Spin-label studies of tropomyosin.
Spin-label studies of tropomyosin.
复制标题
原肌球蛋白的自旋标记研究。
作者：
Y. Chao;A. Holtzer
报道了氮氧自由基自旋标记原肌球蛋白的研究。标记物附着于巯基和氨基。氨基的自旋是高度移动的，巯基的则要少得多。自旋计数研究显示平均约0.5个标记的巯基/原肌球蛋白分子，仅约0.15个标记的氨基/分子。利用这些光谱研究了盐酸胍对原肌球蛋白的变性作用。所获得的信息揭示了巯基附近位点的变性过程。据发现，这些网站是更容易胍比大部分的分子，变性的巯基网站是由1.5 M胍完成，而光学研究表明，分子作为一个整体是不完全变性，直到浓度达到3.5 M。光谱也显示了不同方向的原肌球蛋白纤维相对于所施加的磁场。强烈的取向效应，这些表明，巯基连接的自旋（但不是氨基连接的自旋）有一个明确的方向在纤维中。光谱的解释表明，氮氧平面的法线与纤维轴成50度角倾斜。酪氨酸区域的圆二色性研究也揭示了胍变性的剧烈变化，证实了变性在β碳处产生显著增加的迁移率（如在巯基凯西中）的想法。在酪氨酸不带电荷的pH值下，仅存在于螺旋原肌球蛋白中的强负带似乎是由于酪氨酸与螺旋骨架的相互作用，而在高pH值（约11）下在250 nm处出现的强正CD带似乎归因于带电酚基与不对称骨架α-碳原子之间的相互作用。
Studies are reported on nitroxide spin-labeled tropomyosin. The labels attach to sulfhydryl groups and to amino groups. The amino spins are highly mobile, the sulfhydryl much less so. Spin count studies show an average of approximately 0.5 labeled sulfhydryl/tropomyosin molecule and only approximately 0.15 labeled amino group/molecule. The spectra are used tostudy the denaturation of tropomyosin by guanidine hydrochloride. The information obtained reveals the course of denaturation at sites near the sulfhydryl group. It is found that these sites are more susceptible to guanidine than the bulk of the molecule; denaturation at the sulfhydryl sites is complete by 1.5 M guanidine, whereas optical studies indicate the molecule as a whole is not completely denatured until the concentration reaches 3.5 M. Spectra are also shown of tropomyosin fibers oriented variously with respect to the applied magnetic field. Strong orientation effects are seen and these indicate that the sulfhydryl-attached spins (but not the amino-attached spins) have a definite orientation in the fiber. Interpretation of the spectra reveals that the normal to the nitroxide plane is inclined to the fiber axis at an angle of 50 degrees. Circular dichroism studies in the tyrosine region also reveal drastic changes with guanidine denaturation, confirming the idea that denaturation produces pronounced increase in mobility at the beta carbon (as in the sulfhydryl casey). A strong negative band existing only in helical tropomyosin at pH's where the tyrosines are uncharged appears to be due to interaction of tyrosines with the helical backbone, whereas the appearance of a strong positive CD band at 250 nm at high pH (approximately11) seems to be ascribable to interaction between the charged phenolic groups and the dissymmetric backbone alpha-carbon atom.