Abstract
This chapter describes the preparation, properties, and conditions for assay of lipoamide dehydrogenase apoenzyme from pig heart. The apoenzyme is obtained by removal of the flavine-adenine dinucleotide (FAD) in addition to the D-amino acid oxidase apoenzyme is useful in testing flavin analogs for their coenzymatic properties and their affinities for the protein. The enzyme activity is assayed spectrophotometrically by recording the decrease in absorbancy at 340 nm. The diaphorase activity is determined spectrophotometrically recording the reduction of 2, 6-dichlorophenolindophenol (DCIP) at 600 nm in the reaction. The specific activity with lipoate expressed as change in absorbancy units per milligram of protein per minute is also calculated. Apoenzyme preparations of bacterial lipoamide dehydrogenases are also discussed. The removal of the flavin without denaturation of the apoenzyme is more difficult in the case of the pig heart enzyme. The effect of temperature and time on the development of the activity with oxidized lipoate upon the addition of FAD to the apoenzyme of lipoamide dehydrogenase is graphically represented.
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