Micro-Determination Of Protein Using Some New Polarographic Methods

Protein is a very important substance in life body. It participates in every reaction and activity of organism. The quantitative determination of protein is a considerable factor in biochemical and clinical assays. Over recent years, electrochemical methods have been widely used in bioanalytical chemistry. This thesis studies the determination of protein using some new polarographic mthods. The thesis is divided into five chapters.Chapter one summarizes some major quantitative analysis methods of proteins and hemin. Furthermore, this chapter also summarizes the theory and researches of polarographic adsorptive complex wave and parallel catalytic wave on the dropping mercury electrode.In chapter two, the determination of serum albumin is studied by polarographic adsorptive complex wave. In HAc-NaAc buffer solution at pH 6.3, Zn(II)- 2,2'-bipyridine complex produces an adsorptive wave at about–1.2 V(vs.SCE) by using single sweep polarography. Adding bovine serum album in (BSA )/human serum album in (HSA ) into the solution, it decreases the current of the adsorptive peak of Zn(II)- 2,2'-bipyridine complex. TheΔip'is linearly proportional to the protein concentration from 0.5 to 40 mg/L for BSA and from 0.5 to 50 mg/L for HSA. The detection limit is 0.2 mg/L for BSA and HSA. The method has been applied to the determination of total protein content in samples of human serum sample. The result is satisfactory.In chapter three, hemin is determined based on parallel catalytic wave of porphyrin ring of hemin. In an NH3-NH4Cl buffer solution at pH 9.5, hemin produced an insensitive reduction wave at about–0.70 V(vs.SCE) by using single sweep polarography. Adding 0.01mol/L K2S2O8 to the solution, this reduction wave was catalyzed, yielding a parallel catalytic wave. The derivative peak height is linearly proportional to the hemin in the range of 7.5×10-8 mol/L～4.5×10-6 mol/L,the detection limit is 5.0×10-8 mol/L. The formation's mechanism of polarographic parallel catalytic wave of hemin was discussed. Chapter four discusses polarographic catalytic prewave of ternary-complex of proteins and its sensitive determination. In Na3C6H5O7·2H2O-HCl buffer solution containing 2.5×10-3mol/L Co(II), 3.2×10-5mol/L 2,2'-bipyridine at pH 3.5, an extremely sensitive adsorptive wave of BSA at about–1.06V(vs.SCE) has been obtained by using single sweep polarography. The mechanism shows that proteins reacts with Co(II)- 2,2'-bipyridine complex forming a ternary-complex with a compostition of 1:1:1 and the adsorption efficiently accumulates the electrochemical active complex onto the DME. The polarographic catalytic prewave is due to the reduction of 2,2'-bipyridine in ternary-complex. At the optimal conditions, the derivative peak height is linearly proportional to the BSA or HSA concentration in the range of 0.005～16 mg/L. The detection limit for BSA or HSA is 0.002 mg /L. Most of common amino acids and metal ions have no interference with the protein determination. The new method could be useful in protein studies.In chapter five, in Na3C6H5O7·2H2O-HCl buffer solution at pH 3.5, Co(II)- Phenanthroline complex produce an adsorptive wave at about–1.10 V(vs.SCE) by using single sweep polarography. Adding BSA or HSA to the solution, an new extremely sensitive adsorptive wave is obtained at about–1.06V(vs.SCE) containing 2.0×10-4mol/LCo(II), 2.0×10-5mol/LPhenanthroline. At the optimal conditions, the derivative peak height is linearly proportional to BSA concentration in the range of 0.02～18 mg/L, to HSA 0.05～20 mg/L,. The detection limit for BSA is 0.01 mg /L and for HSA is 0.02 mg /L. The new method could be useful in protein studies.