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. 2005 Jan 20;3(1):1.
doi: 10.1186/1477-3155-3-1.

Examination of Cholesterol oxidase attachment to magnetic nanoparticles

Gilles K Kouassi  1 , Joseph Irudayaraj, Gregory McCarty
Affiliations

Examination of Cholesterol oxidase attachment to magnetic nanoparticles

Gilles K Kouassi et al. J Nanobiotechnology. .

Abstract

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.

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Figures

Figure 1
Figure 1
Transmssion Electron micrographs of Fe3O4 magnetic nanoparticles (a) and Fe3O4-CHO (b).
Figure 2
Figure 2
Distribution of the particle sizes on the electron micrographs. The values denote the averages of duplicate measurements.
Figure 3
Figure 3
FTIR spectra of Fe3O4 magnetic nanoparticles (a) in nanopure water and Fe3O4-CHO (b), and pure CHO (c) prepared in phosphate buffer and then dissolved in nanopure water for FTIR analysis.
Figure 4
Figure 4
Lineweaver Burk plots of the initial rates of CHO (■) and (◆) Fe3O4-CHO at pH 7.4, from experimental data.
Figure 5
Figure 5
Effect of pH on the activities of free (■) and bound CHO (◆).
Figure 6
Figure 6
Thermal stability of free CHO (■) and Fe3O4-CHO (◆) at pH 7.4. The samples were stored at 50, 60, or 70°C for 40 min and the activities were then measured at 25°C.
Figure 7
Figure 7
Effect of various temperatures on the activity Fe3O4-CHO at pH 7.4.
Figure 8
Figure 8
Arrhennius plots of the initial plots of the oxidation rates of cholesterol by free CHO (■) and Fe3O4-CHO (◆) for samples at 50, 60, or 70°C.
Figure 9
Figure 9
Storage stability of free CHO (■) and Fe3O4-CHO (◆). The activities measurements were performed at pH 7.4, at 25°C
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