Examination of Cholesterol oxidase attachment to magnetic nanoparticles
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Magnetic nanoparticles (Fe3O4) were synthesized by thermal co-precipitation of ferric and ferrous chlorides. The sizes and structure of the particles were characterized using transmission electron microscopy (TEM). The size of the particles was in the range between 9.7 and 56.4 nm. Cholesterol oxidase (CHO) was successfully bound to the particles via carbodiimide activation. FTIR spectroscopy was used to confirm the binding of CHO to the particles. The binding efficiency was between 98 and 100% irrespective of the amount of particles used. Kinetic studies of the free and bound CHO revealed that the stability and activity of the enzyme were significantly improved upon binding to the nanoparticles. Furthermore, the bound enzyme exhibited a better tolerance to pH, temperature and substrate concentration. The activation energy for free and bound CHO was 13.6 and 9.3 kJ/mol, respectively. This indicated that the energy barrier of CHO activity was reduced upon binding onto Fe3O4 nanoparticles. The improvements observed in activity, stability, and functionality of CHO resulted from structural and conformational changes of the bound enzyme. The study indicates that the stability and activity of CHO could be enhanced via attachment to magnetic nanoparticles and subsequently will contribute to better uses of this enzyme in various biological and clinical applications.
KeywordsFe3O4 Magnetic Nanoparticles Fe3O4 Nanoparticles Prussian Blue Free Enzyme
List of Abbreviations used
Transmission electron microscopy
Fourier Transform Infrared
Bovine serum albumin
Magnetic materials have been used with grain sizes down to the nanoscale for longer than any other type of material . This is attributable to a number of factors including a large surface area to volume ratio and the possibility of immobilizing a biological entity of interest . In the last decade increased investigations and development were observed in the field of nanosized magnetic particles . Here the term nanoparticles is used to designate particulate systems that are less than 1μm, and effectively below 500 nm .
Due to their magnetic character, magnetite (Fe3O4) nanoparticles can be attracted by a magnetic field and are easily separable in solution. Similarly, substances to which they have been attached can be separated from a reaction medium, or directed by an external magnetic field to site specific drug delivery targets . Magnetic nanoparticles have been widely used in the immobilization of many bioactive substances such as proteins, peptides, enzymes [3, 4, 5, 6], and antibodies . Magnetite is one of the most commonly used magnetic materials because it has a strong magnetic property and low toxicity .
The binding of magnetic particles to bioactive substances involves a number of interactions including the interactions between organic ligand, and the interactions between the amino acid side chains of proteins and the metals centers. Such bindings pave the way for the coupling of biomolecular entities of enhanced stability. Recently reported work in the area of enzyme immobilization has described the catalytic activity of yeast alcohol dehydrogenase  and lipase  directly bound to magnetite nanoparticles, via carbodimiide activation without the use of a ligand. This binding method offers tremendous scope because of its simplicity and high efficiency.
Cholesterol oxidase is a flavin-enzyme (with a FAD prosphetic group) that produces hydrogen peroxide according to the reaction 1.
Cholesterol + O2 → 4 - Cholesten - 3 - one + H2O2 (1)
The structure of cholesterol oxidase reveals deeply buried active sites occupied by water molecules in the absence of its substrate steroids . Cholesterol oxidase is industrially and commercially important for application in bioconversions for clinical determination of total or free serum cholesterol [9, 10, 11, 12] and in agriculture . Its activity can be determined by following the appearance of the conjugated ketones, the formation of hydrogen peroxide in a coupled test with peroxidase, or by measuring the oxygen consumption polarographically . Several studies on its kinetic properties have appeared [13, 14, 15]. More recently, Cholesterol biosensor based on entrapment of cholesterol oxidase in a silicic sol-gel matrix at a Prussian Blue modified electrode has been developed . However, this method of enzyme immobilization raises concerns on reduced surface area for enzyme binding and pore-diffusion resistance . Immobilization of enzymes onto inorganic material surfaces is of vital importance in enzymatic reactions, especially in biosensor applications. Information on the activity and availability of cholesterol oxidase bound to Fe3O4 magnetic nanoparticles will contribute to the basic understanding of its activity and function.
The present study proposes to investigate the direct binding of cholesterol oxidase to Fe3O4 magnetic nanoparticles. The sizes and structure of the nanoparticles were characterized using TEM and FTIR spectroscopy. The stability, activity, and kinetic behavior of bound cholesterol were also examined.
