A colorimetric assay of lipoyl-N-ε-lysine hydrolysis activity using 2,6-dibromoquinone-4-chlorimide

Lipoic acid is a coenzyme for pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, branched chain-ketoacid dehydrogenase, and the glycine cleavage system. Lipoic acid is covalently attached through an amide to the ε-amino group of specific lysine residues of these enzymes. Lipoamidase hydrolyzes the amide bond of lipoyl-N-ε-lysine. Because of the difficulty in quantitating lipoic acid or lysine released by hydrolysis of lipoyl-N-ε- lysine, a sensitive assay of lipoamidase activity was developed based on quantitation of lipoic acid liberated from lipoyl-ε-lysine using 2,6- dibromoquinone-4-chlorimide (DBQC). This method involves acidification of the assay mixture with HCl and separation of lipoic acid from lipoyl-N-ε-lysine by extraction into ethyl acetate where it can react with DBQC. This method is as sensitive as methods based on the reaction of lipoic acid with dinitrothiobenzoate and requires only a single extraction, but does not require reduction of the disulfide and the color reagent does not need to be prepared daily. Results obtained using this assay to quantitate lipoic acid released from lipoyl-N-p-aminobenzoate correlated excellently with results obtained using the Marshall-Bratton reaction to quantitate p-aminobenzoate. We have detected lipoyl-N-ε-lysine hydrolysis activity that is distinct from that of biotinidase and bile salt-stimulated lipase in lymphoblasts from a patient with biotinidase deficiency. This assay can be used to measure lipoyl-N-ε-lysine hydrolysis activity in tissues, especially those with little or no biotinidase activity.