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PubMed · 810804

Competitive protein binding assay for methotrexate.

C E Myers·M E Lippman·H M Elliot·B A Chabner
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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 Ka of 2.1 X 10(8) 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.

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Binding SitesBinding, CompetitiveHumansLacticaseibacillus caseiMethotrexateTetrahydrofolate DehydrogenaseJournal Article

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BibTeXRIS

C E Myers, M E Lippman, H M Elliot, B A Chabner. 1975. Competitive protein binding assay for methotrexate.. https://doi.org/10.1073/pnas.72.9.3683

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