Reduction of ferricyanide by thiamine or thiamine pyrophosphate

The colorimetric assay for the activity of pyruvate dehydrogenase (EC 1.2.4.1) developed by Itokawa is based on a coupled reduction of ferricyanide and the formation of Prussian blue (Brian Research 94, 475-484). In this assay system, we found that the coenzyme, thiamine pyrophosphate, itself reduced ferricyanide independent of both the enzyme and the substrate. Similar effect was also observed with thiamine, but not with thiochrome. The reduction of ferricyanide by thiamine or thiamine pyrophosphate was blocked by trichloroacetic acid. Measurement of the activity of purified pyruvate dehydrogenase based on monitoring the reduction of ferricyanide as described by Schwartz et al (Biochem. Biophys. Res. Commun. 31, 495-500) has been widely used. Our findings clearly disprove the assumption that reduction of ferricyanide is dependent on pyruvate dehydrogenase and invalidate assays for thiamine-dependent dehydrogenases based on the reduction of ferricyanide.