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Magnetic Proteinase K Reactor as a New Tool for Reproducible Limited Protein Digestion Marcela Slováková,* Jean-Michel Peyrin,† Zuzana Bílková, Martina Juklícˇková, 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 Prpc and PrPSc, the preparation and use of immobilized Proteinase K (PK) is described. An accumulation of PrPSc 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 ζ 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 PrPc 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 immobilization, 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 immobilization depends on the type of carrier (biopolymer or synthetic, magnetic, size, porosity, etc.), the enzyme, and the immobilization 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 concentrations. 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 * 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
[email protected]. † Present address: CNRS, Neurobiologie des Processus Adaptatifs, UMR7102 Boite 12, 9 quai saint Bernard, 75005 Paris.
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 utilization 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 optimizations 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 ζ 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 PrPc from the Mov mouse cell line. Mass spectrometry and
10.1021/bc7004413 CCC: $40.75 2008 American Chemical Society Published on Web 03/13/2008
Magnetic Proteinase K Reactor for Protein Digestion
Western blotting analysis were used to confirm digestion by the newly prepared enzyme reactor.
EXPERIMENTAL PROCEDURES Chemicals. Proteinase K (E.C.3.4.21.64, lot 104K8622, 33 U/mg of solid protein), succinyl-Ala-Ala-Ala-p-nitroanilide (EC 257-823-5; substrate for PK), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC), sodium azide, and the surfactants polyoxyethylsorbitane monolaurate (Tween 20), Triton X-405, and Triton X-100 were purchased from SigmaAldrich. The surfactant Pluronics F127 NF (PF 127) was purchased from the BASF Corporation (New Jersey, USA). The sodium salt of N-hydroxysulfosuccinimide and trifluoric acid were obtained from Fluka (Buchs, Switzerland). A Micro BCA Protein Assay Kit (Perbio Science, Pierce France) was used for complete protein determination. All other reagents, of analytical or electrophoresis quality, were obtained from international suppliers (Merck, Darmstadt, Germany; Bio/Rad, Hercules, CA, USA; Sigma, St. Louis, MO, USA). The mouse overexpressed PrPc cells (Mov cell line) were lysated in a lysis buffer (0.5% sodium deoxycholate, 0.5% Triton X100, 50 mM Tris-HCl pH 7.4). The PK immobilized to Eupergit C was purchased from Sigma-Aldrich (USA). Magnetic Carriers. The 500A5 (626 nm mean diameter) and 145TTa (980 nm mean diameter) magnetic latex beads used in this study were donated by Ademtech SA (Pessac, France), both functionalized with -COOH groups. The A120 magnetic alginate microparticles (-COOH) were donated by the Academy of Sciences (Czech Republic) (2–5 µm diameter). Magnetic latex microparticles (-COOH) were purchased from Estapor (Merck, France) (160 nm mean diameter). All stock solutions were stored in an aqueous suspension containing 0.09% NaN3. Carriers were washed extensively with the enzyme buffer (10 mM phosphate buffer pH 7.3) before each use. Equipment. A magnetic separator (Dynal, USA) was used for manipulations with the magnetic carriers. All microscopic observations were made using an inverted microscope (Zeiss Axiovert 100) with 10×, 5× (Zeiss), and 100× oil immersion (Olympus) objectives, and an intensified CCD camera (Lhesa, Cergy Pontoise, France). Spectrophotometers (Shimadzu Corporation, Japan, and NanoDrop, Nyxor Biotech, France) were used for measuring the optical density for catalytic activity quantification. A Voyager-DE STR MALDI-TOF spectrophotometer (Applied Biosystems, Germany) was utilized for mass spectrometry analysis. Immobilization of PK (PK) on Magnetic Particles. Generally, 1 mg of magnetic particles (functionalized by –COOH) was washed with 10 mM of phosphate buffer (pH 7.3). Carboxyl groups were activated via the method developed by Staros et al. (13) using water-soluble carbodiimide EDAC [1-ethyl-3(3dimethylaminopropyl) carbodiimide] at a concentration of 79 mM. Addition of N-hydroxysulfosuccinimide [sulfo-NHS], at a concentration of 12 mM, enhances the yield of amide bond formation (14). The chosen amount (from 1 to 4 mg) of PK was dissolved in 10 mM phosphate buffer (pH 7.3) with 5 mM CaCl2; the total concentration of Ca2+ ions in the mixture was 1 mM according to (11). The immobilization time depends on the chosen temperature: at room temperature, the grafting reaction was maintained for 6 h; at 4 °C the reaction was maintained overnight. Blocking the unreacted activated sites on the particle surface was done with 100 mM Tris, 100 mM MES, and 52 mM EDAC at room temperature for 2 h. The PK reactor was washed and stored in 50 mM Tris/HCl buffer (pH 7.8) with 5 mM CaCl2 and the addition of 0.02% sodium azide. Immobilized PK Activity Assay. The specific enzyme activity was assayed using succinyl-Ala3-p-nitroanilide as a substrate (11, 15). 1 I.U. of the activity is expressed as the
