TY - JOUR
T1 - A fluorescence study of substrate and inhibitor binding to bovine liver dihydrofolate reductase
AU - Degan, Paolo
AU - Carpano, Patrizia
AU - Cercignani, Giovanni
AU - Montagnoli, Giorgio
PY - 1989
Y1 - 1989
N2 - 1. 1. Covalent coupling of fluorescein to methotrexate (MTX) by a 5-carbon spacer yields a dihydrofolate reductase (DHFR) inhibitor (FMTX) with Ki = 11 nM. 2. 2. FMTX shows a fluorescence quenching with respect to fluorescein which is relieved by binding to the enzyme. 3. 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. 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. 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.
AB - 1. 1. Covalent coupling of fluorescein to methotrexate (MTX) by a 5-carbon spacer yields a dihydrofolate reductase (DHFR) inhibitor (FMTX) with Ki = 11 nM. 2. 2. FMTX shows a fluorescence quenching with respect to fluorescein which is relieved by binding to the enzyme. 3. 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. 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. 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.
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U2 - 10.1016/0020-711X(89)90186-9
DO - 10.1016/0020-711X(89)90186-9
M3 - Article
C2 - 2744204
AN - SCOPUS:0024506177
VL - 21
SP - 291
EP - 295
JO - International Journal of Biochemistry
JF - International Journal of Biochemistry
SN - 0020-711X
IS - 3
ER -