Immobilization of Cytochrome P450 2C9 on Magnetic Nanoparticles
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| Year of publication | 2013 |
| Type | Conference abstract |
| MU Faculty or unit | |
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| Description | Studies of the affinity of a drug candidate compounds to drug-metabolizing enzymes, e.g. cytochromes P450, and the investigation of potential drug-drug interactions constitute a routine part of a new drug development processes. Due to the extensive numbers of tested candidates, there is ongoing trend towards the rationalization and hastening of screening assays. Enzyme immobilization represents a useful approach in this field because of repetitive use of a single batch of enzymes and simplification of sequential analyses by providing protein-free samples. Moreover, immobilized enzymes typically have greater thermal and operational stability compared to their soluble forms. The goal of this study was to develop method for immobilization of clinically important cytochrome P450 2C9 isoform (CYP2C9) on magnetic nanoparticles SiMAG. In addition to above mentioned list of advantages, utilization of magnetic support enables easy enzyme positioning inside reaction vessel and fast removal of enzyme and thus reaction termination using magnet. Parameters of immobilization procedure such as attachment scheme, concentration of support surface activators, composition, concentration and pH of immobilization buffer, attachment time and temperature and concentration of immobilized CYP2C9 were optimized with respect to its activity and stability. After the method optimization was complete, there was experimentally proved that immobilized CYP2C9 can be repetitively used for 12 hours without considerable loss of activity. Performed kinetic study of CYP2C9 reaction with diclofenac used as a probe substrate proved that the binding have no negative effect on enzyme activity. Obtained apparent values of Michaelis constant (Km = 2.57 +- 0.15 uM) and Hill coefficient (n = 1.13 +- 0.08) were in a good agreement with literature data determined using homogeneous assays [1–3]. What is more, obtained apparent value of maximal reaction velocity (Vmax = 28.26 +- 0.72 nmol/min/nmol) was significantly higher than it was reported in the reference. In the next study, presented system will be used for construction of in-capillary reactor enabling screenings of drug metabolism by means of on-line capillary electrophoresis. This work was supported by grant No. P206/10/0057 from the Grant Agency of the Czech Republic and by project CZ.1.07/2.3.00/20.0182 from the European Regional Development Fund. [1] R.L. Walsky, R.S. Obach, Drug Metab. Dispos. 32 (2004) 647-660. [2] A. Di Marco, I. Marcucci, A. Chaudhary, M. Taliani, R. Laufer, Drug. Metab. Dispos. 33 (2005) 359-364. [3] J. Konečný, J. Juřica, J. Tomandl, Z. Glatz, Electrophoresis 28 (2007) 1229-1234. |
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