Control of Enzymatic Activities by Magnetite Nanoparticles
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0950-D15-19
Control of Enzymatic Activities by Magnetite Nanoparticles Hui Zhou1, Marie-Eve Aubin-Tam2, and Kimberly Hamad-Schifferli2,3 1 Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139 2 Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA, 02139 3 Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139
ABSTRACT Enzymes are proteins that catalyze chemical reactions, participating in almost all processes in the cell to achieve significant reaction rates and serving a wide variety of functions inside living organisms. Here, we intended to control enzymatic activities by applying external radio frequency magnetic field (RFMF) that heat nanoparticles (NPs) bound to the enzymes. Ribonuclease A (RNase A), a relatively small protein that cleaves single-stranded RNA, was conjugated to 10nm Fe3O4 NPs non-covalently. External RFMFs were applied to solutions of the conjugates, and the enzymatic activities of RNase A were affected. We studied the effect of varying the field frequencies and incubation time of conjugation. Results were compared to bulk thermal heating.
INTRODUCTION Magnetic nanoparticles (NPs) have been found numerous biological applications. Magnetic NPs can be heated by external magnetic fields. This has been exploited for hyperthermia in cancer treatment [3] to burn tumor tissues. This ability to heat NPs by external fields can be used to denature proteins. In our work, we study how Fe3O4 NPs can be used to denature the protein Ribonuclease A (RNase A). Ribonucleases (RNases) are the general names for RNA depolymerases. Ribonuclease A (RNase A) is the predominant form of the enzyme in the pancreas of Bos Taurus [1]. RNase A is stable at room temperature and will not denature at relatively high temperatures (up to 64oC) [2]. 10nm Fe3O4 NPs coated with negatively charged ligands were incubated with RNase A, and conjugates were formed by electrostatic attraction or by ligand exchange where RNase A molecules might attach directly onto the surface of Fe3O4 NPs by carboxyl groups. External RFMF was applied after a certain time of incubation, elevating the temperature of the solutions. Activities of RNase A were affected by magnetic heating, and mechanism will be discussed.
EXPERIMENTAL DETAILS Water soluble Fe3O4 NPs (EMG 705) were obtained from Ferrotec Corporation with an average diameter of 10 nm. RNase A was incubated with Fe3O4 NPs in 1X PBS at 25oC for 1 hour, 18 hours, 60 hours and 120 hours. The molar ratio of Fe3O4 NPs : RNase A was kept 1:10. 150 µL of the complex solutions were placed in a coil of 25 turns and exposed to external magnetic fields for 10 minutes. External RFMFs of approximately 1kA/m were generated by a current from a signal generator and amplified by a 100W amplifier [4]. Activity of the NP-RNase A was measured using fluorescence detection of the cleavage of a DNA oligo with a central rU (5í-(6-FAM)-(dA)3rU(dA)4-(6-TAMRA)-3í, obtained from Proligo)
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