Determination of cytochrome c and other heme proteins using the reduction wave of mercury protoporphyrin IX groups generated by a hydroxylamine induced replacement reaction.

We have found that in the presence of hydroxylamine, the heme prosthetic group of the heme protein adsorbed at the mercury electrode surface reacts with mercury ion produced by the electrochemical oxidation of mercury and is quantitatively converted into the mercury protoporphyrin IX group using single-sweep polarography. As a result, the small redox peak P(0) of the heme prosthetic group at about -0.46 V (vs SCE) disappears and a large new reduction peak P of mercury protoporphyrin IX group at -0.89 V comes out in a pH 9.6 NaHCO(3)-Na(2)CO(3) solution. Peak P is extremely sensitive to heme protein concentration. On the basis of the reduction peak P, a unique electrochemical method for heme protein assays is constructed. For the cytochrome c determination, the peak height is linearly proportional to the concentration in the range of 0.005-15 mg L(-1) (correlation coefficient 0.999). The detection limit is 0.003 mg L(-1). In contrast with peak P(0), the detection limit of cytochrome c is only 0.6 mg L(-1). The voltammograms of heme proteins in the absence and presence of hydroxylamine can serve as a reliable qualitative analytical method. The chemical reaction is peculiar to the heme prosthetic group. Without hydroxylamine it cannot occur. Thereby the method is highly specific and free from interference. The performance takes only a few minutes. These advantages make the method attractive for heme protein detecting.