Magnetic proteinase K reactor as a new tool for reproducible limited protein digestion.
As an aid to differentiating between the prion proteins Prp(c) and PrP(Sc), the preparation and use of immobilized Proteinase K (PK) is described. An accumulation of PrP(Sc) in the central nervous system is the one of the causes of neurodegenerative disease. Current routine diagnosis is based on the postmortem detection of the distinct neuropathological lesion profiles of CNS and by the presence of the PK-resistant core of the prion protein isolated from brain lysates. An assay with PK immobilized to magnetic -COOH micro- and nanoparticles can offer a convenient as well as economic method. The individual immobilization steps were verified by measuring the zeta potential of the particles. The stability of the newly developed PK magnetic reactor, observed during kinetics measurements, was highly satisfactory. The calculated values of the apparent Michaelis constant (4.25 mM for native enzyme and 1.28 mM for immobilized enzyme) were determined from Lineweaver-Burk plots. Human growth hormone was digested using the newly prepared magnetic PK reactor and MALDI-TOF-MS analysis of the digests showed satisfactory efficiency. Controlled digestion of PrP(c) from the Mov mouse cell line was demonstrated with Western blot detection.
Slováková
Marcela
M
Laboratoire Physicochimie-Curie, Institut Curie, Paris Cedex 5, France. Marcela.Slovakova@upce.cz
Peyrin
Jean-Michel
JM
Bílková
Zuzana
Z
Juklícková
Martina
M
Hernychová
Lenka
L
Viovy
Jean-Louis
JL
D000818:Animals; D001203:Ascomycota / Q000201:enzymology; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D019382:Human Growth Hormone / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008280:Magnetics; D051379:Mice; D011328:Prions / Q000378:metabolism; D015203:Reproducibility of Results
D004800:Enzymes, Immobilized; D011328:Prions; D019382:Human Growth Hormone; D019286:Endopeptidase K