22G06
Biochemical Reaction of Amino Acid
by Plasma-Induced Chemical Processing in Liquid
プラズマ誘起液中化学プロセスによるアミノ酸の生化学反応
Tsuyoshi Kitamura1, Satoshi Ikawa2, Hideya Kawasaki1, Ryuichi Arakawa1 and Katsuhisa Kitano3
北村 豪1, 井川 聡2，川崎 英也1，荒川 隆一1, 北野 勝久3
1

Faculty of Chemistry, Materials and Bioengineering, Kansai Univ., 3-3-35 Yamatecho, Suita, Osaka, 564-8680, Japan
2
Technology Research Institute of Osaka Prefecture, 3Eng. Osaka Univ.
1
関西大学化学生命工学部、〒111-222 吹田市中央区２－１2
大阪府立産業技術総合研究所、3大阪大学工学研究科
Plasma medicine is one of attractive new research areas, but fundamental reaction mechanism is still notmade clear enough. In this study, we investigated the chemical effects of low-temperature atmosphericpressure plasma on amino acid in liquid using tyrosine and glutathione as a model. After plasma treatment,
glutathione, which is known as an antioxidant agent, was confirmed to be changed into oxidized
glutathione, which is chemically cross-linked by disulfide binding of bimolecular thiol group, from mass
spectrometry. This research is an elementary step of plasma medicine to understand fundamental reactions,and would bring many thought of important knowledge about biochemical reaction between plasma andbiomacromolecule.

1. Introduction
In recent years, novel application researches ofatmospheric pressure plasmas such as medicalapplication or synthesis of biomaterial have been
conducted intensively [1].
For the purpose of plasma disinfection of human
body, Ikawa and Kitano have successfully foundthat efficient bactericidal activity can be achieved if
the solution is sufficiently acidic [2]. It isinteresting to note that there is a critical pH value ofabout 4.7 for the bactericidal effects, below which
the bacteria are efficiently inactivated and above
which the bacteria are hardly affected by the plasma
application. It has been found experimentally thatthe presence of superoxide anion radicals (O2-•) inthe solution is essential for bacterial inactivation by
the plasma application [3]. The critical pH value
may be associated with pKa of the dissociation
equilibrium between O2-• and hydroperoxy radicals(HOO•), which is known to be approximately 4.8.
Other experimental results indicated that notphysical reaction but biochemical modificationinduced inactivation. To progress like this basicresearch to applications, it is necessary to makeclear the reaction mechanism to biomacromolecures.
In this paper, plasma treated amino acid in solution
by using LF plasmajet [4,5], which is one of theatmospheric low temperature plasma, is analyzed toevaluate the interaction between plasma and aminoacid by using mass spectrometry.
Tyrosine is one of amino acids which acomponent of protein and play an important role forneural signaling. In vivo, it is known that tyrosine
has a strong reactivity with peroxynitrite (ONOO−).
ONOO− is one of reactive oxygen speciesgenerated from the reaction of NO• with O2− •.
ONOO− can induce nitration of protein andnitrotyrosine residue of protein is seen around bloodvessel of arteriosclerosis and ischemia, andinflamed area [6].
Glutathione is known as a typical antioxidant andis easily oxidized under oxidant stress to protect
living cells. Most of glutathione within a cellusually exists as a reduced form and the ratio of
reduced glutathione to oxidized glutathione isuseful as indicator of cellular toxicity [7].

Fig.1. Schematic diagram of the plasma exposuresystem. LF/HV pulses are applied to the single
electrode around the quartz glass tube.

2. Experimental Setup
A schematic illustration of the experimental
setup is shown in Fig. 1. The system consists of adielectric tube through which He gas flows(2000sccm) and a single electrode to which LF
high-voltage pulses (~10kV, ~10kHz) are applied.
Amino acid in pure water was prepared at
1mM. The distance between front end of theglass tube and the solution surface was set to20mm (plasma and water were contact) or30mm (non-contact). After plasma treatment,the solution was diluted by the solvent (purewater : acetonitrile = 1 : 1) to 10uM, and thenanalyzed by electro spray ionization (ESI) mass
spectrometry (MS).
3. Experimental Results
3-1.Plasma Treatment of Tyrosine
As the result of plasma treatment under thecondition where plasma and solution werecontact, mass spectrum of tyrosine (Fig.2) waschanged as shown in Fig.3. While protonatedtyrosine molecule ion (m/z=182) was seen
before treatment, new peaks of ion species wereat m/z=198, 214, 227, 243 after treatment.
Analysis result of these shows that 198 is one
oxygen atom addition ion (+16), 214 is twooxygen addition ion, 227 is two oxygen and one
nitrogen addition ion, 243 is three oxygen andone nitrogen addition ion.
This result represents thatplasma treatment induces
not only oxidation but alsonitration.
Fig.2. Chemical structural formula of L-Tyrosine(Mw 181.19 g/mol).

Fig.3. Positive ions mass spectrometry of tyrosine(A) plasma exposure time 0min (B) plasma exposure
time 7.5min.

3-2. Plasma Treatment of Glutathione
Same experiment was conducted against thesolution of glutathione (Fig.4(a)) under plasma
contact condition and mass spectrum was obtainedin negative ion mode. Before plasma treatment, thepeaks of deprotonated glutathione molecule ion(m/z=306) and deprotonated dimer molecule ofglutathione (m/z=613) were seen. After treatment,
peaks of m/z=354, 611 were newly seen. They wereidentified to three oxygen addition ion (m/z=354)
and oxidized form glutathione (m/z=611). The latteris brought by cross-linking reaction at disulfide
binding of bimolecular thiol groups (Fig.4(b)). Here,the production of oxidized glutathione was seenin slightly different way under non-contactcondition.
(a)

(b)

Fig.4. Glutathione in (a) reduced and (b) oxidizedform.

4. Summary
It is important subject to figure out thereactions of plasma to biomacromoleculeconsidering
plasmainducedchemicalprocessing in liquid. In this paper, weevaluated the interaction between plasma andamino acid solution by using mass spectrometry.
It was found that chemical modification wasbrought to both tyrosine and glutathione.
Especially in the case of plasma process insolution, like this biochemical evaluation isimportant because kinetic momentum ofplasma produced active species is practicallynought at collisional atmospheric pressure.
References[1] 北野勝久、現代化学、7 月号、p25-31、(2009).
[2] 井川聡、日本国特許第 4408957 号.
[3] S. Ikawa, Plasma Process. Polym., 7, pp.33, (2010).
[4] 北野勝久、応用物理学会誌、４月号、(2008).
[5] 北野勝久、日本国特許第 4677530 号.
[6] Beckman, Chem. Hoppe-Seyler 1994, 375, 81-88.
[7] A. Pastore, Clinical Chemistry 47 (2001) No.8.