Characterization of a tryptic fragment isolated from the insoluble tropomyosin of Pinna nobilis

Abstract The tryptic digestion of Pinna paramyosin yields, in addition to soluble products, a high-molecular-weight fragment which precipitates with 3% trichloroacetic acid. This non-dialysable fraction has been isolated by a combination of isoelectric precipitation and ammonium sulfate fractionation, and has been subjected to chemical and physico-chemical studies. The amino acid composition of the fragment is very similar to that of the native protein. Despite the fact that it still contains a high proportion of lysine and arginine, the fragment is resistant to the further action of trypsin (EC 3.4.4.4.), even when the molecule is initially exposed to a denaturing treatment. Unlike the native protein, the fragment possesses and N-terminal amino acid, glutamic acid. The physico-chemical properties of the tryptic resistant core have been established. The molecule is characterized by an s 20, w 0 of 2.88 and a (η) of 0.5 dl/g, as compared with corresponding values of 3.1 S. and 2.5 dl/g for the native molecule. The molecular weight of the fragment is 100000, by both light scattering and Archibald ultracentrifugation. The shape of the molecule, on the basis of viscosity and lightscattering dissymetry measurements, is best represented in terms of a coil model of length 525–550 A. Optical rotatory dispersion measurements suggest that the frament is about 38% helical, considerably less than the native molecule. Despite this lower helical content, the fragment is not attacked by trypsin, which may also be an indication of the globular character of the fragment as compared with the native protein which seems to be more exposed to enzyme action. This suggests that the tertiary structure must play a very important role in protecting the tryptic fragment from digestion.