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amount of enzyme which can hydrolyze casein to produce the color equivalent to 1.0 µmol of tyrosine per minute at pH 7.5 and 37 °C (colored by the Folin-Cocteau reagent). The assays were routinely carried out at a substrate concentration of 1.05 mM and in 50 mM Tris/HCl buffer (pH 7.8) with 5 mM CaCl2 (total volume 1 mL). Assay mixtures were composed of 50 mM Tris/HCl buffer (pH 8.0) CaCl2 (5 mM), N-succinyl-Ala3-pnitroanilide (1.1 mM), and PK (immobilized or 2–10 µg/mL). The mixture was incubated for 15 min at room temperature. The reaction was terminated by adding 0.2 mL glacial acetic acid for the native enzyme or by the separation of the PK particles with a magnetic separator (Dynal, USA), and the optical density at 405 nm of free pNA was measured against the substrate solution as a reference. The final PK activity was estimated from the linear increase in optical density. Except for steps investigating thermal stability, all experiments were carried out at 25 °C. Measurement of ζ Potential. A Zetasizer (Malvern Instrument, U.K.) was used in the ζ measurememt protocols. The ζ potential of the beads was measured in a phosphate buffer of strengths 10 and 100 mM and with pHs ranging from 3 to 11; the pH was adjusted with HCl and NaOH. The total volume for measurements was 1 mL, and the concentration of the beads was 3 × 10-3%. Determination of Apparent KM and Vmax for PK. The Michaelis–Menten kinetic parameters KM and Vmax for the hydrolytic reactions between succinyl-Ala-Ala-Ala-p-nitroanilide and PK were determined from Lineweaver–Burk (L-B) plots. All kinetic experiments were carried out in 50 mM Tris/HCl buffer (pH 7.8) at 25 °C (tempered solution). The concentration range of the substrate N-succinyl-Ala3-p-nitroanilide was (0.275-4.8) × 10-3 mol/L. Proteolysis of Proteins by Immobilized PK. The protein (human growth hormone, molecular size 22 kDa) was dissolved in 50 mM ammonium acetate (pH 7.8) and digested with soluble PK or an immobilized PK reactor in a microtube. Procedure: 100 µL of protein (1 mg/mL) was added to the appropriate amount of PK reactor (E:S 1:16), and digested over 4 h at room temperature with slow rotation. Mass Spectrometry Analysis of Digested Protein by MALDI-TOF-MS. The digest of HGH was desalted using RP ZipTipC18 (Millipore). 1 µL of each sample was applied to a MALDI plate and covered with 1 µL of 2,5-dihydroxybenzoic acid (DHB) (LaserBio Laboratories) as the matrix. For the matrix solution, DHB was dissolved in a solution containing 50% ACN and 0.3% trifluoracetic acid (Fluka Biochemika) to a DHB concentration of 50 mg/mL. The Voyager-DE STR MALDI-TOF mass spectrometer (Applied Biosystems) was used in all experiments. The MALDI-TOF measurements were carried out in positive ion reflectron mode with a mass range of m/z 110-2500. The list of detected monoisotopic masses (m/z) was used for protein identification with the PeptideMass tool of the ExPASy proteomic server (http://www.expasy. org/). Digestion of Cell Lysate Overexpressing PrPc. Mov cells are immortalized neuroglial cells isolated from mice expressing the ovine gene for PrPc. Cells were grown as in (16). Once confluency was reached, cells were lysed in buffer (0.1% Triton X100, 0.1% sodium deoxycholate, 50 mM Tris, pH 7.2). Cell lysates were centrifuged for 2 min at 1000 g to remove cell debris, and stored at -20 °C until used. Cell lysates were then incubated with the proteinase K reactor for 2, 5, 10, 30, and 60 min at RT (0.7 µg of PK reactor for 100 µL of protein). Pefabloc SC (0.4 mM) was then added to stop the reaction, and proteins were precipitated with 4 volumes of cold methanol. Western Blotting. Pellets were resuspended in sample buffer (50 mM Tris/HCl pH 7.8), boiled, subjected to 10% SDS/PAGE
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Table 1. Influence of Temperature on Soluble PK assay with the Substrate N-Succinyl-Ala-Ala-Ala-p-NA (1.05 mM in volume 1 mL), Measured in 50 mM Tris/HCl, pH 7.8 temperature [°C]
PK amount [µg/mL]
25 37 50
4.84 5.16 4.88
Table 4. Monitoring Effects of Temperature and Reaction Time on Grafting Efficiency of PK to 500A5 (Ademtech)a temperature
reaction time
amount of PK [µg/mg carrier]
4 °C
overnight 6h overnight 6h
14.8 5.5 1.1 10.1
RT
a Immobilization occurred in 10 mM sodium phosphate buffer, pH 7.3, 6 h at RT, slow rotation.
