Study On The Electrochemical Behavior Of Heme Proteins, Hemin And Protoporphyrin IX

Heme proteins is a kind of important substance which are consisted of heme prosthetic group. Biological systems rely on them to carry out versatile biological functions essential for their survival, such as cytochrome c can transfer electron, hemoglobin transport and store oxygen. The quantitative and qualitative analysis of heme proteins are regular contents in life science, also are important guide lines of disease diagnoses and curative effect's test in clinic inspection. Hemin is a typical porphyrin compound with natural red pigment and iron ion. It exerts a remarkable function in foodstuff production and anaemia treatment. Protoporphyrin IX is one part of hemin. The content determination of protoporphyrin IX shows great clinic significance for preventing and curing disease related with porphyrin.This thesis studies the electrochemical behavior of heme proteins with single-sweep polarography, and sets up a new quantitative and qualitative analysis method of heme proteins. In the presence of hydroxylamine, the iron protoporphyrin IX group of heme proteins adsorbed at the electrode surface is quantitatively converted to the mercury protoporphyrin IX group when mercury electrode is used. The method shows good selectivity and sensitivity. Meanwhile, this paper study the electrochemical behavior of hemin and protoporphyrin IX, then create new determination methods about them.The study has three parts, the primary contents are described as follows:1. A new chemical reaction of heme proteins in the surface of dropping mercury electrodeA sensitive reduction wave of cytochrome c and other heme proteins at about - 0.89 V(vs. SCE) has been observed by using single sweep polarography in pH 9.6 NaHCO₃ - Na₂CO₃ and hydroxylamine solution. The second derivative wave height are good linearly proportional to the concentration of heme proteins. Non-heme proteins do not have such a reduction wave. Mechanism research shows this reduction wave is generated by a special chemical reaction related with mercury ion produced in dropping mercury electrode. This special chemical reaction only happens in the solution with hydroxylamine coexistence. The method has good selectivity and sensitivity. It can be used as quantitative and qualitative analysis of heme proteins.2. A sensitive polarographic reduction wave of hemin in the presence of hydroxylamine hydrochloride.A new polarographic reduction wave of hemin at about - 0.89 V(vs. SCE) has been found in NaHCO₃ - Na₂CO₃ buffer solution (pH 9.7) adding NH2OH·HCl. The second derivative wave height is linearly proportional to the concentration of hemin in the range of 7.7×10^-9 - 3.1×10-6 mol·L^-1 (correlation coefficient 0.998). The detection limit is 3.8×10^-9 mol·L^-1. Non-iron metalloporphyrin compounds do not have such electrochemical behavior. The formation mechanism of reduction wave has been studied from different aspects. It shows that the electrogenerated mercury ion can takes the place of iron ion of iron protoporphyrin IX and quantitatively forming a mercury protoporphyrin IX at the electrode surface. The peak P is attributed to the reduction of mercury protoporphyrin IX group. It is interesting that the hydroxylamine hydrochloride palys a key role in this reaction. It has been proved that the wave has the property of adsorbability. The cyclic voltammogram of hemin declares that it is an irreversible reductive wave.3. The determination of protoporphyrin IX by single-sweep polarographyProtoporphyrin IX yields a sensitive reduction wave in NaHCO₃ - Na₂CO₃ buffer solution (pH 9.6). Its first derivative wave height shows good linearly proportional to the concentration of protoporphyrin IX in the range of 3.6×10^-7- 5.3×10^-5mol·L^-1 (correlation coefficient 0.998). The detection limit is 8.9×10^-8 mol·L^-1. The method is sensitive, simple and has been used to determine the protoporphyrin content of human urine sample while adding standard substance, the result shows satisfactory.