In Situ Observation of Chymotrypsin Catalytic Activity Change

Mar 23, 2018 - This work demonstrates two approaches to control activity of an ... Imaging Action Potential in Single Mammalian Neurons by Tracking th...
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In situ Observation of Chymotrypsin Catalytic Activity Change Actuated by Non-Heating Low-Frequency Magnetic Field Maria V. Efremova, Maxim M. Veselov, Alexander V. Barulin, Sergey L. Gribanovsky, Irina M. Le-Deygen, Igor V. Uporov, Elena V. Kudryashova, Marina Sokolsky-Papkov, Alexander G. Majouga, Yuri I. Golovin, Alexander V. Kabanov, and Natalia L. Klyachko ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b06439 • Publication Date (Web): 23 Mar 2018 Downloaded from http://pubs.acs.org on March 23, 2018

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In situ Observation of Chymotrypsin Catalytic Activity Change Actuated by Non-Heating LowFrequency Magnetic Field Maria V. Efremova 1,2, Maxim M. Veselov 1, Alexander V. Barulin 1, Sergey L. Gribanovsky 3, Irina M. Le-Deygen 1, Igor V. Uporov 1, Elena V. Kudryashova 1, Marina Sokolsky-Papkov 4, Alexander G. Majouga 1,2,5, Yuri I. Golovin 1,3, Alexander V. Kabanov 1,4*, Natalia L. Klyachko 1,2,4

1

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Laboratory for Chemical Design of Bionanomaterials, Chemistry Department, M.V.

Lomonosov Moscow State University, Moscow, Russian Federation; 2 National University of Science and Technology MISIS, Moscow, Russian Federation; 3 G.R. Derzhavin Tambov State University, Tambov, Russian Federation; 4 Center for Nanotechnology in Drug Delivery, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; 5 D. Mendeleev University of Chemical Technology of Russia, Moscow, Russian Federation * [email protected] [email protected]

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ABSTRACT

Magneto-mechanical modulation of biochemical processes is a promising instrument for bioengineering and nanomedicine. This work demonstrates two approaches to control activity of an enzyme, α-chymotrypsin immobilized on the surface of gold-coated magnetite magnetic nanoparticles (GM-MNPs) using non-heating low-frequency magnetic field (LF MF). The measurement of the enzyme reaction rate was carried out in situ during the exposure to the magnetic field. The first approach involves α-chymotrypsin-GM-MNPs conjugates, in which the enzyme undergoes mechanical deformations with the re-orientation of the MNPs under LF MF (16 – 410 Hz frequency, 88 mT flux density). Such mechanical deformations result in conformational changes in α-chymotrypsin structure, as confirmed by infrared spectroscopy and molecular modeling, and lead to 63% decrease of enzyme initial activity. The second approach involves α-chymotrypsin-GM-MNPs/trypsin inhibitor–GM-MNPs complex, in which the activity of the enzyme is partially inhibited. In this case the re-orientation of MNPs in the field lead to disruption of enzyme-inhibitor complex and almost two-fold increase of enzyme activity. The results further demonstrate the utility of magneto-mechanical actuation at the nanoscale for the remote modulation of the biochemical reactions.

KEYWORDS enzyme, α-chymotrypsin, trypsin inhibitor, magnetic nanoparticles, gold-coated magnetite nanoparticles, non-heating low-frequency magnetic field

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Enzymes, immobilized on nanostructured carriers, 1–4 such as functionalized magnetic nanoparticles (MNPs), 5–8 are promising tools in organic synthesis, biocatalysis and nanomedicine. 1, 2, 4, 5, 8 Successful applications of immobilized enzymes in these areas depend on the ability to precisely alter their catalytic activity. Since the enzyme activity is strongly dependent on the structure of the protein molecule, 9 studies of alterations of the structure and function in enzyme molecules through external influences, for example, mechanical forces are highly significant. Effects of mechanical deformations on structure and chemical properties of cells and single biomacromolecules have been successfully researched in recent decades using various eloquent techniques. 10–13 One example of such mechanochemical techniques is single molecule force spectroscopy (SMFS), allowing quantitative measurements of forces and deformations that alter biochemical properties of both subcellular structures within the cells 10–15 and separate biomacromolecules, 11–13 in particular, enzymes. 9, 16–19 It was found that the critical forces triggering alterations of some of the most important biochemical processes are in the range of several to hundreds of pN. 12–16 In addition to studying the responses of biomacromolecules to applied forces and determining the critical values of these forces, the mechanochemical approaches could potentially be used in various bioengineering and nanomedicine applications. However, many such applications would require remote and simultaneous effect upon a large number of biomacromolecules. In the present work a possibility of mechanochemical modulation of biocatalysis is demonstrated using MNPs remotely actuated by non-heating low-frequency (f < 1 kHz) magnetic fields (LF MF). It has been proven theoretically 18–21 that 20-30 nm magnetite MNPs can generate forces ranging from 0.1 to 1000 pN in uniform LF MF of flux density B = 50 – 300 mT. In such fields

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heating effect of MNPs is negligible (