Homogeneity of myosin subfragments by equilibrium centrifugation

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Abstract
A number of enzymes are currently used to obtain proteolytic subfragment of rabbit skeletal muscle myosin. Subfragment-1 can be obtained by papain digestion of polymeric myosin in the presence (Mg .cntdot. S1) or absence (EDTA .cntdot. S1) of divalent cations. Subfragment-1 prepared by chymotrypsin is readily fractionated according to its alkali L chain content into S1 (A1) and S1 (A2). Digestion of soluble myosin by trypsin or chymotrypsin leads to heavy meromyosin (HMM) and light meromyosin (LMM). Many of these subfragments show extensive cleavages in the H and/or L chain region by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In view of the widespread use of proteolytic subfragments in kinetic and structural studies, it was of interest to establish the extent of heterogeneity of these preparations under nondenaturing conditions by equilibrium centrifugation. Analysis of the fringe displacements by computer programs showed that, for 3 initial loading concentrations, the MW averages Mn, Mw and Mz were superimposable across the entire solution column for all S1 and HMM species. The same applied for the initial MW averages of LMM and rod, except that with these highly asymmetric molecules a small drop in MW was observed toward the cell bottom as expected from excluded volume effects. The subfragments of myosin apparently are remarkably homogeneous in benign solvents, despite the existence of some cleavages in their primary structure.