Competitive protein binding assay for methotrexate

C. E. Myers, Marc E Lippman, H. M. Eliot, B. A. Chabner

Research output: Contribution to journalArticle

105 Citations (Scopus)

Abstract

A competitive protein binding assay has been developed for methotrexate based on the tight binding of this drug to Lactobacillus casei dihydrofolate reductase (= tetrahydrofolate dehydrogenase; 5,6,7,8 tetrahydrofolate: NADP+ oxidoreductase; EC 1.5.1.3). Free drug may be separated from that bound to reductase by adsorption with dextran albumin coated charcoal. Scatchard plot analysis of the enzyme drug interaction confirmed the presence of a single homogeneous class of binding sites with an association constant K(a) of 2.1 x 108 M-1. This high affinity binding permits detection of methotrexate in the range of 0.3-30 pmol with a coefficient of variation of 15% or less. The predominant circulating folate, 5 methyl tetrahydrofolate, and the clinically useful rescue agent leucovorin (5 formyl tetrahydropteroyl glutamic acid) do not interfere with the assay, nor does the methotrexate metabolite 4 amino 4 deoxy 10 methylpteroic acid. Assay of clinical samples, including plasma and cerebrospinal fluid, showed close agreement between the previously described enzyme inhibition assay and the more rapid competitive binding method.

Original languageEnglish
Pages (from-to)3683-3686
Number of pages4
JournalProceedings of the National Academy of Sciences of the United States of America
Volume72
Issue number9
StatePublished - Dec 1 1975
Externally publishedYes

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Tetrahydrofolate Dehydrogenase
Competitive Binding
Protein Binding
Methotrexate
Lactobacillus casei
Leucovorin
Charcoal
Enzyme Assays
Dextrans
Drug Interactions
Pharmaceutical Preparations
Adsorption
Cerebrospinal Fluid
Glutamic Acid
Albumins
Oxidoreductases
Binding Sites
Acids
Enzymes

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

Myers, C. E., Lippman, M. E., Eliot, H. M., & Chabner, B. A. (1975). Competitive protein binding assay for methotrexate. Proceedings of the National Academy of Sciences of the United States of America, 72(9), 3683-3686.

Competitive protein binding assay for methotrexate. / Myers, C. E.; Lippman, Marc E; Eliot, H. M.; Chabner, B. A.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 72, No. 9, 01.12.1975, p. 3683-3686.

Research output: Contribution to journalArticle

Myers, CE, Lippman, ME, Eliot, HM & Chabner, BA 1975, 'Competitive protein binding assay for methotrexate', Proceedings of the National Academy of Sciences of the United States of America, vol. 72, no. 9, pp. 3683-3686.
Myers, C. E. ; Lippman, Marc E ; Eliot, H. M. ; Chabner, B. A. / Competitive protein binding assay for methotrexate. In: Proceedings of the National Academy of Sciences of the United States of America. 1975 ; Vol. 72, No. 9. pp. 3683-3686.
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