Isolation and partial renaturation of proteolytic fragments of the myosin head

Methods have been devised for isolating two of the tryptic fragments (those termed '20K' and '50K') of myosin 'subfragment 1' in pure form. Fragment 20K was examined for renaturation after removal of denaturants used in its preparation. It generated a CD spectrum corresponding to ca. 64% formed structure (roughly what would be expected from its amino acid sequence) and a red-shifted UV spectrum such as arises when phenylalanine and tyrosine are perturbed by structural interactions. Actin affinity of fragment 20K was tested by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide cross-linking, inhibition of the actin-activated ATPase of subfragment 1 containing light chain 3, cosedimentation with actin, and light scattering; the affinity exceeded 5 x 10⁶ M^-1. The foregoing suggests that moeity 20K has a sovereign existence in (i.e., is a domain of) myosin subfragment 1. Preliminary work indicates that fragment 50K also binds actin, but with a lesser affinity.