Table 2. Influence of Buffering System on Soluble PK Assay with the Substrate N-Succinyl-Ala-Ala-Ala-p-NA (1.05 mM in volume 1 mL) buffering solution
PK amount [µg/mL]
50 mM MES pH 6 10 mM sodium phosphate pH 7.3 50 mM TRIS/HCl pH 7.8
1.83 1.9 3.9
Table 3. Immobilization of PK to Microparticles 500A5 (Ademtech) in Various Buffering Solutionsa cross-linker
buffer, pH
EDAC, S-NHS 10 mM phosphate, pH 7.3 EDAC, S-NHS 50 mM MES, pH 6 EDAC, S-NHS 100 mM TRIS/HCl, pH 7.8
conditions
amount of PK [µg/mg carrier]
p6h,RT 6 h,RT 6 h,RT
9.92 4.81 3.29
a 10 mM sodium phosphate buffer at pH 7.3, 50 mM MES solution at pH 6.0, and 100 mM TRIS/HCl at pH 7.8, in the presence of 1 mM CaCl2, 6 h at RT, slow rotation.
electrophoresis, and electrotransferred (1 h) onto nitrocellulose membranes. PrPc was visualized with either ICSM18 or SAF 84.3 mAb overnight at 4 °C; both directed to the C-terminal half-of PrPc. Western blots were developed by AgNO3 (Amersham Pharmacia) in a darkroom.
RESULTS Proteinase K Activity Assay. With regard to the proteinase K activity assay, we focused on common conditions for assays of the soluble enzyme to verify that these conditions are usable for the immobilized enzyme. Further, we wanted to verify that there is no change in the basic kinetic parameters of the enzyme assay. To find the optimal conditions for the immobilized PK activity assay, various pH levels, buffering systems, and temperatures were examined for the soluble enzyme (Tables 1 and 2). The syntheticsubstrateN-succinyl-Ala-Ala-Ala-p-NAwasused(11,15). The temperatures for both the soluble and immobilized enzyme were compared, and the results showed identical courses (data not shown). The activity was determined in three buffering solutions with differing pH levels (5, 11). Optimizations of PK Immobilization. Though commercial kits for binding proteins or enzymes to various carriers are now readily available, extensive optimizations are essential in testing newly developed carriers. Optimizations are needed to improve enzyme performance, for example, activity and stability. Appropriate conditions for PK immobilization to 500A5 magnetic microparticles (Ademtech, France) were first proposed and subsequently optimized. The following paragraphs describe the experiments in which the composition of the immobilization mixture, addition of calcium ions, ionic strength in the mixture, temperature, and duration were examined. Three buffers were tested for the immobilization procedure: 10 mM sodium phosphate buffer at pH 7.3, 50 mM MES at pH 6.0, and 50 mM MES at pH 7.3, all with 1 mM CaCl2. The resulting enzyme activities suggested differences between the individual buffers during the grafting procedure (Table 3). The 10 mM phosphate buffer at pH 7.3 was determined to be the most suitable, then came 50 mM MES at pH 6.0 and pH 7.3.
