A new enzymatic method for the determination of protein-bound lipoic acid was established. Bound lipoyl groups were liberated in the form of lipoyllysine by protease digestion and assayed by lipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.8.1.4)-mediated NADH oxidation. NADH oxidation was coupled to reduction of the lipoyl disulfide group. Fluorescence kinetics of NADH oxidation were markedly enhanced by the addition of glutathione disulfide, recycling the enzyme-mediated lipoyl/dihydrolipoyl conversion. In the presence of a large excess of glutathione disulfide, NADH oxidation follows pseudo-first-order kinetics in terms of lipoyllysine concentration. A good linear correlation is obtained between the oxidation rate and lipoyllysine concentration up to 5 microM and the calibration curve indicates that the detection limit could be 100 nM lipoyllysine. The method was applied to protease lysates of bovine, rat, and rabbit tissues to determine lipoyllysine levels. Kidney and liver were found to have the highest content of lipoyllysine in the range of 3.9 to 4.6 nmol/g rat or rabbit wet tissue or 11.6 to 13.1 nmol/g bovine acetone powder.
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