Capillary isoelectric focusing of the scrapie prion protein.
Prion diseases or transmissible spongiform encephalopathies belong to a group of neurodegenerative diseases that infect both animals and humans. These diseases are associated with an accumulation of fibrils in the brains of infected individuals. These fibrils are composed of an abnormal isoform of a host-encoded glycoprotein that is characterized by its insolubility and partial resistance to proteases. Another characteristic of the scrapie prion protein (PrPsc) is the wide range of isoelectric points (pI values) that have been observed on conventional isoelectrofocusing gels. In this study, we explored the use of capillary isoelectric focusing (cIEF) to characterize the pI values for PrPsc isolated from sheep and hamster brain. We used a Beckman 5500 P/ACE using UV detection at 280 nm. A cIEF 3-10 Kit from Beckman Instruments was used to perform the analysis. The PrPsc was solubilized in 0.01 M Tris-HCl, pH 8.00 containing 2 mM EDTA. 5% SDS and 10% hexafluoroisopropanol at 100 degrees C for 10 min. The solubilized PrPsc was placed over a high-performance hydrophilic interaction column. After elution, the peaks were concentrated and assayed for immunoreactivity with specific antisera. The peaks that contained immunoreactivity were then placed on the cIEF capillary. The samples containing PrPsc were solubilized in 1% n-octylglucoside before isoelectric focusing. The scrapie infected sheep sample had peaks with pI values ranging from 5.2 to 3.00 with a major peak at 3.09. The normal sheep brain had pI values that were higher. The hamster adapted scrapie strain had peaks with pI values ranging from 6.47 to 3.8. These pI values were slightly higher than those obtained for the sheep samples. The use of cIEF to determine the pI values of PrPsc led to the identification of a major species of PrPsc from sheep with a very acidic pI.
Schmerr
M J
MJ
US Department of Agriculture, National Animal Disease Center, Ames, IA 50010, USA.
Cutlip
R C
RC
Jenny
A
A
D000818:Animals; D001923:Brain Chemistry; D002138:Calibration; D006224:Cricetinae; D019075:Electrophoresis, Capillary; D007525:Isoelectric Focusing / Q000379:methods*; D018620:PrPSc Proteins / Q000032:analysis*; D012608:Scrapie / Q000473:pathology*; D012756:Sheep
D018620:PrPSc Proteins