Figure 1. Binding efficiency of PK to 500A5 microparticles (Ademtech). Composition of the reaction mixture: 1 mg of magnetic particles, specified amount of PK, 79 mM EDAC, 12 mM S-NHS per 1 mg of the carrier, 6 h at RT, slow rotation.
One or two-step carbodiimide activations of -COOH particles using EDAC and/or S-NHS are known from the literature (14). In order to ensure the highest levels of PK reactor activity, both methods were tested (at pH 6.0). An immobilization protocol using only EDAC leads to a 24% lower enzyme activity (data not shown). It was proven that the combined use of EDAC and S-NHS activation is required for high PK reactor performance with 500A5 microparticles (Ademtech). It was previously shown that the ratio of the protein and EDAC/S-NHS concentrations plays a role in the grafting efficiency and also in unwanted particle agglomeration (17). Increasing the concentration of EDAC/S-NHS produces a significant change in the charge of the beads, which then causes their repulsion or agglomeration. For these reasons, we looked for a compromise level of EDAC/S-NHS with a proven optimal ratio. A decrease in EDAC/S-NHS concentrations by half in the subsequent experiments using PK reactor prepared with 500A5 microparticles led to 30% lower levels of catalytic activity. The stability of the enzyme reactor was also dramatically lower. In the next experiments, temperature and reaction time were altered; results of PK activity after immobilization are shown in Table 4. The effect of the amount of PK per mg of particles added to the reaction volume was studied. Increasing quantities of PK added to 1 mg of 500A5 microparticles were incubated under fixed binding conditions: 6 h at room temperature. The enzyme activity was determined with the substrate succinyl-Ala3-pnitroanilide in 50 mM Tris/HCl at pH 7.8 with 5 mM CaCl2. The highest activity was obtained using 3 mg of PK per mg of the carrier. Additional increases in initial PK concentration led to a decrease in final proteolytic activity (Figure 1). Since calcium ions are involved in enzyme activity and are needed for enzyme stability (11), we studied the effect of Ca2+ on the covalent bond (Figure 2). Zeta Potential Measurement. Zeta potential is a measure of the magnitude of the repulsion or attraction between particles. Its measurement provides detailed insight into the dispersion mechanism and is the key to electrostatic dispersion control. The most important factors that affects zeta potential are the pH and molar strength of the buffering solution used. Particles
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Figure 2. Effect of Ca2+ in binding solution on binding efficiency of PK reactor (500A5 microparticles). Table 5. Zeta Potential Measurement for 500A5 particles (2%) in 10 mM and 100 mM Phosphate Buffer at pH 7.3 and Addition of 0.3% PF127 Surfactant buffering
10 mM phosphate buffer pH 7.3
zeta potential value (mV)
-37.12
10 mM phosphate 100 mM phosphate buffer pH 7.3 buffer pH 7.3 with 0.3% PF127 -23.1
-16.68
Figure 3. Zeta potential curves of magnetic 500A5 particles and of magnetic PK reactor (prepared with 500A5 particles) in 10 mM phosphate buffer at pH 7.3.
are considered to have a lower stability in the range -30 to 30 mV (18). By measuring the zeta potential (Table 5, Figure 3) of the 500A5 microparticles and preparing the PK reactor using the same particles, the optimal conditions were found to be as follows: 10 mM phosphate buffer at pH 7.3 with the addition of 0.3% PF127 surfactant. Under these conditions, the particles showed only a slight agglomeration, which affected neither the binding nor digestion processes. The optimal conditions for the PK assay and for preparation of the PK reactor with 500A5 microparticles were used for other types of magnetic carriers and commercially available carriers in order to find the best working system. In general, 1 mg of magnetic particles was activated by the above EDAC and S-NHS cross-linking two-step method at pH 7.3 and the immobilization procedure occurred at 4 °C overnight. Due to the different sizes and material compositions of the chosen microparticles, the obtained enzyme activities varied significantly (Table 6). The achieved activities of the PK reactors were also compared to a commercially available nonmagnetic PK reactor, consisting of PK bonded to Eupergit C macroporous acrylic beads (Sigma, USA). In comparison, the activity of the commercial nonmagnetic PK reactor only reached 6% of that of 500A5 particles (Ademtech). Characteristics of PK Reactor. Stability of PK Reactor. Its storage and operational stabilities were analyzed. The activity
