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 microM 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.
Li
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Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143, USA.
Kenyon
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GL
D002087:Butyrates / Q000378:metabolism*; D007651:Keto Acids / Q000032:analysis*; D007659:Ketones / Q000032:analysis*; D011810:Quinoxalines / Q000032:analysis; D019998:Racemases and Epimerases; D012015:Reference Standards; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D013997:Time Factors
D002087:Butyrates; D007651:Keto Acids; D007659:Ketones; D011810:Quinoxalines; C005087:alpha-ketobutyric acid; D019998:Racemases and Epimerases; C021540:mandelate racemase