Magnetic Proteinase K Reactor as a New Tool for Reproducible Limited Protein Digestion Marcela Slováková,* Jean-Michel Peyrin, † Zuzana Bílková, Martina Juklíc ˇková, Lenka Hernychová, and Jean-Louis Viovy Laboratoire Physicochimie-Curie, Institut Curie, Paris Cedex 5, France, Unité de Virologie Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France, Department of Biological and Biochemical Sciences, University of Pardubice, 53210 Pardubice, Czech Republic, and Institute of Molecular Pathology, University of Defence, Hradec Králové, Czech Republic. Received December 3, 2007; Revised Manuscript Received January 31, 2008 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  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. INTRODUCTION In general, proteolytic enzymes covalently bonded on solid carriers have a wide range of practical applications. Although their hydrolytic activity usually decreases slightly upon im- mobilization, they possess important advantages over dissolved enzymes, e.g., the possibility of recovery and reuse, simple operation, enhanced stability, and limited protease autolysis. The activity and stability of such biocatalysts after their immobiliza- tion depends on the type of carrier (biopolymer or synthetic, magnetic, size, porosity, etc.), the enzyme, and the immobiliza- tion method (1, 2). The choice of matrix is a key factor in the operating range of the final application and degree of process automation (3, 4). The magnetic form of the particles provide the best simple, reproducible, and economic approaches, which enable a highly efficient and gentle separation, during either an immobilization procedure or biochemical applications (2), e.g., on-chip technology (3, 4). Due to their small size (up to 1 µm in mean diameter), they offer a very large specific surface area. The specific surface area and size of the polymeric particles used in this research work (Ademtech, France) were within narrowly defined ranges. The composition of the particles is stable in solutions of widely ranging pH and at moderate salt concentra- tions. The surface charge density is negative at slightly basic pH, according to the manufacturer. Proteinase K (PK) is a subtilisin family serine protease isolated from Tritirachium album Limber. This highly active extracellular alkaline serine endopeptidase was so named in the past because of its ability to digest native keratin (5–7). PK has an immense range of applications in pure and applied research. One of the fundamental fields of application of PK is its use together with a prion protein immunoblot for the detection of the pathological form of prion protein in various fluids and tissues. Other important applications of PK are in the scientific and biomedical fields: the removal of biomolecules, e.g., inhibitors, DNA molecules, highly resistant proteins, and glycoproteins, and so forth, from biological materials (8–10). The main characteristics of PK have already been determined: molecular mass 28 930 (11), isoelectric point 8.9, optimal pH range for enzyme activity from 7.5 to 12 (5). Calcium ions are involved in this enzyme’s activity, and additional calcium ions in solution are also required for the stability of the enzyme (11). This proteinase has unusual stability at low concentrations of SDS and urea. PK is primarily specific against aromatic or hydrophobic amino acid residues on the carboxyl side of the splitting point (12). In this paper, we tested new magnetic carriers for utiliza- tion in a proteinase K reactor: 500A5 and 145TTa magnetic beads (styrenic copolymer with hydrophilic monomer, Ademtech, France), A120 magnetic alginate microparticles (Academy of Sciences, Czech Republic), Estapor magnetic latex microparticles (Merck, France). Along with optimiza- tions of the grafting conditions, i.e., temperature, time, type and strength of buffer, the characterization of the enzyme reactor was a further aim of this work. The stability of the PK reactor was investigated in terms of multiple uses. The  potential and apparent Michaelis–Menten constant of the magnetic PK reactor were determined. The newly prepared enzyme reactor was used for the proteolytic digestion of human growth hormone (HGH) (22 kDa) and PrP c from the Mov mouse cell line. Mass spectrometry and * Corresponding author. University of Pardubice, Department of Biological and Biochemical Sciences, Strossova 239, 53003 Pardubice, Czech Republic, Phone number +420 466 037 721. E-mail address Marcela.Slovakova@upce.cz. † Present address: CNRS, Neurobiologie des Processus Adaptatifs, UMR7102 Boite 12, 9 quai saint Bernard, 75005 Paris. Bioconjugate Chem. 2008, 19, 966–972 966 10.1021/bc7004413 CCC: $40.75  2008 American Chemical Society Published on Web 03/13/2008