remained the same as the initiatial value after 10 weeks. The catalytic activity per mg of carrier temporarily increased at the beginning of the observation period. One possible explanation is the conformation of the enzyme molecule was restored after the immobilization procedure (3, 19). Operational Stability. By repeated use of the same aliquot at 25 °C with 1.05 mM substrate, up to 50% of the activity of the immobilized PK was maintained after 8 batches. In comparison, the reusability of the nonmagnetic commercially available PK reactor maintained 68% of its original activity after 8 repeats (data not shown). Kinetic Constants. Enzyme reactor was characterized by measurement of Michaelis constant (KM) which reflects the affinity between enzyme and substrate. The method used here was based on the frontal analysis of enzymatic reaction products, which was performed by the monitoring of the absorption at 405 nm of p-nitroaniline from the hydrolysis of succinyl-AlaAla-Ala-p-nitroanilide (20). Our experimentally obtained data were processed in terms of Michaelis–Menten kinetics by the Lineweaver–Burk plots of V versus [S] as shown in Figure 4, where [S] is concentration and V the velocity of enzymatic reaction (21). Initial conditions of the reaction (ratio E:S, reaction volume) were chosen in agreement with Michelis-Menten kinetics. The apparent KM of the PK reactor observed was 1.29 mM, and that of native PK 4.23 mM. The obtained KM is influenced by the chemical composition of the carrier and by the microenvironment of the enzyme reactor from the bonded enzyme molecules, and is called apparent KM (Figure 4). Maximum velocity Vi of the reaction catalyzed by the PK reactor was 0.36, which is 57% in comparison to native PK catalysis (value 0.63). Application of Magnetic PK Reactor. Digestion of Model Protein: Human Growth Hormone. The efficiency of digestion was confirmed using a model protein human growth hormone (HGH) with a MW of 22 kDa by peptide mass fingerprinting. Proteolytic digestion was carried out in batch mode at room temperature for 15, 60, and 180 min and 24 h in 50 mM ammonium acetate (pH 7.8). The HGH was subsequently analyzed by Tricine/SDS PAGE (Figure 5) and VoyagerDE STR MALDI-TOF (Figure 6). The sequence coverage for the 15 min digestion of HGH, calculated from the list of obtained peptides, was 75%. It was proven that the proteolytic activity and specificity of PK were maintained after its immobilization to magnetic particles. SDS PAGE and Western Blot of Digested PrPc Peptides. Having shown that the PK reactor reproducibly digests a model protein, we thought to test its efficiency on a more complex medium such as a cell extract. We next digested proteins extracted from the immortalized Mov cell line overexpressing prion protein with the appropriate amount of the immobilized PK reactor (Figure 7). Our results showed a visible decay of the proteolysis-sensitive cellular prion protein with digestion time.
DISCUSSION PK from Tritirachium album Limber (3.4.21.64) is reported as one of the most active serine proteases with a strong proteolytic activity on denatured proteins. The most interesting property of the enzyme is its capacity to degrade native proteins (5), which is commonly employed for differentiating between the physiological and pathological forms of prion protein (22). This study reports the immobilization of PK on newly developed magnetic latex microparticles (Ademtech, France) and other chemically and structurally similar types of magnetic carriers. 500A5 microparticles were developed to have a reduced tendency to agglomerate and low levels of nonspecific sorption. Various conditions for coupling were utilized with the aim of
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Table 6. Enzyme Activities of PK Magnetic Reactors Prepared with Carriers Differing in Polymer Coating and Size label of carrier 500A5 145Tta A120 00–39 PK-reactor
material used (where known) magnetic, styrenic copolymer with hydrophilic monomer magnetic, styrenic copolymer with hydrophilic monomer magnetic, alginate magnetic, latex acrylic, macro-porous
mean diameter (nm)
PK amount [µg/mg carrier] average from 3 immobilizations
Ademtech, Pessach, FR
626
12.09
Ademtech, Pessach, FR
980
3.56
2500 160 not shown
8.48 8.35 0.54
manufacturer
Academy of Sciences, CZ Estapor (Merck) FR Sigma, USA
Figure 4. Lineweaver–Burk plots for native and immobilized PK using static method and succinyl-Ala-Ala-Ala-p-nitroanilide as substrate.
