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21世紀COE外国旅費補助・出張報告会 （2006年6月12日） Hydration Number of Biomolecules Evaluated with THz Time-Domain Attenuated Total Reflection Spectroscopy 物理学第一教室 光物性研究室D1 有川 敬 会議名称：CLEO/QELS and PhAST 2006 開催期間：2006年5月21～26日 開催場所：アメリカ、カリフォルニア州、ロングビーチ 1. CLEO/QELS and PhASTについて 2. 分野の説明 3. 発表内容、成果 CLEO/QELS and PhASTについて •３つの会議の合同開催（昨年の参加者5281人） CLEO : レーザー、電気光学 QELS : 量子エレクトロニクス、レーザーサイエンス PhAST : 光技術応用 •CLEO Topics •固体レーザー、半導体レーザー •ＬＥＤ、有機ＬＥＤ •ファイバーレーザー、増幅器 •非線形光学応用 •高強度レーザー、物理 •超高速光学、電気光学、応用 •テラヘルツ技術、応用 •薬学、生物学応用 •光通信、ネットワーク •レーザー加工 •光センシング、計測 など 分野の説明（テラヘルツ電磁波とは） •テラヘルツ電磁波（1テラ＝1012）は、ミリ波と赤外線の中間の周波数帯に位置する電磁波。 •この周波数領域には様々な物理現象が見られます。 配向緩和 300MHz 3GHz 1m 100mm 電波 プラズマ振動 30GHz 10mm マイクロ波 分子間振動 300GHz 3THz 1m 100mm 遠赤外 分子内振動 30THz 10mm 300THz 1mm 電子遷移 3PHz 100nm 中赤外 近赤外 紫外 X線回折 10nm 1nm X線 可視光 電波 THz領域 光 0.1nm g線 テラヘルツ領域の物理現象 ●孤立した分子（気体） －回転準位 ●巨大分子 －分子間振動モード（有機分子） －生体機能に関係する集団運動（生体高分子） ●固体 －フォノン振動 －強誘電ソフトモード（誘電体） －電子集団励起（半導体） －超伝導ギャップ（超伝導体） ●液体 －水素結合ネットワーク（水） －水和（水溶液） テラヘルツ電磁波を用いた研究例① Build-up of collective behavior in an electron-hole plasma in GaAs Nature Vol. 414 (2001) テラヘルツ電磁波を用いた研究例② 違法薬物、危険物の透視 60 50 40 30 20 10 0 0 10 20 30 40 50 60 mm Ｘ線と補完的なイメージング光源 本研究の対象 ●孤立した分子（気体） －回転準位 ●巨大分子 －分子間振動モード（有機分子） －生体機能に関係する集団運動（生体高分子） ●固体 －フォノン振動 －強誘電ソフトモード（誘電体） －電子集団励起（半導体） －超伝導ギャップ（超伝導体） ●液体 －水素結合ネットワーク（水） －水和（水溶液） 水和水 生体分子 バルク水 Hydration Number of Biomolecules Evaluated with THz Time-Domain Attenuated Total Reflection Spectroscopy Takashi Arikawa*, Masaya Nagai, Koichiro Tanaka Department of Physics, Kyoto University, Japan *JSPS Research Fellow CLEO/QELS ’06 Long beach, California, USA (May 21-26, 2006) Background of this study Bulk water ~ps Hydrated H2O water •Protein folding •Expression of biological function What is the role of water? 10ms ~ ns Biomolecule H2O Interaction between biomolecule and water Hydrated water ↑Hydration model of biomolecule1 One indicator of the strength of interaction between biomolecule and water [1] H. S. Frank and Wen –Y. Wen, Disc. Faraday Soc., 24, 133 (1957) Hydration of Sucrose(C12H22O11) Method Hydration Number Time Scale Calorimetric Measurement1 6.33 ~100s Ultrasound Measurement2 13.8 ~10-6s MD calc3 20.8 Discrepancy Bulk Water Whole number of hydrated water molecules Solute = Polarizability (Im) Reason: Different time scales of the experimental perturbation methods Decrement of bulk water molecules Hydrated Water MHz Ultrasound GHz THz [1] H. Kawai et al., Cryobiology 29, 599 (1992) [2] C. Branca et al., J. Phys. Chem. B 105, 10140 (2001) [3] A. Lerbret et al., J. Phys. Chem. B, 109, 11046 (2005) Purpose of This Study ❑Evaluate the whole hydration number of biomolecules from THz dielectric properties of water solution. Solute Molecule (molecular formula ) Disaccharide Method : THz Time-domain Attenuated Total Reflection Spectroscopy1 Sucrose (C12H22O11) Trehalose (C12H22O11) Amino acid Alanine （C3H7O2N） Arginine (C6H14O2N4) THz wave Dove Prism (MgO) [1] H. Hirori et al., Opt. Express 13, 10801 (2005) K. Tanaka CLEO/IQEC 06, CMCC1 Dielectric Constant of Sucrose Solution Characteristics of dielectric response in water solution 6 Dielectric Constant 4 2 Crystal r Molecule Molecule •A solution with higher concentration has smaller absorption (i) 0 Water 0.149 mol/l 0.305 mol/l 0.633 mol/l 0.989 mol/l 6 4 2 0 0.0 i → Decrease of bulk water & Increase of hydrated water •No dispersion around 1.45 THz Crystal 0.5 •High frequency tail of debye relaxation of bulk water → Sucrose