Birefringence as a probe of crossbridge orientation in demembranated muscle fibres.

Birefringence measurements were used to investigate crossbridge orientation in demembranated muscle fibres of frog and rabbit. Birefringence depends on the interfilament spacing as well as on crossbridge orientation, so conditions were chosen such that changes in interfilament spacing were either eliminated or known from previous studies. At sarcomere length 2.3 micron there was a large birefringence decrease on putting relaxed fibres into rigor; at 3.7 micron this gave no change in birefringence. A simple model for crossbridge structure was used to interpret the birefringence data; it seems likely that reorientation of subfragment-1 (S-1) is responsible for the observed change. Decoration of rigor fibres with exogenous S-1 gave a birefringence increase corresponding to S-1 binding with its long axis at about 50 degrees to the fibre axis. The corresponding mean S-1 angle in relaxed muscle was estimated as about 35 degrees. When relaxation of rigor fibres was initiated by photolysis of caged-ATP in the absence of Ca2+ the birefringence increase showed two components, one 20 times faster than the other. The fast component was accompanied by a decrease of rapid stiffness, suggesting that it is caused by some crossbridges detaching to take up an orientation more parallel with the fibre axis. However the mechanical measurements indicated the presence of some active force generating crossbridges at this time, even in the absence of Ca2+, and these may also make a contribution to the fast birefringence component. Birefringence transients following ATP release in the presence of Ca2+ suggest that crossbridges are on average more perpendicular to the fibre axis during active force generation than in the rigor state.