Figure 5. Tricine-SDS-PAGE analysis of digested HGH (PK reactor prepared with magnetic latex microparticles, Estapor). Positions: 1, molecular marker; 2, HGH; 3, HGH digested for 15 min; 4, HGH digested for 60 min; 5, HGH digested for 180 min; 6, HGH digested for 24 h. Conditions of electrophoresis: constant potential 30 V, current 0.03 A per gel for 30 min, then constant potential 100 V, current 0.03 A per gel for 60 min at 4 °C.
preparing a magnetic enzyme reactor with sufficient enzyme activity and stability. From the surface chemistry, it is known that a pH slightly below the isoelectric point of the protein positively influences the grafting efficiency. The experimentally determined isoelectric point of PK was at the value 8.4 (data not shown), which is in general agreement with the value 8.9 in (5) and the theoretical value 8.25 from (23). At pH 7.3, the positively charged enzyme and negatively charged magnetic particles lead first to adsorption of the enzyme to the particle surface based on ionic forces and second to covalent linkage after carbodiimide activation in the immobilization procedure. By selecting a specific binding buffer, the subsequent adsorption of the enzyme molecules onto the particle surface potentiates a local amount of molecules in the immediate vicinity of the functional groups of the carrier, which enhances binding efficiency. In the coupling reaction, N-substituted carbodiimide EDAC reacted with a carboxylic group of the 500A5 microparticles to form a highly reactive and short-lived acylisourea derivative. The activated species then reacted with the primary amines of the ligands to form a stable peptide bond. The binding efficiency was substantially increased by the presence of S-NHS as
described in the paper by Staros (24). The two-step protocol at pH 7.3 in 10 mM phosphate buffer exhibited the highest binding yieldsandwaschosenasoptimalforfurtherimmobilizations(25,26). Temperature and reaction time have a considerable influence on the resulting enzyme reactor activity, especially with enzymes that have proteolytic and autolytic activities. Experiments with various immobilization procedures showed that using a low temperature of 4 °C with prolonged incubation overnight ensured a high binding efficiency of the enzyme reactor. The most possible explanation for the activity decrease after prolonged incubation overnight was autolytic activity (Table 4). If the immobilization was truncated from 6 to 2 h at room temperature, the resulting PK activity was very low and not sufficient for further applications. To find a compromise between immobilization yield and catalytic activity, we optimized the amount of protein to 3 mg per mg of activated beads (500A5), corresponding to a reactor with high catalytic activity. The X-ray structure study showed that two bound Ca2+ ions were required for the catalytic activity of PK (11). In the absence of Ca2+ ions, the catalytic activity of PK dropped to 20%, while the presence of Ca2+ slows down PK autolysis and also enhances thermal stability. In our experiments, increasing Ca2+ concentrations from 1 mM to 2.5 mM and 5 mM (the recommended concentration) only slightly increased the activity of the PK reactor (Figure 2). A Ca2+ increase to 5 mM negatively influenced the colloidal stability of the 500A5 microparticles. A total concentration of Ca2+ of 1 mM during the immobilization was considered to be sufficient. Under all the studied and optimized conditions, this newly developed magnetic PK reactor prepared with 500A5 microparticles exhibited a more than sufficient storage and operational stability. These parameters are usually important for using an enzyme in routine laboratory work. The “apparent” KM of immobilized PK compared to the soluble enzyme exhibited a threeford higher affinity to the substrate. Such a decrease in the KM values of immobilized enzymes has been recognized previously (20, 27), and has been ascribed to the presence of areas of increased substrate concentration around the particle due to electrostatic attraction or hydrophobic adsorption of the substrate to the solid. From a reaction rate point of view, the maximal velocity of substrate change was calculated as a change in optical density (formation of product) units per time unit. The lower rate of enzyme reaction with immobilized PK, shown by its maximum velocity Vi, can possibly be explained as due to limited diffusion of the substrate molecules to the surface of the particles and to the active sites of the immobilized enzyme. The 500A5 microparticles exhibited slight bead agglomeration during manipulations, which may complicate reactor application and efficiency. The zeta potential measurement confirmed the necessity of adding the surfactant. The surfactant PF127 at concentration of 0.3% was considered optimal. The magnetic PK reactor was applied in the proteolytic cleavage of high-molecular-weight substrates, the model substrate human growth hormone, and the cellular prion protein. Both HGH and PrPc were digested by limited proteolysis, and
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Figure 6. MALDI mass spectra of 15 min digested HGH by PK reactor (prepared with magnetic latex microparticles, Estapor). The framed masses correspond to identified peptides by the Expasy software (PeptideMass) with 0 or 1 (*) miscleavages. The nonframed masses arise from further miscleavages and contamination. Conditions of MS analysis: 1 µL of every digested sample was dried on MALDI-target before covering with 1 µL of DHB in ACN (50%) and TFA (0.3%) as the matrix (“dried droplet” technique). Calibration was performed with a standard peptide mixture with an m/z range of 800 to 3500 Da (LaserBio Laboratories, FR).
possibly be used for the digestion other resistant proteins with all features of covalently bonded enzymes.
