COMPETITIVE-BINDING RADIOASSAY FOR 5-FLUORODEOXYURIDINE 5'-MONOPHOSPHATE IN TISSUES

  • 1 January 1979
    • journal article
    • research article
    • Vol. 39  (7) , 2471-2475
Abstract
A competitive binding radioassay was developed for 5-fluorodeoxyuridine 5''-monophosphate [5-dUMP] based on the tight binding of this potent inhibitor to thymidylate synthetase (EC 2.1.1.45) [Lactobacillus casei]. Unbound ligand may be separated from that bound to enzyme by precipitating the intact inhibitor-enzyme complex with trichloroacetic acid. Scatchard plot analysis using a 2-site model for binding yielded apparent dissociation constants of 1.2 .times. 10-11 and 1.7 .times. 10-10 M from a least-squares computer fit of the data. 5-dUMP could be detected in the range of 0.02-2.0 pmol with no apparent interference by other substances. Assay of 5-dUMP levels in L1210 ascites tumor [mouse] following 5-fluorouracil [an antitumor drug of which 5-dUMP is believed to be the active metabolite] in vivo revealed peak levels occurring within the 1st h with a subsequent disappearance half-life of 3.9 h. Close agreement was found between the previously described enzyme inhibition assay and the more rapid and sensitive competitive binding method.