A new spectrophotometric assay for measuring pyruvate dehydrogenase complex activity: a comparative evaluation
Pyruvate dehydrogenase complex (PDHc) plays a key role in pyruvate decarboxylation, the transformation of pyruvate to acetyl-CoA. In this study, a new assay for measuring PDHc activity has been developed. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is adopted as the electron acceptor in this method, and the change in absorbance caused by the reduction of MTT by hydroxyethyl-TPP, which is the catalyzed product of pyruvate dehydrogenase (PDH, E1), within a certain period of time is measured to reflect PDHc activity. For further impartial evaluation of the characteristics of this newly developed assay, a series of comparative studies were also conducted with several commonly used assays of PDHc activity, including the conventional spectrophotometric assay of NADH accumulation, the p-iodonitrotetrazolium violet (INT)-coupled assay, the 2,6-dichlorophenolindophenol (2,6-DCPIP) assay, and the potassium ferricyanide assay. Results have proved that the spectrophotometric assay using MTT is highly sensitive and inexpensive with little interference from the sulfhydryl compound. In conclusion, the newly established assay is applicable for activity measurement not only in purified PDH and PDH in purified PDHc, but also in crude PDH solution prepared under certain conditions; hence, the assay can indirectly reflect the activity of PDHc activity.