Martina Koneracka, Peter Kopcansky, Milan Timko,
Chenyl Nynitapal Ramchand, Zainul M. Saiyed, Michael Trevan, and Anil de Sequeira
Magnetic particles have been increasingly used as carriers for binding proteins, enzymes, and drugs. Such immobilization procedures for proteins, enzymes, antibodies, and other biologically active compounds have a major impact in different areas of biomedicine and biotechnology. The immobilized biomolecules can be used directly for a bioassay or as affinity ligands to capture or modify target molecules or cells. This chapter details immobilization procedures for proteins and enzymes onto various magnetically responsive carriers such as naked magnetic particles, carboxyl-modified microspheres, and amino-modified microspheres using direct binding procedure in the presence of coupling agents such as carbodiimide. The physical and chemical properties of freshly prepared magnetic particles were determined by magnetic measurements (VSM magnetometer), transmission electron microscopy (TEM), and atomic force microscopy (AFM). The extent of immobilization and enzyme activities were spectrophotometrically measured in order to find the retained activity after immobilization onto magnetic particles. The binding of proteins and enzymes was also confirmed by TEM microscopy.
Key Words: Albumin; glucose oxidase; chymotrypsin; streptokinase; dispase; carbodiimides.
Immobilization of proteins, enzymes, antibodies, and other biologically active compounds onto inert solid support has found several applications in the field of biotechnology and biomedicine. Conventionally, enzymes and cells were immobilized on a solid support, such as synthetic polymeric matrices, or entrapped within the gel matrix formed of different polysaccharides such as alginate, cellulose, or agarose. The use of synthetic polymers as supports for enzyme immobilization provides several advantages (e.g., inertness to microbial attack, higher chemical resistance, and the option to use complex buffer components). Of the many synthetic polymers, polystyrene (PS) is inexpensive, readily available in different par-
From: Methods in Biotechnology: Immobilization of Enzymes and Cells, Second Edition Edited by: J. M. Guisan © Humana Press Inc., Totowa, NJ
ticle sizes, and possesses mechanical rigidity (1). Nonporous PS-based adsorbents have been employed for reverse-phase (2), anion-exchange (3), and affinity chromatography of proteins (4-6). Whereas the use of gel matrices for enzyme immobilization offers a relatively inert aqueous environment within the matrix, the high gel porosity allows for high diffusion rates of macromolecules. Alginate beads have been used for a variety of biotechnological applications (7).
In recent years, several magnetic separation procedures have been developed to isolate and purify biomolecules (8). The advantage offered by such techniques is the ease of recovery, speed, and extreme specificity by which a biomolecule can be isolated from a complex mixture. Over the past decade, systems containing micrometer- or submicrometer-sized magnetic particles coated with a suitable stabilizer have been used as carriers for binding proteins, enzymes, and drugs. For example, heparin-stabilized colloidal magnetite has been used for binding of cells from whole blood; dextran-coated magnetite has been used as a drug carrier, whereas silane-coated ferrite particles are used for radioimmunoassays (9). Magnetic particle-based systems have also been used as an affinity matrix for separation of enzymes. One-step purification of horse liver alcohol dehydrogenase by magnetic Sepharose® beads coupled with 5'-AMP has been successfully accomplished (10). Magnetic alginate microsphere have been used for affinity purification of a-amylase (11).
Superparamagnetic microspheres preconjugated to various types of generic binding proteins (such as Protein A and streptavidin) and antibodies are rapidly becoming the solid-phase support of choice in many areas, including immunological applications, nucleic acid work, and cell separation and visualization. These monodisperse spheres are composed of magnetite in a polymer matrix. The polymer beads are then coated with functional polymers that provide reactive groups, such as COOH and NH2. Proteins can be adsorbed readily onto PS microspheres or co-valently coupled to carboxylic acid or other functional groups on the surface of microspheres. Covalent coupling is, however, preferable for many applications. A covalently bound ligand is likely to be more stable, to cover the surface more completely or more evenly, and to be more efficient in terms of consumed reagents. Furthermore, addition of spacers or linkers will allow biomolecules to be presented in a more flexible fashion, and careful chemistry can attach ligands in a specific orientation (12). Antibodies can be modified in a similar manner. Antibody-coated microspheres form the basis for particle capture enzyme-linked immunosorbent assay (ELISA) tests and assays. Antibodies immobilized onto magnetic beads have been utilized extensively in diagnostics and other research applications for the purification of cells and biomolecules. Magnetite labeled antibodies is expected to be applicable clinically as therapeutic agents for the induction of hyperthermia (13).
