High level of heterogeneity seems to be a ubiquitous feature of mammalian PrPs (prion proteins) and may be relevant to the pathogenesis of prion diseases. In the present study, we describe the heterogeneity of PrPC (cellular form of PrP) from porcine brain. It was disclosed and characterized by a combination of one-dimensional PAGE and two-dimensional PAGE analyses with enzymic deglycosylation and copper-affinity experiments. We found that the identified two main populations of porcine PrPC consist of diglycosylated forms and correspond to the full-length (molecular mass 32–36 kDa) and proteolytically modified protein (molecular mass 25–30 kDa), known as C1. The two populations were fully separated during Cu2+-loaded immobilized metal affinity chromatography, indicating different affinity for copper ions. The more basic forms, migrating as species of higher molecular mass, exhibited stronger affinity for copper ions, whereas those with more acidic pI and of lower molecular mass were low-affinity Cu2+-binding molecules and thus could represent N-terminally truncated PrPC. Size-exclusion chromatography revealed that most of the PrPC molecules in porcine brain extracts exist in the form of high-molecular-mass complexes (probably with other proteins). The heterogeneity of porcine PrPC, resulting from proteolytic modification and glycosylation, influences its ability to assemble into these complexes. N-truncated molecules dominate over full-length PrPC in fractions of molecular mass over the range 65–130 kDa, whereas the full-length species are the major forms of PrPC present in the monomeric fraction and in complexes above 130 kDa. Two-dimensional PAGE analysis indicated that the complexed PrPC differs in the composition of pI forms from the monomers.

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