is surrounded by hydrated water 1.0 Frequency [THz] 1.5 10 12 Binary Mixture Model (Onsager’s Local Field theory) ~ 1 P 0E ( Pw 0E ~ Ps H2O H2O H2O Sucrose Pw H2O H2O H2O H2O Ps ) H2O Pi N i i F Ni : Number density i : Polarizability F : Local field Approximate calculation of local field ●Lorentz field ~ 2 ~ F ( ) E 3 E ●Onsager’s theory1 Considering the effect of permanent dipole ~ F ( ) r a 1 r 3 ~ ~ g ( ) m 3 ~ 3 E ~ a 1 r ( ) a [1] N. E. Hill “Dielectric Properties and Molecular Behaviour” Polarizability [10 4 Real Part Different value of polarizability depending on the concentration Sucrose 2 Physically inconsistent 0 Reason : Hydration effect is not considered -2 0 Imaginary Part -2 0.149 mol/l 0.305 mol/l 0.633 mol/l 0.989 mol/l -4 -6 -8 0.0 0.5 1.0 12 Frequency [10[THz] Hz] Frequency 1.5 Polarizability (Im) 2 -1 Cm-12V ] 2 -1 2 10 -12 -1 2 10 Orientational Polarizability [10 Cm V-1] Orientational Polarizability of Sucrose Bulk water Solute Hydrated water MHz GHz THz Modified Binary Mixture Model Bulk water H2O H2O H2O H2O H2O Assumption H2O Sucrose H2O H2O H2O H2O H2O H2O Volume of hydrated sucrose װ Volume of sucrose Hydrated water : set orientational polarizability to zero Solution = Hydrated sucrose (Sucrose & nH hydrated water molecules) + Bulk water (a)Real Part 2 0 -1 2 10 -2 2 Orientational Polarizability [10-12Cm2V-1] 4 -12 10 -1 2 2 -1 Polarizability V -12 ] Cm2V-1] [10 Cm[10 Polarizability Orientational Determination of Hydration Number & Polarizability Sucrose •Hydration number is 19.3 (MD calculation : 20.8) •Orientational polarizability is zero (b)Imaginary Part = 19 nnHH=19.3 0 -2 -4 0.0 0.5 1.0 1.5 Frequency [THz] 10 12 Hydration Number Molecule Sucrose (C12H22O11) Disaccharide Trehalose (C12H22O11) Alanine （C3H7O2N） Amino acid Arginine (C6H14O2N4) H2 O D2O Alanine Sucrose 19.3±0.2 24.7±0.1 (20.81) 20.6±0.1 26.35±0.1 Arginine 1 Trehalose (22.9 ) 5.47±0.01 6.99±0.01 (3.482) 14.0±0.1 17.9±0.1 [1] (MD calculation) A. Lerbret et al., J. Phys. Chem. B 109, 11046 (2005) [2] (Monte Carlo method) T. K. Kim et al., J. Mol. Liquids 59, 179 (1994) D2O/H2O 1.28 1.28 1.28OH OH HOH2C o 1.28 OH 10 -1 3 -12 10 -1 3 2 -1 Polarizability V -12] Cm2V-1] [10 Cm [10 Polarizability Orientational Determining Condition of hydration number 0.44 0.33 0.22 Zero-Polarizability condition (a) Real Part (Onsager’s Model) H2O Sucrose + water numberHydrated is determined Hydration 0.11 so that the vector sum of 0 permanent dipoles of solute and (b) Imaginary Part 0.33 hydrated water becomes zero nH m s m w,i 0 i ms 0.22 0.11 0 0.0 Solute 0.5 1.0 mw 1.5 Frequency [THz] 10 12 *J. Higo et al., Proc. Natl. Acad. Sci. U.S.A. 98 (11) 5961 (2001 ) Formula for Hydration Number and Isotope Effect High frequency polarizability of solute and water Volume of a solute molecule a 3 s 2 ( s 1 ) (2 s 1 ) s nH 4 Static dielectric constant of Solution w , D 2 O w , H 2 O 1 . 28 nH , D 2 O 1.28 nH , H 2 O 0 w Summary Hydration of biologically-relevant molecules studied by THz TD-ATR spectroscopy 1. Hydration number is determined so that the vector sum of permanent dipoles of solute and hydrated water becomes zero. 2. Hydration number is determined by static dielectric constant of solution s, volume of a solute a3, and high frequency polarizability of solute and solvent s(w). 3. Evaluated hydration numbers are in good agreement with MD calculation results (in the case of disaccharides). 発表成果 •テラヘルツ電磁波を水溶液中の生体分子の水和の研究に用いた研究例は他にはない •大きな会議で、多くの聴衆の前で、内容的にインパクトのある講演ができたと思う