Determination of Cytochrome c and Other Heme Proteins Using the Reduction Wave of Mercury Protoporphyrin IX Groups Generated by a Hydroxylamine Induced Replacement Reaction

Abstract

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.

Thermodynamic mechanism of free heme action on sickle cell hemoglobin polymerization

“…The overwhelming difficulty in the determination of free heme in erythrocytes is its separation from hemoglobin without additional heme release or loss of existing free heme; even advanced spectroscopy methods fail to quantify free heme in the presence of hemoglobin . Most of the recent work on heme quantification has focused on the determination of the total heme content, including the one bound to globins, often in the context of forensics . Earlier methods were mostly based on the spectroscopic response of heme and heme‐containing proteins 38 ; recent studies rely on HPLC, capillary electrophoresis and other electrochemical methods, and, most recently, mass spectrometry …”

The free heme concentration in healthy human erythrocytes

“…Most of the recent work on heme quantification has focused on the determination of the total heme content, including quantities bound to globins, often in the context of forensics [19]. The methods employed in these studies were mostly based on the spectroscopic response of heme and heme-containing proteins [20]; HPLC [21], electrochemical methods such as capillary electrophoresis [22,23], and mass-spectrometry [19,24] have been used in recent work.…”

Characteristics of the Protoporphyrin IX Binding Sites on Human Serum Albumin Using Molecular Docking

“…Damage of the mitochondrial membrane, liposomes, Golgi apparatus, cytoplasmatic reticulum and nuclear membrane has been observed. This reaction cannot occur without a photosensitizer [3,4]. Photodynamic diagnosis (PDD) and therapy (PDT) take advantage of this phenomenon.…”