不同处理条件对乳铁蛋白构象的影响研究

运用荧光、圆二色谱及紫外光谱手段,研究了六种不同物理或化学条件处理后,牛乳铁蛋白(Bovine lactoferrin,bLF)三级、二级结构及二硫键的变化。荧光结果显示:6mol.L-1盐酸胍、8mol.L-1尿素和50mmol.L-1二硫苏糖醇三种处理后bLF溶液的最大发射波长从333nm红移至354nm,疏水基团大量暴露,三级结构发生明显变化;100℃加热5min、超声(450W,5s,6个脉冲)、1%巯基乙醇处理后,bLF溶液荧光强度明显减弱,最大发射波长几乎无变化。圆二色谱结果表明:经盐酸胍处理,bLF中α-螺旋结构消失,其余五种处理,二级结构的变化较小。紫外光谱数据表明:二硫苏糖醇对bLF二硫键破坏最严重,超过总二硫键含量的55%,超声次之,盐酸胍、巯基乙醇和加热破坏较少。结果对进一步明确乳铁蛋白的构效关系提供了一定的依据。

A comparison of different treatment conditions on the conformation changes of bovine lactoferrin

In this study, the tertiary, secondary structures and disulfide bond changes of bovine lactoferrin (bLF) under 6 differents physico-chemical treatments were investigated by fluorescence, circular dichroism(CD) and UV-Vis absorption. A red shift from 333 to 354 nm in the fluorescence emission maximum (lambda(max)) was observed in the bLF treated by 6 mol x L(-1) GdnHCl, 8 mol x L(-1) Urea and 50 mmol x L(-1) DTT simultaneously, meanwhile a large number of exposed hydrophobic groups were detected. However, there was no marked shift in lambda(max) of bLF treated by heating (100 degrees C, 5 min), Ultrosonic(450 W, 5 s, 6 pulses) or beta-ME (1%), of which fluorescence intensity decreased significantly compared with the untreated bLF. The results indicated that the mechanism of changes in tertiary structure of the former three methods were different from the latter three. The detection by CD showed that the alpha- helix structure vanished completely in the bLF treated by GdnHCl. However, there was no remarkable change in the secondary structure of the bLF treated by the other five methods. In addition, UV-Vis absorption suggested that disulfide bond was seriously destructed in the bLF treated by DTT and Ultrosonict, but GdnHCl, beta-ME and heating induced a little damage merely. This study is instructive and meaningful to the further research on relationship between structure and activity of bLF.