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.

Quantitative analysis of cytochrome C released from rice mitochondria using the adsorptive polarographic wave of guanidine modified Co(II)-cytochrome C complex.

A new, simple and sensitive method for the quantitative analysis of cytochrome C (Cyt C) based on the reduction wave of guanidine modified Co(II)-Cyt C complex at about -1.74 V (vs. SCE) by single sweep polarography in the solution containing 8 x 10(-6) mol L(-1) CoCl2, 0.04 mol L(-1) guanidine hydrochloride, 0.2 mol L(-1) NaOH and 0.5% Na2SO3. The peak height is linearly proportional to the concentration of Cyt C in the range of 0.005 approximately 1.500 mg L(-1) (correlation coefficient 0.999). Common amino acids, saccharide, organic acid and metal ions of appropriate concentrations have no interference on the Cyt C determination. The released Cyt C in the process of mitochondrial permeability transition of Hong-Lian cytoplasmic male sterile line of rice has been measured by the method, and the result is satisfactory.

[Polarograhic adsorptive wave of protein hydrolysate in Pb2+ and sodium hydroxide solution and its application].

AIM: To propose a new simple and sensitive voltammetric method for determination of proteins. METHODS: Protein with sulfhydryl or disulfide bond in 0.5 mol x L(-1) NaOH, 1.5 x 10(-4) mol x L(-1) Pb2+ and 0.02% tetrabutylammonium iodide was heated in boiling water for 5 minutes. The reactive product gave a well defined reductive adsorption wave at -0.66 V (vs SCE) by means of single sweep polarography, and the height of derivative wave was proportional to the concentration of proteins. RESULTS: The peak height was linearly proportional to bovine serum albumin (BSA) or human serum albumin (HSA) concentration in range of 7.5 x 10(-10) -3.0 x 10(-7) mol x L(-1) (r(BSA) = 0.9995, and r(HSA) = 0.9990). The detection limit of BSA or HSA was 3.0 x 10(-10) mol x L(-1). For lysozyme (Lyso), the concentration range was from 1.4 x 10(-8) to 1.3 x 10(-6) mol x L(-10 (r(Lyso) = 0.9997) and the detection limit was 7.0 x 10(-9) mol x L(-1). CONCLUSION: The method is simple, rapid, sensitive and applicable to the assay of diluted human serum albumin samples.

Polarographic behavior of Co(II)-BSA or -HSA complex in the presence of a guanidine modifier.

An extremely sensitive adsorptive wave of BSA at about -1.73 V (vs SCE) has been obtained in the solution containing 8 x 10(-)(7) mol/L CoCl(2), 0.2 mol/L guanidine hydrochloride, and 0.2 mol/L NaOH by using single-sweep polarography. The interaction of BSA with guanidine in an alkaline solution results in its Co(II) complex with a positive excess charge. Thus, the complex is strongly adsorbed by the DME as a result of static electrical attraction. The adsorption efficiently accumulates the electrochemical active complex of Co(II)-BSA onto the DME. In the following potential scan, the complex produces a sensitive adsorptive reduction peak, which can be used to determine low-level BSA. In the absence of guanidine, the complex of Co(II)-BSA with a negative excess charge is repulsed by the DME. The reduction current is very small. The peak current depends on both BSA and Co(II) ion concentrations. HSA is similar to BSA in polarographic behavior. At the optimal conditions, the peak height is linearly proportional to the BSA or HSA concentration in the range of 0.005-20 mg/L (correlation coefficient 0.999). The detection limit for BSA or HSA is 0.002 mg/L. Lysozyme, common amino acids, and metal ions have no interference with the protein determination. The new method could be useful in protein studies.