An assay for transaminase C is presented in which the pyruvate formed via transamination between cY-ketoisovalerate and alanine is determined using p-dimethylaminobenzaldehyde. This reagent reacts with pyruvate in alkaline solution to form a yellow chromophore having an absorption peak at 420 nm, but produces very little color with a-ketoisovalerate. This differential reactivity allows pyruvate to be determined in the presence of o-ketoisovalerate.
Transaminase C catalyzes the reversible transfer of amino groups between valine and pyruvate or cu-ketobutyrate. The existence of this enzyme was first reported in Escherichia coli by Rudman and Meister (1) in 1953. However, since then, the enzyme has been little studied, probably because a convenient assay for it is lacking. In a paper on the regulation of transaminase C in E. coli, McGilvray and Umbarger (2) used an assay in which the pyruvate formed was converted to its 2,4-dinitrophenylhydrazone derivative and was separated from the hydrazone of cY-ketoisovalerate (ar-KIV) by its preferential extraction into ethyl acetate. The ethyl acetate layer was then re-extracted with a sodium carbonate solution, and the color formed by the pyruvate hydrazone in alkaline solution was measured. This procedure suffers from certain disadvantages. The several extractions required make the method rather laborious, and the ethyl acetate extraction is not completely specific for the hydrazone of pyruvate.
In this paper we present an assay which permits the determination of pyruvate in the presence of a-ketoisovalerate and thus eliminates the need for separating the two keto acids.
Methods
Bacterial strains. The following bacterial strains were used: Bacillus subtilis 746, a prototrophic derivative of strain 168; B. subtilis 672, a derivative of strain W23; Escherichia cofi HfrC (met-); Salmonella typhimurium 1619 (lys-).