Immobilized ATP and actin columns as a tool for the characterization and separation of different myosins and active myosin fragments.

A comparative affinity chromatography study using agarose-ATP columns revealed differences between heavy meromyosin subfragment 1 (HMM S-1) preparations obtained from rabbit white skeletal, rabbit red skeletal, bovine cardiac, and chicken gizzard muscle myosins. The characteristic patterns were markedly affected by Ca2+ and Mg2+ ions in a manner typical for each myosin species. Similar differences were also observed on comparing the intact myosins of red and white muscles. It thus became possible to separate, at least partially, mixtures of myosin (or HMM S-1) of different origins. Muscle "acetone-dried powder" was used as chromatographic medium for active myosin fragments in affinity chromatography columns. At low ionic strength the columns bound appreciable amounts of heavy meromyosin (HMM) and of HMM S-1. Binding was reversible and the myosin fragments could be eluted by ATP or magnesium pyrophosphate. The absorption peak of bovine cardiac HMM S-1 was found to be less symmetrical than that of the rabbit white skeletal analog. Chemical modification by trinitrophenylation of white skeletal HMM S-1 was found to affect the affinity of binding and the shape of the absorption peak, thus enabling a partial separation of trinitrophenylated fragment from the unmodified protein using an actin column. The resolving power of ATP columns for the separation of red and white skeletal myosins was increased after trintrophenylation of the proteins.