Rs. Li et Gl. Kenyon, A SPECTROPHOTOMETRIC DETERMINATION OF ALPHA-DICARBONYL COMPOUNDS AND ITS APPLICATION TO THE ENZYMATIC FORMATION OF ALPHA-KETOBUTYRATE, Analytical biochemistry, 230(1), 1995, pp. 37-40
A simple, rapid, and sensitive spectrophotometric method has been deve
loped 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. Applic
ation to the determination of alpha-ketobutyrate and the rate of its f
ormation from isomerization of vinylglycolate catalyzed by mandelate r
acemase is described. Under optimal conditions 1 to 4 mM o-phenylenedi
amine in 80% acetic acid was incubated at 50 degrees C for 10 min with
the alpha-dicarbonyl compounds. Solutions of alpha-ketobutyrate as lo
w as 5 mu M in the buffer solution can be measured quantitatively, Thi
s method has also been used for determining the rate of formation of t
he phenylglyoxalate from (S)-mandelate catalyzed by (S)-mandelate dehy
drogenase. (C) 1995 Academic Press, Inc.