Activity increase of horseradish peroxidase in the presence of magnetic particles

Article abstract of DOI:10.1021/ja7102263

We report evidence for the effect of very low magnetic fields on a biological reaction. The presence of magnetite particles (with permanent magnetic moment of 1 emu/g) in the assay had a 30-fold increase on the rate of reaction of horseradish peroxidase.

Full text of DOI:10.1021/ja7102263

Published on Web 02/15/2008
Activity Increase of Horseradish Peroxidase in the Presence of Magnetic
Particles
Nikolaos G. Chalkias,*,† Patarawan Kahawong,‡ and Emmanuel P. Giannelis‡
School of Chemical and Biomolecular Engineering, Cornell UniVersity, Ithaca, New York 14853, and Department of
Materials Science and Engineering, Cornell UniVersity, Ithaca, New York 14853
Received November 11, 2007; E-mail: nc68@cornell.edu
Magnetic field effects (MFE) on enzymatic reactions have been
investigated as early as the 1960s, and most of the experiments
carried out initially reported no effect on the reaction rates.1 In the
mid-1980s focus was turned to radical pairs;2,3 in the context of
the radical pair recombination theory,3 the effect of magnetic fields
on biological reactions with paramagnetic species was studied.4
Recently, a number of publications examined the effect of an
external magnetic field and that of spin orbit coupling (SOC) on
the kinetics of enzymatic reactions.5 In particular, evidence was
found for the existence of a magnetosensitive step in the catalytic
cycle of horseradish peroxidase (HRP).6,7
Here, for the first time, we report evidence for the effect of very
low magnetic fields on HRP. The presence of magnetite particles
(Fe3O4) in the enzyme assay had a 30-fold increase on the rate of
Figure 1. Activity of HRP in the presence of magnetic and super-
paramagnetic iron oxide particles. Values normalized with the activity of
neat HRP. The error bars are the standard deviation of four repetitions.
reaction of horseradish peroxidase.
During the catalytic cycle, native HRP, its’ intermediate com-
pounds I and II, and the oxidized organic substrate radicals are all
paramagnetic species.7 A combination of any two can form a
paramagnetic (radical) pair that can undergo spin selective pro-
cesses.7 The presence of magnetic particles in the assay is
hypothesized to have an effect on the spin states of geminate pairs
(G-pairs) and on random-encounter diffusing radicals (F-pairs) via
an accelerated intersystem crossing (ISC) mechanism that can alter
the overall kinetics of the enzyme reaction.3,8 To test the effect of
the magnetite particles on enzymatic activity we used iron oxide
particles with varying magnetic strengths. Specifically we used a
series of iron oxide particles with permanent magnetic moments
of 1, 0.48, and 0 emu/g denoted B4, B3, and superparamagnetic
(SPM), respectively. Given the average diameter of the B4 particles
(∼300 nm) and their magnetic strength, an estimated magnetic
field was calculated for the vicinity of such a particle. The mag-
netic field ranged from 3 G, 200 nm from the particle’s surface, to
0.1 G for 1 µm from the surface (details in the Supporting
Information).
The catalytic activity of horseradish peroxidase increased 30-
fold, when magnetite particles were present in the assay at low
concentrations. These results were reproduced using different
batches of enzyme and different vendors (Fischer, Pierce). In
Figure 1 the activity of HRP is shown with varying particle
concentrations in the assay (the phenol-aminoantipyrine (AAP)
chromogen was used).9 In contrast, the enzyme activity did not
change when commercial super-paramagnetic particles with no
permanent magnetic moment were present in the assay.
to observe any positive or negative effect on the activity when
magnetic particles were present in the assay.
The iron oxide particles appeared to have no reducing or
oxidizing effect on the substrates, and no activity was found when
the protocol was tested in the absence of HRP. The protocol was
also tested in the presence of Fe3+ ions; likewise no effect on the
measured activity of HRP was observed.
In addition, the possibility of a redox process between the
enzyme’s active site and the particles was examined, and the Soret
band of the enzyme was probed. The characteristic peak of the
heme group at 403 nm was not shifted, and the intensity of the
peak did not change in the presence of iron oxide particles.
We also found that the effect of the particles was reversible;
namely, the enzymatic activity returned to the original value once
the particles were removed with a strong Nd magnet. Furthermore,
the activity increase was not observed when smaller particles (<80
nm) of the B3 batch were tested in similar concentrations. The above
result ruled out the possibility of a surface effect responsible for
the observed activity increase. Thus, the observed activity increase
appears to be related to the magnetic properties of the iron oxide
particles.
To obtain a better understanding of the enzyme behavior in the
presence of magnetic particles, we examined the apparent Michae-
lis-Menten kinetics parameters of the enzyme with and without
magnetic particles. The neat enzyme had an activity maximum at
0.3 mM hydrogen peroxide. Further increase of substrate in the
assay resulted in a decrease of the catalytic activity. This last
observation has been well documented for HRP, where at relatively
high concentrations of peroxide the enzyme undergoes substrate
inhibition.10,11 The activity of HRP with B4 particles reached a
plateau at high peroxide concentrations, and substrate inhibition
was registered only at concentrations close to 50 mM (compare to
0.3 mM in the absence of particles).
We examined various other redox enzymes with and without a
heme group (cytochrome c, chloroperoxidase, catalase, and glucose
oxidase) under the same limiting conditions, but we were unable
† School of Chemical and Biomolecular Engineering.
‡ Department of Materials Science and Engineering.
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10.1021/ja7102263 CCC: $40.75 © 2008 American Chemical Society

