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.
Harvey et al. (1977) studied this question.