Competitive protein binding assay for methotrexate.
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.
D001665:Binding Sites; D001667:Binding, Competitive; D006801:Humans; D007780:Lacticaseibacillus casei / Q000201:enzymology; D008727:Methotrexate / Q000032:analysis* / Q000097:blood / Q000134:cerebrospinal fluid; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism
D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate