Harvey S C, Cheung H C
Biochemistry. 1977 Nov 29;16(24):5181-7. doi: 10.1021/bi00643a004.
The single photon counting method has been used to measure the decay of fluorescence polarization anisotropy of myosin rods labeled with extrinsic fluorophores. Rods labeled with 8-anilino-1-naphthalenesulfonate (ANS) or 5-dimethylaminoaphthalene-1-sulfonyl chloride (DNS-Cl) exhibit negative rotational correlation times; the anisotropy increases with time. Possible artifactual causes for the negative decay times are ruled out. It is shown that such curves are to be expected for rigid rods when the fluorophore is bound so that the absorption and emission dipoles each make a small angle with the long axis of the molecule and lie on opposite sides of the rod. At pH 4 and below, rapid decay of the anisotropy (positive correlation times) indicates the presence of a freely bending region in the rod. This is probably the proteolytically sensitive region between light meromyosin and heavy meromyosin subfragment 2. At pH 8, no such free bending is observed, even at temperatures as high as 50 degrees C. From this observation and other physical properties of the rod, we conclude that, at pH 8, the hinge region has considerable resistance to bending. It is more like a spring than a free hinge. The rotational diffusion about the rod axis is faster than would be predicted for a rigid, smooth molecule.
单光子计数法已被用于测量用外在荧光团标记的肌球蛋白杆的荧光偏振各向异性的衰减。用8-苯胺基-1-萘磺酸盐（ANS）或5-二甲基氨基萘-1-磺酰氯（DNS-Cl）标记的杆表现出负的旋转相关时间；各向异性随时间增加。排除了负衰减时间的可能人为原因。结果表明，当荧光团结合使得吸收和发射偶极子各自与分子的长轴成小角度并位于杆的相对侧时，对于刚性杆来说，这样的曲线是预期的。在pH 4及以下，各向异性的快速衰减（正相关时间）表明杆中存在自由弯曲区域。这可能是轻酶解肌球蛋白和重酶解肌球蛋白亚片段2之间的蛋白水解敏感区域。在pH 8时，即使在高达50摄氏度的温度下也未观察到这种自由弯曲。根据这一观察结果和杆的其他物理性质，我们得出结论，在pH 8时，铰链区域具有相当大的抗弯曲性。它更像一个弹簧而不是一个自由铰链。围绕杆轴的旋转扩散比刚性、光滑分子预期的要快。
