Reinvestigation of some physicochemical and chemical properties of human ceruloplasmin (ferroxidase).

Abstract

The original molecular weight determinations and hydrodynamic data on ceruloplasmin have later been contradicted by crystallographic and other investigations. In order to arrive at the correct copper stoichiometry of the protein we have undertaken a careful reinvestigation of some aspects of the physical chemistry and chemistry of ceruloplasmin with particular attention directed towards molecular weight and copper content. Human ceruloplasmin form I (the major form) was isoalted from fresh normal or retroplacental serum by a procedure designed to avoid proteolysis. The molecular weight of the protein, determined by meniscus depletion sedimentation equilibrium, was found to be 134 000+/-3 000. The sedimentation coefficient (7.25 S) and diffusion coefficient (4.46 10(-7) cm2/s) are consistent with this molecular weight. The frictional ratio calculated from these data, 1.41, and the observed intrinsic viscosity of 4.5 ml/g indicate that ceruloplasmin has a slightly more extended shape than a typical globular protein. This might at least partly be ascribed to its carbohydrate moiety. The amino acid composition, carbohydrate composition, and copper content of ceruloplasmin were determined. The copper content of several preparations varied between 6.0 and 6.6 atoms per mol. The copper stoichiometry of ceruloplasmin is discussed.