Results and discussions
Particle size and structure
The unbound enzyme was determined by assaying the protein content in the supernatant. It was found that the percentage of cholesterol oxidase bound was between 98 and 100%, irrespective of the amount of particles. The amounts of Fe3O4 nanoparticles used were 14.4, 17.2 and 20 mg/mL, corresponding to CHO/Fe3O4 weight ratios of 0.01, 0.08 and 0.007, respectively. These results show that in all the binding operations, there were sufficiently available amount of particles to bind the enzymes till complete saturation. In a previous study , it was found that increasing the amount of Fe3O4 nanoparticles, that is reducing the weight ratio of CHO to Fe3O4 below 0.033 caused an increase in lipase binding up to 100%. This was not observed in this study, possibly because of the difference in the binding mechanism, due to differences in the structure of the enzyme. However, the percentage of bound CHO (98–100%) shows that the binding process was successful.
Cholesterol oxidase activity and binding kinetics
Effect of pH
Inactivation rate constants (k) of the "bare" and bound CHO at various temperatures
3.4 × 10-2
4.6 × 10-3
9.3 × 10-2
5.6 × 10-2
2.8 × 10-1
1.9 × 10-2
Effect of temperature on enzyme activity and stability
Materials and methods
Cholesterol oxidase (EC 126.96.36.199), Nocardia sp. was purchased from VWR international (Pittsburgh, USA). Carbodiimide-HCl (1-ethyl-3-(3-dimethyl-aminopropyl), ammonium hydroxide reagent, Triton X-100, TRIS (Hydroxymethyl) aminomethane HCL, 4-cholesten-3-one, bovine serum albumin (BSA), iron (II) chloride tetrahydrate 97 %, and iron (III) chloride hexahydrate 99% were obtained from Sigma-Aldrich, St Louis (USA). The Biorad Protein Assay Dye Reagent Concentrate was purchased from Biorad Laboratories (Hercules, CA). Acetonitrile was obtained from EMD Chemicals, (New Jersey, USA).
Preparation of magnetic nanoparticles
Magnetic nanoparticles (Fe3O4) were prepared by chemical co-precipitation of Fe2+ and Fe3+ ions in a solution of ammonium hydroxide followed by a treatment under hydrothermal conditions [4, 5]. Iron (II) chloride and iron (III) chloride (1:2) were dissolved in nanopure water at the concentration of 0.25 M iron ions and chemically precipitated at room temperature (25°C) by adding NH4OH solution (30%), at a control pH (10–10.4). The suspensions were heated at 80°C for 35 min under continuous mixing and separated by centrifuging several times in water and then in ethanol at 2800 rpm. The purification step was used to remove impurities from Fe3O4 nanoparticles. The particles were finally dried in a vacuum oven at 70°C. The dried particles exhibited a strong magnetic attraction to a magnetic rod.
Attachment of cholesterol oxidase onto magnetic nanoparticles
50–70 mg of magnetic nanoparticles was added to 1 mL of phosphate buffer (0.05 M. pH 7.4). The mixture was sonicated for 15 min after adding 0.5 mL of carbodiimide solution (0.02 g/mL in phosphate buffer (0.05 M. pH 7.4). Following the carbodiimide activation, 2 mL of cholesterol oxidase (0.25 mg/mL) was added and the reaction mixture was sonicated for 30 min at 4°C in a sonication bath and the mixture was centrifuged at 3000 rpm . The precipitates containing Fe3O4 nanoparticles and Fe3O4bound cholesterol oxidase (Fe3O4-CHO) were washed with phosphate buffer pH 7.4 and 0.1 M Tris, pH 8.0, 0.1 M NaCl and then used for activity and stability measurements. NaCl was added to enhance the separation of the magnetic nanoparticles .
Determination of immobilization efficiency
The amount of protein in the supernatant was determined by a colorimetric method at 595 nm using the Biorad Protein Assay Reagent Concentrate with bovine serum albumin (BSA) as the protein standard. The amount of bound enzyme was calculated from:
A = (C i - C s )*V (2)
Where A is the amount of bound enzyme, Ci and Cs is the concentration of the enzyme initially added for attachment, and in the supernatant, respectively (mg-mL-1), V is the volume of the reaction medium (mL).
The size of Fe3O4 nanoparticles and Fe3O4-CHO was characterized by transmission electron microscopy (TEM, JEM 1200 EXII, JEOL USA) and structure by Fourier Transform Infrared (FTIR) spectroscopy (Biorad FTS 6000, Cambridge, MA). The samples for TEM analysis were prepared by placing a drop of the magnetic nanoparticles dispersed in nanopure water onto a copper grid and evaporated in air at room temperature. Before preparing a sample onto the copper grid, the dispersed solution was sonicated for 4 min to obtain better particle dispersion. The binding of CHO onto the magnetic nanoparticles was investigated using FTIR. CHO and Fe3O4-CHO samples in phosphate buffer and Fe3O4 particles were dissolved in nanopure water for FTIR analysis.
