Partial Purification and Evidence for Multiple Molecular Forms of the Scrapie Agent

A procedure for the partial purification of the scrapie agent from mouse spleen was developed based on its sedimentation profile. Differential centrifugation and deter- gent treatment with sodium deoxycholate yielded a fraction designated "Pj" which was enriched for scrapie infectivity -20-fold with respect to cellular protein. The P5 fraction was devoid of cellular membranes but heavily contaminated with ribosomes as judged by electron microscopy. On centrifugation of the fraction Pj to near equilibrium in a sucrose gradient scrapie infectivity was distributed over a range of densities from Because the assay for the scrapie agent is so unusually slow, development of a rational strategy for purifying the agent seemed of utmost importance. Although the choice of purifi- cation techniques is empirical, differential centrifugation is generally found to be quite effective in the initial stages of isolating most biological macromolecular structures. Thus, preparatory to developing a purification format utilizing dif- ferential centrifugation, we determined the sedimentation characteristics of the scrapie agent in fixed-angle rotors as described in the preceding communication (Prusiner et al., 1977; 1978). From these observations a scheme for partially purifying the scrapie agent from spleen was developed using differential centrifugation in combination with detergent treatment. The purification procedure yields approximately 20-fold enrichment of the agent with respect to total cellular protein. As predicted from the sedimentation profile data, the partially purified preparations of scrapie agent contained many ribosomal structures. Electron microscopy of the preparations confirmed the presence of ribosomes and the virtual absence of organized membranous structures. Sucrose gradient centrifugation was employed to separate the scrapie agent from contaminating ribosomes. After partial purification and storage at -70 OC, the agent exhibited het- erogeneity with respect to both size and density. Heat treat- ment at 37 or 80 "C increased the apparent size of the agent suggesting aggregation. The data are consistent with the possibility that the scrapie agent contains nonpolar regions or hydrophobic patches on its surface. Indeed, the physical be- havior of the scrapie agent appears similar to that observed for