Most manufacturers provide variously sized particles that have a variety of different chemical terminations. For example, dextran-based biocompatible magnetic nanoparticles are available commercially from Miltenyi Biotec, Germany. These magnetic beads (^MACS) are available with many covalently immobilized molecules such as protein A, protein G, streptavidin, and antibodies against various CD markers (14). Likewise, Polysciences, Inc., manufactures and supplies Biomag® magnetic beads consisting of an iron-oxide core with an inert silane coating. The particle surface is functionalized with amine or carboxyl groups for the covalent attachment of proteins, glycoproteins, and other ligands with retention of biological activity. Biomag particles are supplied covalently attached to a wide variety of monoclonal antibodies (MAbs) specific for human and murine cell surface markers or secondary antibodies such as goat antimouse, goat antirat, and sheep antifluorescein (15).
Biologically active compounds immobilized on magnetic carriers can also be used directly for a bioassay or as affinity ligands to capture or modify target molecules or cells. For example, Abudiab and Beitle (16) have developed a magnetic immobilized metal affinity separation media for isolating proteins from complex mixtures. The method involved coupling of iminodiacetic acid (IDA) to the surface of magnetic agarose, this when charged with metal ions (Cu+2 or Zn+2) is capable of binding model proteins that display metal affinity and of separating protein mixture (16). Similarly, Qiagen Inc., Germany has developed protein purification and assay protocols based on Ni-NTA magnetic agarose beads to suit different applications. Ni-NTA (nitriloacetic acid) tagged magnetic agarose beads have been used for versatile magnetocapture assays using 6x His-tagged proteins (17). The method employs both metal affinity and magnetism as the basis of purification. The procedure involves use of metal chelating NTA groups covalently bound to the surface of agarose beads, which contain strong magnetic particles. Nickel ions immobilized on NTA have a high affinity for a tag of six consecutive histidine residue, thereby allowing capture of 6x His-tagged proteins for sensitive interaction assays or microscale purification. Thus, using this technique purification of protein based on Ni-NTA metal chelate affinity followed by direct use of purified protein for microplate-based assays is possible (18). Other procedures for magnetic affinity separations of various proteins have been summarized recently (19).
Mehta et al. (20) have shown that proteins and enzymes can be bound covalently to freshly prepared magnetite in the presence of carbodiimide. Several clinically important enzymes and proteins that include bovine serum albumin (20), streptokinase, chymotrypsin, dispase, and glucose oxidase (GO) have been immobilized based on this method. The immobilized enzymes showed between 50 and 80% of the original added enzyme activity. The direct coupling method for enzymes or bioactive molecules to the magnetic particles is a result of the presence of hydroxyl group on the magnetic support. Bacri et al. (21) have shown that hydroxyl group will remain on the particles at pH between 6.0 and 10.0. Thus, the free hydroxyl group on the surface of the particles is responsible for the binding of the enzymes.
The direct coupling method for enzymes or bioactive molecules to the magnetic particles has a number of potential advantages. Because these magnetite particles are not coated with any polymer materials, the overall size is smaller, thus increasing the ratio of surface area to volume, allowing a greater response to any magnetic field. Moreover in case of cell separation using magnetic particles it is shown that the larger the particle size used for separation, the higher the extent of nonspecific entrapment in the larger aggregates of magnetic particles. Thus smaller magnetic particles hold the promise of greater specificity; because they can exist as stable colloidal suspensions that will not aggregate, thus allowing for uniform distribution in a reaction mixture (22). This chapter details immobilization procedure for proteins and enzymes onto magnetically responsive carriers.
1.1. Immobilization of Enzyme Protein to Magnetic Particles Using Direct Binding Procedure
A water-soluble carbodiimide derivative can be used to activate hydroxyl group on the surface of magnetic particles and thereby couple the amino group on the enzymes to the hydroxyl group through an amide linkage (see Fig. 1). It is important that the pH of the reaction medium is kept neutral. At a pH that is too low, the amine is protonated and does not react readily, whereas at a higher pH the carbodiimide may decompose. The coupling procedure is simple to perform and has the advantage of occurring under very mild conditions and of conjugating the enzymes directly to magnetic particles without interposing additional groups between two (20,22).
1.2. Physical and Chemical Properties of Magnetic Particles
The magnetic properties of magnetic particles are described as superparamagnetic (having zero coercivity and remanence) and the classical theory of Langevin paramagnetism, modified to include a particle size distribution, can be used. So the magnetization as a function of magnetic field in liquid phase can be expressed by following equation:
where L(y) = coth(y) - 1/y is the Langevin function, a = nIsDf:^oH/6kBT, Is is the saturation magnetization of the bulk material, x = D/Dr is the reduced diameter (Dr is the mean diameter of particles) and f(x) is the particle size distribution function. For the system of fine magnetic particles the particle size distribution function is log normal (23):
where f(y) is log-normal distribution function and o is standard deviation.
The particle size distribution is possible to estimate from magnetization measurements and consecutively then relations for Dv and G can be expressed as:
where x is initial susceptibility.
