CAPILLARY ISOELECTRIC-FOCUSING OF THE SCRAPIE PRION PROTEIN

Citation
Mj. Schmerr et al., CAPILLARY ISOELECTRIC-FOCUSING OF THE SCRAPIE PRION PROTEIN, Journal of chromatography, 802(1), 1998, pp. 135-141
Citations number
20
Categorie Soggetti
Chemistry Analytical","Biochemical Research Methods
Journal title
Volume
802
Issue
1
Year of publication
1998
Pages
135 - 141
Database
ISI
SICI code
Abstract
Prion diseases or transmissible spongiform encephalopathies belong to a group of neurodegenerative diseases that infect both animals and hum ans. These diseases are associated with an accumulation of fibrils in the brains of infected individuals. These fibrils are composed of an a bnormal isoform of a host-encoded glycoprotein that is characterized b y its insolubility and partial resistance to proteases. Another charac teristic of the scrapie prion protein (PrPsc) is the wide range of iso electric points (pI values) that have been observed on conventional is oelectrofocusing gels. In this study, we explored the use of capillary isoelectric focusing (cIEF) to characterize the pI values for PrPsc i solated from sheep and hamster brain, We used a Beckman 5500 P/ACE usi ng UV detection at 280 nm. A cIEF 3-10 Kit from Beckman Instruments wa s used to perform the analysis. The PrPsc was solubilized in 0.01 M Tr is-HCl, pH 8.00 containing 2 mM EDTA, 5% SDS and 10% hexafluoroisoprop anol at 100 degrees C for 10 min. The solubilized PrPsc was placed ove r a high-performance hydrophilic interaction column. After elution, th e peaks were concentrated and assayed for immunoreactivity with specif ic antisera. The peaks that contained immunoreactivity were then place d on the cIEF capillary. The samples containing PrPsc were solubilized in 1% n-octylglucoside before isoelectric focusing. The scrapie infec ted sheep sample had peaks with pI values ranging from 5.2 to 3.00 wit h a major peak at 3.09. The normal sheep blain had pI values that were higher. The hamster adapted scrapie strain had peaks with pI values r anging from 6.47 to 3.8. These pI values were slightly higher than tho se 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. (C) 1998 Elsevier Science B.V.