Determination of lipoyllysine derived from enzymes by liquid chromatography.

Lipoyllysine was liberated from the commercial enzymes bovine heart alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase. After incubation of the enzymes with pronase for 4 h, the lipoyllysine liberated was determined by high-performance liquid chromatography with ultraviolet detection at 340 nm. Standard lipoyllysine was synthesized in our laboratory. The specific determination of lipoyllysine with ultraviolet detection only at 340 nm could be utilized for the enzyme hydrolysate samples. Recoveries of lipoyllysine added (5.0 micrograms) to a reaction mixture containing protease and bovine serum albumin or ovalbumin model proteins (1.0 mg) were 116.8 and 119.5%, respectively. The lipoyllysine content in beef heart alpha-ketoglutarate dehydrogenase was 0.55 microgram/mg of enzyme and 0.83 microgram/mg of enzyme protein in beef heart pyruvate dehydrogenase.