For example, the saturation magnetization Is was measured to be 14 mT, as using the above-mentioned method the log-normal parameters of the particle size distribution have been calculated to be: mean particle diameter Dv, of 9.3 nm, and standard deviation o of 0.29. Using the relation m = IsVr (Vr is mean volume of particle calculated from mean particle diameter and Is is saturation magnetization of particle) the magnetic moment of the individual particle can be estimated. In our case for Dv = 9.3 nm the magnetic moment was estimated as to be 1.84 x 104 is elementary Bohr magneton, which has the value = 9.2740789 x 10-24 Am-2). Figures 2 and 3 provide the results from atomic force microscopy (AFM) and transmission electron microscopy (TEM) measurements, respectively.
From the results obtained by the various measurement techniques, it is clear that for the mean particle diameter values: Dv/VSM (9.3 nm) < Dv/TEM (12.2 nm) < Dv/AFM (13.1 nm) and for the standard deviation values: oVSM (0.29) > oTEM (0.22) > oAFM (0.21). However, the smaller mean particle diameter observed from VSM data (9.3 nm) in comparison with value obtained from TEM and AFM data can be explained by the presence of a nonmagnetic surface layer around the magnetite particles and small differences in standard deviation can be caused by method of sample preparation (24).
The estimation of particle size and its distribution is very useful, as the physical and chemical properties of magnetic fluids depend on the detail of particle size distribution mainly at biological application of magnetic fluids.
2.1. Preparation of Magnetic Particles
1. Ammonium hydroxide, iron (II) sulfate heptahydrate, and iron (III) chloride hexahydrate were procured from Sigma-Aldrich (St. Louis, MO).
2. Vibrating sample magnetometer (VSM) was used for estimation of volume concentration of magnetite particles, saturation magnetizations, and cosequently for particle size diamater.
2.2. Immobilization of Proteins Onto Naked Magnetic Particles
1. Bovine serum albumin (BSA), GO, chymotrypsin, streptokinase, dispase, and 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide hydrochloride (CDI) were obtained from Sigma-Aldrich.
2. Coupling buffer: 3 mMsodium phosphate buffer, pH 6.3.
2.3. Immobilization of Proteins Onto Carboxyl-Modified Microspheres (25)
1. Carboxyl-modified microspheres (often supplied at 10% solids; see Note 2).
3. Coupling buffer, pH 7.2 to 8.5: 25-100 mM phosphate-buffered saline (PBS) (Buffers containing free amines, such as Tris of glycine should be avoided.)
4. Water soluble carbodiimide (WSC; e.g., 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide hydrochloride and/or 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate).
5. Protein or other biomolecule that is to be immobilized.
6. Quenching solution with primary amine source (30-40 mM; e.g., hydroxylamine, ethanolamine, glycine, or other) and 0.05-1% (w/v) blocking reagent (BSA, casein, pepticase, polyethylene glycol [PEG] ).
7. Storage buffer, pH 7.0 to 7.5: 25-100 mM PBS, containing 0.01 to 0.1% blocking reagent (BSA, casein, pepticase, polyethylene glycol).
2.4. Immobilization of Proteins Onto Amino-Modified Microspheres (25)
1. Amino modified microspheres (often supplied at 10% solids; see Note 2).
2. Amine reactive homobifunctional cross-linker (e.g., glutaraldehyde, imidoesters, or NHS esters).
3. Wash/coupling buffer, pH 6.0 to 9.0: 25-100 mM PBS.
4. Protein and other biomolecules that is to be immobilized.
5. Quenching solution with primary amine source, 30-40 mM (e.g., hydroxylamine, ethanolamine, or glycine) with 0.05-1% (w/v) blocking reagent.
6. Storage buffer, pH 7.0 to 7.5: 25-100 mM PBS, pH 7.4, containing 0.01 to 0.1% blocking reagent (BSA, casein, pepticase, polyethylene glycol).
Magnetic particles (Fe3O4) were prepared by co-precipitating ferric and ferrous salts in an alkaline solution followed by washing in hot water (see Fig. 4 and Notes 3 and 4).
1. Dissolve 27.8 g of iron (II) sulfate heptahydrate (FeSO4 C 7H2O) and 54 g of Iron (III) chloride hexahydrate (FeCl3 C 6H2O) in 100 mL double distilled water.
2. Mix the above solution thoroughly.
3. Add drop-wise to 8 M NH4OH with constant stirring at room temperature.
4. Heat the obtained precipitate at 80°C for 30 min.
5. Black particles were obtained that exhibited strong magnetic response.
6. Wash the particles with copious amounts of hot distilled water.
7. Finally, magnetic particles were suspended in slightly alkaline medium, pH 8.9.
3.2. Immobilization of Different Proteins Onto Naked Magnetic Particles Using Carbodiimide (see Fig. 5)
Bovine serum albumin (BSA), GO, chymotrypsin, streptokinase, and dispase were immobilized onto magnetic particles using 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide hydrochloride (CDI).
