A fluorescence study of substrate and inhibitor binding to bovine liver dihydrofolate reductase

Paolo Degan, Patrizia Carpano, Giovanni Cercignani, Giorgio Montagnoli

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)291-295
Number of pages5
JournalInternational Journal of Biochemistry
Volume21
Issue number3
DOIs
Publication statusPublished - 1989

ASJC Scopus subject areas

  • Biochemistry

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