Preparation and some properties of equine platelet tropomyosin

Two methods have been developed for the purification of equine platelet tropomyosin in amounts adequate for chemical and physical characterization. The first method, which avoids denaturing conditions, involves successive extractions with 0.10 M and 1.0 M NaCl buffer at pH 8.0, several isoelectric precipitations at pH 4.6, and ion exchange chromatography on DEAE-cellulose and hydroxylapatite. In the second method, tropomyosin is extracted at low pH, subjected to two isoelectric precipitations, a heating step to 85 degrees C, and chromatography on hydroxylapatite. The yields in the two methods were 20% and 30%, respectively, with a final purity of greater than 95% in both cases. No significant differences were observed in the properties of the products prepared by the two methods. The amounts of actin and tropomyosin as a percentage of the total protein of platelets were estimated to be about 16% and 1.6%, respectively, values which correspond to an approximate molar ratio of actin to tropomyosin of 13-14:1. Sodium dodecyl sulfate gel electrophoresis yielded a subunit Mr of 28,500 for the platelet tropomyosin. Two subunit forms of the protein, designated alpha and beta, in a molar ratio of 1:2, were observed on sodium dodecyl sulfate gel electrophoresis in the presence of 6 M urea, and evidence is presented for the presence of both alpha alpha and beta beta dimers. Circular dichroism, analytical ultracentrifugation, and paracrystal studies all indicate that, like muscle tropomyosin, the platelet protein exists as an asymmetric two-stranded coiled coil of alpha-helices.