22G08
Protein Inactivation in Solution by Plasma-Induced Chemical Processing
プラズマ誘起液中化学プロセスによるタンパク質の失活
Eisuke Takai1, Katsuhisa Kitano2, Junpei Kuwabara3, 4, Tamotsu Zako5,Yoichi Yamada1, Kentaro Shiraki 1*
高井英輔, 北野勝久, 桑原純平, 座古保, 山田洋一, 白木賢太郎
1Institute of Applied Physics, University of Tsukuba, 2Center for Atomic and Molecular Technologies, Graduate School ofEngineering, Osaka University, 3Institute of Materials Science, University of Tsukuba, 4Tsukuba Research Center forInterdisciplinary Materials Science (TIMS), 5Bioengineering Laboratory, Advanced Science Institute, RIKEN

1-1-1 Tennodai,Tsukuba, Ibaraki 305-8573, Japan筑波大院・数理物質 〒305-0005 つくば市天王台1-1-1
Plasma medicine is one of the attractive new research areas, but fundamental understanding related toplasma modification of biomacromolecules in aqueous solution remains elusive. We investigated thechemical effects of low-temperature atmospheric pressure plasma on protein in solution using lysozyme asa model. Plasma treatment decreased enzymatic activity, which is attributed to the increased molecularweight of lysozyme with chemical modification. These effects arise neither from UV light nor from heatload, suggesting the attack of reactive species on lysozyme. This is a fundamental step for elucidatingchemical reactions by plasma for biomedical applications.

1. Introduction
Plasma processing at atmospheric pressure has
attracted a great deal of attention because of its
advantageous features, such as its obviation ofcomplicated and expensive vacuum systems, its
capacity for adequate and controllable gas
temperature, its high concentrations of chemicallyreactive species, and its flexible operation. Such
attributes of atmospheric pressure plasma have been
applied as ionizer of mass spectrometry, eliminationof organic materials, tooth bleaching, andsterilization.
Althoughvariousbiomedical
applications of the atmospheric pressure plasma
have been developed, fundamental understanding ofinteraction between plasma and biomacromolecules
has remained insufficient.
Our plasma system, using a low-frequency (LF)high voltage (HV) power supply and a single-HVelectrode, which is designated as an LF plasma jet,can easily generate low-temperature atmosphericpressure plasma (LTAP) at low cost. The advantageof the LF plasma jet became possible to sterilize
bacteria in solution without heat load, current, andultraviolet (UV) light. [1] To apply the LF plasma jet
to medical applications, this investigation of
enzymatic activity and molecular weight would be
important for understanding fundamental reactionsof complicated biological reactions.

Fig. 1. Schematic diagram of the plasma generation
system. LF/HV pulses are applied to the electrode, which
is wound around the quartz glass tube. The device isHe-based low-temperature atmospheric plasma. Plasma
is generated in He gas flowing (500 sccm) in the quartz
glass tube. Supplying of O2 gas of 150 sccm to thechamber maintains the O2 concentration in the chamber
atmosphere.

2. Experimental Section
Experimental setup is shown in figure 1,
comprising the plasma generator and sample
chamber. The LF plasma jet was generated from theend of the quartz glass tube, in which helium (He)gas flowed, by the application of alternating HVpulses (from -3.5 to +5.0 kV with the frequency of
13.9 kHz) to the electrode, which was a small metal
sheet wound around the tube. He plasma with low
gas temperature was generated with an elongatedshape. This plasma jet was generated inside theairtight chamber from its center port. In thechamber, various active oxygen species produced
from oxygen (O2) gas were supplied to the solution.
The ambient gas of the chamber can be controlledwith the other O2 gas supply port connected to theside of the plasma jet port. The sample of 0.3 ml
containing 0.1 mg/ml lysozyme in 10 mM
phosphate buffer (pH 7.4) was applied to thesolution in the vessel. To exchange the ambient gas,Fig. 2. Residual activity of lysozyme treated by an LF
plasma jet (open circles), hot air treatment with heat(closed circles), and plasma treatment through a quartz
glass plate with UV (open squares).

gases were flowed for 3 min before the plasma
generation. After the plasma treatment, samples
were corrected with pure water to compensate forevaporation.
3. Experimental Results
Experimental result is shown in figure 2 presents
the time course of residual activity of lysozyme
after treatment of the LF plasma jet for therespective periods. The residual activity decreasedwith time, about one-third after the plasma
treatment for 30 min.. To confirm the cause ofinactivation, two control experiments wereperformed; hot-air treatment and plasma treatment
through the quartz glass plate. Briefly, hot-air
treatment was performed as the control experimentwith air at 80 °C instead of the LF plasma jet blown
on the samples. The plasma treatment through aquartz glass plate was performed as the control
experiment that the LF plasma jet irradiated on thesamples through a UV-permeability glass vial toconfirm the effect of UV alone. As expected, theenzymatic activity of lysozyme did not change inthese control experiments (Fig. 2). These data
indicate that the decrease in the residual activity
results from chemical modification by plasma
treatment.
To investigate the chemical reaction on theprotein, mass spectrometry (MS) was used. Figure3(a) shows data of matrix-assisted laser
desorption/ionization time of flight MS of thesamples after or before plasma treatment for 30
min.. The most abundant peak of native lysozyme
was m/z 7153, which corresponds to the doubly
protonated molecule; the singly protonatedmolecule showed a peak at a maximum of m/z
14306. A minor species at m/z 28612 was detected,
which corresponds to the dimer with one
protonation. However, the peaks of the MS

Fig. 3. MS spectrum of lysozyme treated by LF plasma
jet for 0 or 30 min between m/z (a) 5000-35000, (b)7000-7500 and (c) 14000-15000.

spectrum after the plasma treatment for 30 min
were shifted to high molecular weight;
plasma-treated lysozyme increased the mass ofabout 90 (Fig. 3(c)). These results indicate that theLF plasma jet modifies some amino acid sidechains in lysozyme.
4. Discussion
We found that the LF plasma jet on lysozyme
decreased enzymatic activity and increased
molecular weight, which are caused neither by UVlight nor heat from plasma. The data suggest thatreactive species generated by the LF plasma jetaffect on lysozyme. A possible mechanism of theLF plasma jet on protein is biochemical reaction
caused by hydroxyl radical, superoxide anion
radical, hydroperoxy radical and nitric oxide, which
results in the chemical modifications of chemicallyreactive side-chain of the amino acids, such as
cysteine, aromatic rings of phenylalanine, tyrosine,and tryptophan. Further experiments will be
performed to clarify the chemical and physiologicalinfluences of the LF plasma jet on biologicalmolecules. These data imply the interesting
application of the LF plasma jet on protein forbiomedical applications.
References[1] S. Ikawa, K. Kitano, S. Hamaguchi, Plasma
Processes Polym. 7 (2010) 33–42.
[2] E. Takai, K. Kitano, J. Kuwabara, K. Shiraki,
Plasma Processes Polym. in press