C O M M U N I C A T I O N S
extent of disproportionation. A control solution with the same
concentration of ABTS radicals but without iron oxide particles
showed no significant disproportionation in the time scale of the
experiment (the complete data of the full spectra scans of the
samples as a function of time are included in the Supporting
Information).
This increase in the disproportionation rate appears to correlate
with the increasing magnetic strength of the particles. In particular,
the results suggest that the weak magnetic field of the randomly
distributed particles increased the recombination rate of the diffusing
ABTS radicals.
The above observations suggest that magnetic particles could
play a significant role in reactions with spin-correlated radical pairs
and possibly affect paramagnetic species, similar to the species
formed during the HRP catalytic cycle (geminate pairs or diffusing
RERPs).
Figure 2. Extent of ABTS radicals recombination in the presence of iron
oxide particles with increasing permanent magnetic moment.
For HRP, a modified Michaelis-Menten equation is used that
takes into account substrate inhibition.12
Acknowledgment. The authors acknowledge financial support
from Cornell’s Nanobiotechnology Center (NBTC). The authors
would like to thank Prof. Anthony Hay, Prof. Tadhg Begley, Dr.
Magnus Bergkvist, and Dr. Alejandro Becerra-Arteaga for their
comments.
Vmax ‚ [S]
V )
Km + [S] + Ki ‚ [S]2
The presence of 6 µg/mL of magnetic particles in the assay
significantly decreased the substrate inhibition term Ki (close to
10-fold) and increased the turnover rate (kcat) for HRP. The turnover
rate increased almost three times in the presence of B3 and five
times in the presence of B4 particles. The tables of the calculated
Supporting Information Available: Tables with the values of all
kinetic parameters. A reaction scheme of the catalytic cycle of HRP.
Characterization data for B4, B3, and SPM particles. Full spectra scans
of radicals (oxidized) and unoxidized ABTS as well as detailed
experimental descriptions. Reaction schemes for ABTS and ABTS
radicals as well as phenol and AAP. This material is available free of
charge via the Internet at http://pubs.acs.org.
Vmax, Km, and Ki are included in the Supporting Information.
Identical experiments were also conducted with 2,2′-azino-di-
(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS) as the chromogen
substrate. The Michaelis-Menten kinetics parameters with this
chromogen were also estimated (for values see Supporting Informa-
tion). The kcat of the enzyme once again increased significantly in
the presence of magnetic particles. However, in contrast to the
results obtained with the phenol/AAP chromogen system, similar
substrate inhibition was observed when magnetic particles were
present in the assay. The differences between the apparent kinetic
parameters obtained with the two chromogen assays are possibly
due to the different oxidation mechanisms for each substrate.11,13
The phenol/AAP depends on the combination of two radicals to
generate a colored product13 whereas ABTS does not. Secondary
side reactions exist for phenol radicals resulting in formation of
biphenyl products.13,14 Nevertheless, in both protocols an increase
in kcat was observed.
From the data obtained using ABTS, B3 particles appeared to
have a larger effect on the turnover rate than the more magnetic
B4 particles. This observation was somewhat puzzling, as we
expected the more magnetic particles to have a larger effect (as in
the case of phenol/AAP). One possibility might be an increase in
the recombination rate of the generated ABTS radicals that partially
disproportionate back into ABTS in the presence of magnetic
particles (for a reaction scheme, see Supporting Information) To
test the above hypothesis, we designed an experiment that would
track the evolution of a disproportionation reaction between two
radicals, when considered as Random Encounter Radical Pairs
(RERP or F-pairs).
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In Figure 2, the percentage of ABTS radicals recombining back
to ABTS is shown, and particles with an increasing permanent
magnetic moment had an increasingly beneficial role vis-a`-vis the
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