The preparation of tropomyosin and troponin from natural actomyosin

Abstract

The procedure described by Bailey for the preparation of tropomyosin B has been modified to yield either a complex of tropomyosin and troponin or the individual proteins. This complex, the EGTA-sensitizing factor has been implicated in the relaxation mechanism of muscle. The starting material, in the modified procedure, was purified natural actomyosin which had been treated with ethanol and ether. Subsequent high ionic strength extraction of this material yielded the EGTA-sensitizing factor complex. Impurities were removed on ammonium sulfate fractionation, and the complex was isolated in the fraction precipitated between 40 to 60% ammonium sulfate saturation. Troponin was obtained as the isoelectric supernatant of the complex over the pH range of 3.5 to 4.6. Tropomyosin was isolated from the isoelectric precipitate by ammonium sulfate fractionation between the limits of 53 to 60% saturation. This separation depended critically on the protein concentration. In the sedimentation velocity analyses the separation was manifested by a transformation of the hypersharp boundary of the EGTA-sensitizing factor complex into two broader boundaries representative of tropomyosin and troponin. The separation was also monitored by the effect of relevant fractions on the ATPase activity of synthetic actomyosin.