Equilibrium density gradient centrifugation of the scrapie agent in Nycodenz

Plasma membrane-enriched preparations from scrapie-infected and healthy hamster brains were detergent-extracted, then separated by equilibrium density centrifugation in continuous Nycodenz gradients. The highest level of infectivity was always associated with the insoluble residue which sedimented through 40% Nycodenz. The degree of aggregation in these insoluble complexes varied depending upon treatment. Centrifugation in gradients containing 2 M- to 8 M-urea resulted in the formation of large insoluble aggregates which seemed to retain a high level of infectivity when measured by the method of incubation interval assay. However, measurement of infectivity in these same samples by endpoint titration of tenfold dilutions resulted in values a thousand times lower. These observations reinforce previous findings that scrapie infectivity exists as a macromolecular complex and, furthermore, they emphasize the necessity for using non-denaturing conditions for purification of the scrapie agent.