| Instrumental analysis methods are widely used in such fields of biological science as pharmacology physiology, biochemistry. In this experiment, damages of erythrocyte membrane induced by bilirubin free radicals were studied by the means of electrophoretio technical, atomic electron spin spectrum, ultraviolet-visible spectrum , fluorescence- polarization method. People attach more and more importance to the relationship between free radical and biological medicine , which is relative to many illnesses and the causative factor of many diseases considering the fact that pigment gallstone is frequently with inflammation, the blockage of bile duct and the damages of liver, kidneys, stomach, intestines, et al., We did the following work to study the toxic mechanism of cells caused by bilirubin free radical and the role of the bilirubin free radicals in the formation of pigment gallstone: 1. The properties of the bilirubin which was acted upon by free radicals were studied by the means of electron spin resonance (ESR). 2. After the erythrocytes were acted upon by the bilirubin treated withdifferent free radical resources or the same free radical resource of difference densities, the damage of the membrane was observed by the means of SDS-PAGE .It was found that bilirubin free radicals caused the degradation of membrane bound proteins, the fig. of electrophoretic scans showed that the integral area(content) of belt 1 and belt 2 decreased relatively .while the integral area(content) of belt 3 increased relatively . It also showed that many belts of small molecules appeared and the degradation degree of the protein aggravated with the increase of the free radical density .The degradation of protein will affect the erythrocytes acted upon framework of the cell, which makes the cell incapable of maintaining its normal physiological functions. 3. Lipid peroxidation of intact human erythrocytes acted upon by free radicals were studied with TBA method. The result indicated bilirubin free radicals can cause the lipid peroxidation of human erythrocytes and the degree of lipid peroxidation increases as the density of free radical increases. 4. The effect of bilirubin free radicals on the fluidity of human erythrocyte membrane was measured by the means of fluorescence-polarization method. The result indicated that after human erythrocyte membrane was acted upon by bilirubin free radicals, the fluorescence polarization degree (P) of human erythrocyte membrane decreased[BRco60(0. 1887±0. 0312) vs. the control group ( 0. 2932 ± 0. 0095), P<0.01], and the microviscosity ( n) decreased [BRco6 (1. 429±0. 4065) us. the control group (3.533 ± 0.3206), P<0.01]. The membrane fluidity increased as the density of free radicals inscreased.5. Flurescamine method was used to measure the content of free amino acid and the content of membrane bound primary amine in erythrocyte membrane solution. The result showed the decrease of that the amino-group in membrane bound proteins treated with bilirubin free radicals and the increase of free amino acid contentin the relevant solution.The above experiments indicate when active-oxygen free radicals produced either from 60Co or from FeS04+EDTA acted with solid bilirubin, ESR signals appeared (g=2.00375) and kept stable for a long time. In fact, the bilirubin solid on the market has the same table ESR signals which can weakened by 94% with the free radicals acted upon by Vc and mamital. This indicated that bilirubin could form free radicals after it was treated with active-oxygen free radicals under oxygen circumstances. The bilirubin free radicals can cause damages on human erythrocyte membrane. Membrane is a floating structure made up of phospholipids bilayer and inlaid proteins. This floating structure is very essential for maintaining the structure and functions of a cell, such as delivering supplies, cellular recognition, differentiation and immunization,ect. Bilirubin free radicals can degrade the proteins in membrane . This change affects the normal physiologi... |
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