTX) by a 5-carbon spacer yields a dihydrofolate reductase (DHFR) inhibitor (FMTX) with Ki = 11 nM. 2. FMTX shows a fluorescence quenching with respect to fluorescein which is relieved by binding to the enzyme. 3. The dissociation constants (Kd) of MTX, FMTX, NADPH and 7,8-dihydrofolate (DHF) from bovine liver DHFR have been determined by fluorometric titrations. 4. The Kd values for NADPH, MTX and FMTX from the complementary binary complexes (MTX.DHFR, FMTX.DHFR and NADPH.DHFR) were also obtained; these show a 2- to 4-fold decrease with respect to those obtained by titration of the free enzyme. 5. A competitive assay for MTX has been developed by exploiting the fluorescence enhancement of DHFR-bound FMTX. This assay may be useful for the routine determination of MTX in the concentration range from 10(-9) to 10(-7) M.

通过一个含5个碳原子的间隔臂将荧光素与甲氨蝶呤（MTX）共价偶联，得到一种二氢叶酸还原酶（DHFR）抑制剂（FMTX），其抑制常数（Ki）为11 nM。2. FMTX相对于荧光素表现出荧光猝灭，而与酶结合后荧光猝灭得以解除。3. 通过荧光滴定法测定了MTX、FMTX、NADPH和7,8 - 二氢叶酸（DHF）与牛肝DHFR的解离常数（Kd）。4. 还获得了来自互补二元复合物（MTX·DHFR、FMTX·DHFR和NADPH·DHFR）的NADPH、MTX和FMTX的Kd值；相对于通过滴定游离酶获得的值，这些值显示出2至4倍的降低。5. 通过利用与DHFR结合的FMTX的荧光增强，开发了一种MTX的竞争性检测方法。该检测方法可用于常规测定浓度范围为10⁻⁹至10⁻⁷ M的MTX。