摘要
在酸性条件下用硫酸银断裂马心细胞色素c(以下简称cyt.c)的肽链与血红素相连的硫醚键,通过酸性丙酮抽提,硫基乙醇处理及超速离心等步骤纯化得去血红素的cyt.c(以下简称Apo-cyt.c)。Apo-cyt.c与天然cyt.c相比,其酸性电泳迁移率明显降低,紫外-可见光谱在190~220nm处吸收上升,荧光光谱的最大发射峰波长产生红移,同时CD谱中α螺旋的特征峰完全消失,这说明在cyt.c去血红素的过程中,蛋白质已由原来的紧密球状结构变成了较为松散、伸展的无规卷曲构象。因此,血红素对cyt.c天然构象的维持有着重要作用。
The two thioether bonds which link the heme moiety with polypeptide chain of horse heart cytochrome c were cleaved by reaction with silver sulfate in acidic solution. The pure apoprotein was obtained after being extracted with acid acetone,treated with 2-mercaptoethanol and ultracentrifuged. Compared with native cytochrome c, Apo-cyt.c moved slower in acidic electrophoresis. However, the absorbance in ultra-visible spectrum was raised between 190 nm and 220 nm, and the maximum emission wavelength exhibited an apparant red-shifted. In the CD spectrum,the specific absorbance of α-helix was disappeared completely. all these results suggested that the compact and nearly spherical comformatiom of native cytochrome c was distroyed to a randomly coiled comformation when the heme moiety was removed from the protein.Therefore,it can be concluded that the heme moiety is necessary for cyt. c to stabilize its native comformation.
基金
国家自然科学基金