Tritium release assay for oleic acid desaturation

Abstract

The “tritium release” assay for the enzymic conversion of stearic acid into oleic acid introduced by Talamo and Bloch in 1969 (1) represented a major advance in the measurement of enzymic fatty acid desaturation. By measuring the release of tritium from the 9- and 10-positions of erythro-[9,10 3H 2]stearic acid into water instead of isolating the oleic acid chromatographically, the processing time can be reduced from about 2–3 hr to 5–10 min per assay. Recently, Johnson and Gurr (2) showed that the release of tritium does not represent an absolute measure of enzymic activity due to a primary kinetic isotope effect discriminating against tritium. In that paper we recorded the magnitude of the isotope effect, described the synthesis and use of threo-[9,10 3H 2]stearic acid, which increases the sensitivity of the assay, and demonstrated that release of tritium from this substrate was proportional to the formation of oleic acid. Because of the considerable interest in this laboratory in the biosynthesis of linoleic acid in plants, we have developed the principle of the tritium release assay to be applicable to the assay of the plant enzyme which converts oleic acid into linoleic acid. This has necessitated the synthesis of the appropriate oleic acid labeled with tritium in the 12- and 13-positions and this is described in some detail. In this paper we have been less concerned with the mechanistic details of the isotope effect than with the practical details involved in the use of the method and have discussed at some length some of its inherent advantages and disadvantages.