The activity of bound CHO was determined by measuring the initial oxidation rates of cholesterol by cholesterol oxidase at given temperature following the increase of 4-cholesten-3-one concentration at 240 nm, using a Beckman Du Spectrometer. A solution of cholesterol was prepared by dissolving 4.8 g of cholesterol in 10 mL of 2-propanol. A phosphate buffer solution (0.05 M. pH 7.4) containing 4% of Triton-100 was added to the mixture to result in a 0.26 M cholesterol solution. The mixture was gently heated until the solution was clear. To start the enzymatic reaction, 5 ml of cholesterol solution was added to 15 mL centrifuge test tubes containing Fe3O4-CHO, and mixed by vortex. A solution of free CHO of the same concentration was used to evaluate the activity of the free enzyme. The solution was incubated at various temperatures (25–70°C) at specific intervals of time (1 h) and centrifuged at 3000 rpm for 5 min to separate the supernatant from Fe3O4-CHO. 10 μL aliquots of the supernatant were then taken and the concentration of 4-cholesten-3-one was assessed. Before measuring the amount of 4-cholesten-3-one in a sample, the activity of the free enzyme was stopped by adding an equal volume of acetonitrile to the reacting solution . Each kinetic measurement was the average of duplicate replications.
Thermal stability of free and immobilized enzyme
The thermal stability of free and Fe3O4-CHO were determined by measuring the residual activity of the enzyme at 25°C, after being exposed to different temperatures (25–70°C) in phosphate buffer (0.05 M, pH 7.4) for 40 min. Aliquots of the reacting solution were taken at time intervals (every 30 min for 7 hours) and assayed for enzymatic activity as described above. The first order inactivation rate constant, k was calculated from the equation:
In A = In A0 - kt (3)
where A 0 is the initial activity, A is the activity after a time t (min), k is the reaction constant.
Effect of temperature on enzyme activity
The effect of temperature on the free CHO and Fe3O4-CHO was estimated by determining the concentration of 4-cholesten-3-one in samples at various temperatures. A solution of cholesterol was added to the various centrifuge test tubes containing bound or free enzymes. The test tubes were stored in a water bath at specific temperatures (25, 37, 50, 60, and 70°C). At time intervals, the concentration of 4-cholesten-3-one was determined by spectrophotometric analysis.
The storage stability was evaluated by determining the concentration of 4-cholest-en-3-one at room temperature at time intervals (5 days). Test tubes containing Fe3O4-CHO or free enzyme solution were stored at 25°C in phosphate buffer (0.05 M. pH 7.4) for 30 days. Thereafter, 5 mL of cholesterol was added. The storage stability of the free and bound cholesterol oxidase was determined by assaying for their residual activity.
Determination of kinetics parameters
The kinetic parameters of free CHO and Fe3O4-CHO, Km and Vmax were determined by measuring initial rates of oxidation of cholesterol (1.3–5.2 mM) by CHO (0.25 mg/mL) in phosphate buffer pH 7.4 at 25°C.
Magnetic nanoparticles were synthesized by thermal co-precipitation of ferric and ferrous chlorides. The binding of CHO to the particles was confirmed by FTIR spectroscopy and the size characterized by TEM. The binding efficiency was between 98 and 100% irrespective of the amount of particles used. Kinetic studies of the free and bound CHO revealed that the stability and activity of CHO were significantly improved upon binding to nanoparticles. Furthermore, the bound enzyme exhibited a better tolerance to pH, temperature and substrate concentration. The activation energy indicated that the binding of CHO onto Fe3O4 magnetic nanoparticles reduced the energy barrier for CHO activity. As a result of the binding to the magnetic nanoparticles, the storage stability of CHO was considerably enhanced. This higher stability of the Fe3O4-CHO is attributable to its possible fixation on the surface of the particles preventing auto-digestion and thermal inactivity. In addition, the binding on Fe3O4 nanoparticles might allow a better spatial orientation of the FAD prosphetic groups and the side chains of CHO to provide better stability to the enzyme. The overall improvements observed in activity, stability, and functionality of CHO resulted from structural and conformational changes of the bound cholesterol oxidase. The study may be useful in improving the stability and activity of cholesterol oxidase, and will contribute to more efficient use of this enzyme.
The authors acknowledge the 2003 USDA challenge grant program for partial funding of this research. Dr Chen Xu is also acknowledged for the TEM images.
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