title: Development of a trichloroacetic acid precipitation assay for covalent adducts of thymidylate synthase.
abstract: The use of trichloroacetic acid as a protein precipitant and denaturant in the quantitative measurement of covalent complexes of thymidylate synthase is described. Enzyme inactivated with N[3H]ethylmaleimide and inhibitory ternary complex (formed with native enzyme, 5-[6-3H]fluoro-2'-deoxyuridylate, and methylenetetrahydrofolate) served as reagents which were used to establish the conditions under which trichloroacetic acid precipitation, washing, and solubilization steps provided quantitative results. The ternary complex formed by dihydrofolate reductase with [3H]methotrexate and NADPH was used as a control to assess whether tight, but noncovalent, enzyme:ligand complexes survived trichloroacetic acid precipitation. The fact that no counts above background were detected in the pellet of precipitated protein demonstrated that the noncovalent complexes were completely dissociated by this treatment. The dynamic range of linear response for the inhibitory ternary complex of thymidylate synthase spanned five orders of magnitude, and the assay detected levels of enzyme as low as 10 fmol, a value which was essentially limited by the specific radioactivity of 5-[6-3H]fluoro-2'-deoxyuridylate. The ability of the enzyme to bind 5-[6-3H]fluoro-2'-deoxyuridylate specifically, as measured by the trichloroacetic acid assay, generated a specific binding value of 13.4 nmol of enzyme/mg protein (assuming a binding ratio of 1.5 for the inhibitory ternary complex). Specific binding values were compared to specific activity values (obtained from the spectrophotometric assay) at each stage of purification of the enzyme from Lactobacillus casei and were found to give parallel results. The characteristics of the trichloracetic acid assay procedure, which exclusively detects covalent enzyme-ligand adducts, are compared to those for other ligand binding assays for thymidylate synthase.
authors.affiliation: Department of Chemistry, University of South Carolina, Columbia 29208.
authors.forename: R J
authors.initials: RJ
authors.lastname: Cisneros
authors.forename: R B
authors.initials: RB
authors.lastname: Dunlap
mesh_terms: D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism; D011232:Chemical Precipitation; D003856:Deoxyuracil Nucleotides / Q000302:isolation & purification*; D005468:Fluorodeoxyuridylate / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D013763:Tetrahydrofolates / Q000302:isolation & purification* / Q000378:metabolism; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D014238:Trichloroacetic Acid
chemical_list: D001426:Bacterial Proteins; D003856:Deoxyuracil Nucleotides; D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; D005468:Fluorodeoxyuridylate; D014238:Trichloroacetic Acid; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase