The assay of aromatic amino acid transaminations and keto acid oxidation by the enol borate-tautomerase method.

The enol tautomers of the aromatic oc-keto acids combine instantaneously and reversibly with borate to form complexes of a mixed ester-anhydride structure, thereby displacing the apparent keto-enol equilibrium in favor of the enol tautomer. The enol tautomer and its borate complex have strong absorption in the 300 ml* region (1). This strong absorption is the basis of the spectrophotometric methods described here for the assay of five rat liver enzymes which catalyze either the formation or utilization of aromatic pyruvates. The principle is a general one by which reactions forming or removing aromatic ol-keto acids can be followed by changes in the optical density of the enol borate. Brief accounts have been published of the use of this method with p-hydroxyphenylpyruvate oxidase (2) and tyrosine-oc-ketoglutarate transaminase (3). The present paper describes in greater detail the assays of these two enzymes and of three other enzymes: phenylalanine-pyruvate, histidine-pyruvate, and tryptophan-a-ketoglutarate transaminases. Emphasis has been placed on the identification and control of the factors affecting these enzyme activities in crude preparations of rat liver. The separation of the reactions, one from the other and from other transaminations occurring in the same preparations, will be the subject of a separate communication. With the present assays the levels of the enzymes have been measured in crude liver extracts of rats after various experimental treatments (2, 3).