Binding of [3H] methotrexate (MTX) for the radioassay of this drug and study of enzyme inhibitor interaction
The binding of MTX to dihydrofolate reductase has been studied directly using [3H] MTX and L 1210 leukemia lysates rather than by measuring inhibition of enzyme activity. Competitive inhibition of binding by unlabeled MTX has been exploited as a radioassay for the drug and dose response curves with an absolute sensitivity of 10 pg can be obtained. Physiologic folates do not inhibit the binding of [3H] MTX. The mean recovery of MTX added to serums was 93%. MTX has been measured in serum and spinal fluid of patients and cell lysates of L 1210 leukemic mice treated with this drug. Additional studies demonstrated greater binding of [3H] MTX by L 1210 lysates between pH 4.6 and 6.0 in the presence of NADPH and 2 mercaptoethanol. At pH > 7.0 less binding occurred and bound drug more readily dissociated. With a theoretical finite binding capacity for [3H] MTX, a plot of the reciprocal of the fraction bound vs. MTX added should have been linear with a slope of 0.78. Similar plot of experimental data was linear but with a slope of 0.41 indicating more MTX was found as more was added after apparent saturation. These findings suggest additional binding sites for MTX and may explain why more drug is needed to inactivate enzyme activity than calculated from enzyme drug molar equivalence.