Purification and properties of an acidic protein from chromaffin granules of bovine adrenal medulla

Abstract

A soluble protein has been purified from an aqueous extract of bovine adrenal chromaffin granules by chromatography on Sephadex G-200. This protein comprises 25 percent of the total protein of the granules and gave a single band on gel electrophoresis. The protein is unusually rich in acidic amino acids, notably glutamic acid (26. 0 percent, w/w); it is also relatively rich in proline (8. 6 percent, w/w) but poor in cystine (0. 35 percent, w/w). A molecular weight of 77000 was obtained from sedimentation and diffusion measurements on the protein, and approach-to-equilibrium measurements gave apparent molecular weights of the same order. A molecular weight 7 times that given above was estimated from the results of chromatography on a column of Sephadex G-200 that had been calibrated with globular proteins. However, good agreement between the ultracentrifuge and Sephadex experiments was obtained on the assumption that Sephadex chromatography depends on the effective hydrodynamic radii of proteins and not on their molecular weights. The hydrodynamic properties of the protein differed from those of a typical globular protein. Thus the protein had a high intrinsic viscosity, a high fractional ratio and a large effective hydro-dynamic volume. The hydrodynamic properties of the protein, but not its molecular weight, were dependent on the ionic strength of the solvent. Increasing the ionic strength caused an increase in the sedimentation and diffusion coefficients, but a decrease in the intrinsic viscosity and in the f rictional ratio of the protein. Optical-rotatory-dispersion measurements indicated that only a small part of the poly-peptide chain was in an [alpha]-helical conformation. These results are compatible with the protein''s having a conformation approaching that of a random-coil polypeptide, the volume occupied by the molecule being determined by electrostatic repulsion between the excess of negative charges.