ACKNOWLEDGMENT
Figure 7. Western blotting of limited proteolysis of digested cellular prion protein (Mov cell line overexpressing ovine PrPc). Samples were digested with PK reactor (0.7 µg, prepared with 500A5 microparticles, Ademtech) in 100 µL volume of cell lysate, precipitated with methanol, separated on 10% Tris/HCl gel, and blotted to nitrocellulose membrane with specific ICSM18 or SAF 84.3 mAb. Positions: 1, PrPc digested for 2 min; 2, PrPc digested for 5 min; 3, PrPc digested for 10 min; 4, PrPc digested for 30 min; and 5, PrPc digested for 60 min.
mass spectrophotograms, electrophoreograms, and blots confirmed that the proteolysis system was working well. Through these experiments, we confirmed the PK magnetic reactor to be a useful tool for PrP digestion, with the use of a magnetic carrier being published for the first time.
CONCLUSION This study describes (1) the methods used to optimize the PK grafting procedure, (2) the characterization and validation of the newly prepared magnetic enzyme reactor, and (3) the benefits of using immobilized PK in bioanalytical applications, and examples of those applications. We optimized a method for covalently bonding PK to a magnetic carrier with polymer coating and carboxylic derivatization. The carbodiimide chemistry used here to bind the enzyme to the new carrier gave the amide bonds excellent stability. The newly developed carrier prepared by the manufacturer Ademtech served as a good example of magnetic particles with a large range of possible uses. Furthermore, these optimized conditions, when applied to other magnetic nano and microparticles, showed high applicability and reusability. The results of this study also demonstrated that the storage conditions of the PK enzyme reactor are highly compatible with a laboratory routine. As examples of the simple handling of the magnetic PK reactor, the human growth hormone and mouse overexpressed PrPc were digested and verified by the analytical methods MALDI-TOFMS, PAGE, and Western Blot. This magnetic PK reactor can
We gratefully acknowledge the Ademtech Company, France, and Academy of Sciences, Czech Republic, for their donations of magnetic particle batches. This work was supported in part by Czech funding grant MSMT0021627502, the EU project “Neurotas” (contract no. 037953) and Czech Science Foundation 203/05/0241.
LITERATURE CITED (1) Turkova, J. (1999) Oriented immobilization of biologically active proteins as a tool for revealing protein interactions and function. J. Chromatogr., B: 722, 11–31. (2) Spanova, A., Rittich, B., Horak, D., Lenfeld, J., Prodelalova, J., Suvakova, J., and Strumcova, S. (2003) Immunomagnetic separation and detection of Salmonella cells using newly designed carriers. J. Chromatogr., A: 1009 (1–2), 215–21. (3) Bilkova, Z., Slovakova, M., Minc, N., Fuetterer, C., Cecal, R., Horak, D., Benes, M. J., Le Poitier, I., Krenkova, J., Przybylski, M., and Viovy, J. L. (2006) Functionalized magnetic micro- and nanoparticles: optimization and application to µ-chip trypsin digestion. Electrophoresis 27, 1811–1824. (4) Slovakova, M., Minc, N., Bilkova, Z., Smadja, C., Faigle, W., Fütterer, C., Taverna, M., and Viovy, J. L. (2005) Use of self assembled magnetic beads for on-chip protein digestion. Lab on Chip 5, 935–942. (5) Ebeling, W., Hennrich, N., Klockow, M., Metz, H., Orth, H. D., and Lang, H. (1974) PK from Tritirachium album Limber. Eur. J. Biochem. 47 (1), 91–7. (6) Pandhare, J., Dash, C., Rao, M., and Deshpande, V. (2003) Slow tight binding inhibition of PK by a proteinaceous inhibitor: conformational alterations responsible for conferring irreversibility to the enzyme-inhibitor complex. J. Biol. Chem. 278 (49), 48735–48744. (7) Betzel, C., Pal, G., and Saenger, W. (1988) Three-dimensional structure of PK at 0.15-nm resolution. Eur. J. Biochem. 178, 155–171. (8) Horak, D., Rittich, B., Spanova, A., and Benes, M. J. (2005) Magnetic microparticulate carriers with immobilized selective ligands in DNA diagnostics. Polymer 46, 1245–1255. (9) Daly, P., Corcoran, A., Mahon, B. P., and Doyle, S. (2002) High-sensitivity PCR detection of parvovirus B19 in plasma. J. Clin. Mikrobiol. 40 (6), 1958–1962.
