The first enzyme in the combined pathway to the branched-chain amino acids, ,acetohydroxy acid synthetase, simultaneously synthesizes a-acetolactate and a-aceto-.a-hydroxybutyrate, whi'ch are precursors of valine (leucine) and isoleucine, respectively (1).
The a-aceto-a-hydroxy acids may be determined by the method of Westerfeld (2) after decarboxylation by strong acid to the corresponding acyloins. When studying the synthetase in crude cell-free extracts the assay may be complicated by the presence of acetohydroxy acid decarboxylating enzymes and other aceboin forming enzymes. Such sidereactions have been considered in the differential assay procedure of Kuwana et al. (3). When both pyruvate and a-ketobutyrate 'are present as substrates the sum of the two acetohydroxy acids formed is determined by these procedures. A microbiological assay of a-aceto-a-hydroxybutyrate has been developed by Leavitt and Umbarger (4). Recently Ronkainen et al. ( 5) published a gas chromatographic procedure for the separate ,determination of the two acetohydroxy acids in fermentation solutions. The two acids were separated from the fermented medium by column chromatography and analyzed after conversion to a-diketones. The method to be described involves no previous separation from the biological material. It has been used in studies on the metabolic regulation of acetohydroxy acid synthetase from Saccharomyces carlsbergensis (6), and permits a quick and direct ,determination of a-acyl-a-hydroxy acids with 5-7 carbon atoms (C&,-C,) formed by incubation of cell-free enzyme extracts with C&-C& a-keto acids.
PRINCIPLE OF THE METHOD
The method is based upon the eonversion of a-aceto-cu-hydroxy acids, and other 8a-acyl-a-hydroxy acids, to the corresponding a-diketones (7)