1. Dissolve the protein that is to be coupled in phosphate buffer pH 6.3 at a concentration of 20 mg/mL.
2. Separately weigh and dissolve CDI in phosphate buffer, pH 6.3, at a concentration of 20 mg/mL.
3. Mix 1 mL of protein (20 mg/mL) with 1 mL of CDI (20 mg/mL) dissolved in buffer, pH 6.3.
4. Add 3 mL of a given volume of magnetite particles that had previously been washed copiously in buffer, pH 6.3, to the mixture of protein and CDI.
5. Incubate the reaction mixture on a shaker at room temperature for 24 h.
6. After 24 h separate magnetic particles by placing a magnet under the vial containing the reaction mixture.
7. Decant the supernatant, wash magnetic particles three times in buffer solution.
8. Pool the supernatants, measure the left over protein concentration using Bradford's dye binding assay (see Note 1).
9. Calculate the quantity of bound protein as the difference between the total protein added to the immobilization mixture and the total protein recovered from the pooled washings.
10. Confirm the binding of protein to magnetic particles by FTIR spectroscopy and electron microscopy (see Fig. 6).
11. Check the enzyme activity in order to find the retained activity after immobilization onto magnetic particles (see Notes 5-7).
3.3 Immobilization of Proteins
Onto Carboxyl-Modified Polymeric Microspheres (25)
1. Wash 1 mL (100 mg/mL) of microspheres two times in 10 mL of activation buffer (MES buffer is a common choice).
2. After washing, completely resuspend microspheres in 10 mL of activation buffer.
3. Add 100 mg of water soluble carbodiimide (WSC) to the microspheres with constant stirring.
4. Incubate the reaction mixture at room temperature for 15 min.
5. Wash microspheres two times with coupling buffer and resuspend completely in 5 mL of the same.
Fig. 6. The evidence of successful immobilization of BSA can be seen as thin layers around the magnetite particle (B).
Fig. 6. The evidence of successful immobilization of BSA can be seen as thin layers around the magnetite particle (B).
6. Dissolve protein (1-10X excess of calculated monolayer) in 5 mL of coupling buffer.
7. Combine microsphere suspension and protein solution.
8. Incubate at room temperature for 2 to 4 h with constant stirring.
9. Wash, resuspend in 10 mL of quenching solution, and mix gently for 30 min.
10. Wash and resuspend in storage buffer to desired storage concentration (often 10 mg/mL).
3.4. Immobilization of Proteins
Onto Amino-Modified Polymeric Microspheres (25)
1. Wash 1.0 mL (100 mg/mL) of microspheres twice with 10 mL of coupling buffer.
2. After washing, completely resuspend microspheres in 10 mL of glutaraldehyde solution (glutaraldehyde dissolved in wash/coupling buffer to a final concentration of 10%).
3. Incubate at room temperature for 1 to 2 h, with constant stirring
4. Wash twice and resuspend in 5 mL of wash/coupling buffer.
5. Dissolve protein (1-10X excess of calculated monolayer) in 5 mL of coupling buffer.
6. Combine microsphere suspension and protein solution.
7. Incubate the reaction mixture with constant stirring for 2 to 4 h at room temperature.
8. Wash and resuspend in 10 mL of quenching solution and mix gently for 30 min.
9. Wash and finally resuspend in storage buffer to desired storage concentration (often 10 mg/mL).
1. Because of interference of carbodiimide Lowry's method for protein estimation should not be used.
2. Polymeric microspheres, produced commercially by Bangs Laboratories, are available with many surface active functional groups such as carboxyl and amino, as well as hydroxyl, hydrazide and chloromethyl; and silica-silanol. Biomolecules can be effectively linked to these microspheres through a variety of coupling chemistries. As examples, covalent coupling of proteins or other biomolecules using carboxyl and amino-modified microspheres has been described above (TechNote 205 Covalent Coupling; at http://www.bangslabs.com). Recently, Bangs Laboratories also introduced magnetic microspheres with different surface groups that are intended for covalent coupling of ligands such as protein, and nucleic acids.
3. The saturation magnetizations of dehydrated particles were determined with a vibrating sample magnetometer (VSM).
4. The amount of magnetic particles in a given volume of magnetic fluid was estimated thermogravimetrically and by magnetic measurements of magnetization curves (VSM magnetometer).
5. GO activity determined by GO-PO method.
6. Streptokinase activity determined by the smallest amount of streptokinase that causes lyses of a standard fibrin clot within 10 min.
7. Chymotrypsin and dispase assayed according to the Sigma diagnostic procedures (26).
This work was supported by the Slovak Academy of Sciences state research and development order "New materials and components in submicrometer technology."
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