972 Bioconjugate Chem., Vol. 19, No. 4, 2008 (10) Russo, G. L., Nisco, E. D., Fiore, G., Donato, P. D., d’Ischia, M., and Palub, A. (2003) Toxicity of melanin-free ink of Sepia officinalis to transformed cell lines: identification of the active factor as tyrosinase. Biochem. Biophys. Res. Commun. 308, 293– 299. (11) Bajorath, J., Hinrichs, W., and Saenger, W. (1988) The enzymatic activity of PK is controlled by calcium. Eur. J. Biochem. 176 (2), 441–7. (12) Morihara, K., and Tsuzuki, H. (1975) Specificity of PK from Tritirachium album Limber for synthetic peptides. Agric. Biol. Chem. 39, 1489–1492. (13) Staros, J. V., Wright, R. W., and Swingle, D. M. (1986) Enhancement by N-hydroxysulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions. Anal. Biochem. 156, 220–2. (14) Hermanson, G. T. (1996) Bioconjugate Techniques, pp141, 156, 157, 159, 169–173, 470, 570–572, Academic Press, London. (15) Peters, K., Pauli, D., Hache, H., Boteva, R. N., Genov, N. C., and Fittkau, S. (1989) Subtilisin DY–kinetics characterization and comparison with related proteinases. Curr. Microbiol. 18, 171–177. (16) Cronier, S., Laude, H., and Peyrin, J. M. (2004) Prions can infect primary cultured neurons and astrocytes and promote neuronal cell death. PNAS 101 (33), 12271–12276. (17) Bilkova, Z., Castagne, A., Zanusso, G., Faronazzo, A., Monaco, S., Damoc, E., Przybylski, M., Benes, M., Lenfeld, J., Viovy, J. L., and Righetti, P. G. (2005) Immunoaffinity reactors for prion protein qualitative analysis. Proteomics 5, 639–647. (18) Malvern instruments manual (2004).
Slováková et al. (19) Bilkova, Z., Slovakova, M., Lycka, A., Horak, D., Lenfeld, J., Turkova, J., and Churacek, J. (2002) Oriented immobilisation of galactose oxidase to bead and magnetic bead cellulose and poly(HEMA-co-EDMA) and magnetic poly(HEMA-co-EDMA) microspheres. J. Chromatogr., B: 770 (1–2), 25–34. (20) Jiang, H., Zou, H., Wang, H., Ni, J., Zhang, Q., and Zhang, Y. (2000) On-line characterization of the activity and reaction kinetics of immobilized enzyme by high-performance frontal analysis. J. Chromatogr., A: 903, 77–84. (21) Logan, S. R. (1998) Physical Chemistry for the Biomedical Sciences, Vol 77, p 86, CRC Press, London. (22) Prusiner, S. B. (1998) Prions. Proc. Natl. Acad. Sci. U.S.A. 95, 13363–13383. (23) http://www.expasy.org/tools/pi_tool.html (accessed 11/20/ 2007. (24) Staros, V. (1982) N-Hydroxysulfosuccinimide active esters bis(N-hydroxysulfosuccinimide) esters of two dicarboxylic acids are hydrophilic, membrane-impermeant, protein cross-linkers. Biochemistry 21, 3950–3955. (25) Hermanson, G. T., Krishna, M. A., Smith, P. K. (1992) Immobilized affinity ligand techniques, pp 81–85, Academic Press, London. (26) Cuatrecasas, P., Parikh, I. (1972) Adsorbents for affinity chromatography. Use of N-hydroxysuccinimide esters of agarose. Biochemistry 11, 2291–2298.. (27) Chibata, I., Tosa, T., Sato, T., and Mori, T. (1978) Immobilized enzymes, p 126, John Wiley & Sons, London. BC7004413
