A SPECTROPHOTOMETRIC DETERMINATION OF ALPHA-DICARBONYL COMPOUNDS AND ITS APPLICATION TO THE ENZYMATIC FORMATION OF ALPHA-KETOBUTYRATE

A simple, rapid, and sensitive spectrophotometric method has been developed for the determination of alpha-keto acids, alpha-diketones, and alpha-ketoaldehydes using o-phenylenediamine as a derivatizing reagent in acidic media to yield chromophoric quinoxaline derivatives. Application to the determination of alpha-ketobutyrate and the rate of its formation from isomerization of vinylglycolate catalyzed by mandelate racemase is described. Under optimal conditions 1 to 4 mM o-phenylenediamine in 80% acetic acid was incubated at 50 degrees C for 10 min with the alpha-dicarbonyl compounds. Solutions of alpha-ketobutyrate as low as 5 mu M in the buffer solution can be measured quantitatively, This method has also been used for determining the rate of formation of the phenylglyoxalate from (S)-mandelate catalyzed by (S)-mandelate dehydrogenase. (C) 1995 Academic Press, Inc.