Radioactive assay of 2,4-dienoyl-coenzyme A reductase

A radioactive method for assaying 2,4-dienoyl-CoA reductase, also referred to as 4-enoyl-CoA reductase (EC 1.3.1.34), is described. The assay measures the incorporation of tritium from [4B-3H]NADPH into 2-trans,4-cis-decadienoyl-CoA or 2-trans,4-trans-decadienoyl-CoA which, after cleavage of the thioester bond with hydroxylamine, can be separated from the radioactive coenzyme by extraction with toluene. This assay is at least 30 times more sensitive than the spectrophotometric assay, even though rates determined by the radioactive method are 10 times lower than rates obtained spectrophotometrically due to a primary kinetic isotope effect. The linearity of this assay with respect to time and protein concentration is sufficient for determining 2,4-dienoyl-CoA reductase activities in extracts from small samples of human fibroblasts, which were found to contain reductase activities between 1.8 and 5.8 mU/mg of protein.