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本文利用荧光探剂ANS研究了大鼠肝微粒体膜脂流动性和表面负电荷密度的测定方法。所得结果表明:(1)在测定ANS荧光强度和荧光偏振度时,选用的最佳实验条件是:膜蛋白浓度为50μg/ml;在37℃下温育;膜与ANS温育15分钟;ANS在0～4℃下保存两周之内仍可考虑使用。(2)测定膜表面负电荷密度时,ANS滴定的浓度范围为6.25～100μmol/L。(3)测定ANS在膜中的相对荧光量子产率时,选用的膜蛋白浓度范围为12.5～200μg/ml。此外,实验还发现,向肝微粒体膜悬液中加入Cd~(2+)后能明显增加膜与ANS的结合,并能降低膜脂流动性和膜表面负电荷密度。
In this paper, the method of determination of the fluidity and surface negative charge density of rat liver microsomes was studied by using fluorescent probe ANS. The results showed as follows: (1) The optimal experimental conditions for determining the fluorescence intensity and fluorescence polarization of ANS were as follows: membrane protein concentration 50μg / ml; incubation at 37 ℃; membrane incubation with ANS for 15 minutes; ANS Can be considered for use within two weeks of storage at 0-4 ° C. (2) When the negative charge density of the membrane surface was measured, the concentration of ANS titration ranged from 6.25 to 100μmol / L. (3) Determination of the relative fluorescence quantum yield of ANS in the membrane, the concentration of the selected membrane protein ranged from 12.5 to 200 μg / ml. In addition, the experiment also found that the addition of Cd 2+ to the suspension of liver microsome could significantly increase the binding of membrane to ANS and decrease the fluidity of membrane lipid and the negative charge density of membrane surface.