First Observation of Natural Circular Dichroism for Biomolecules in Soft X-ray Region using a Polarizing Undulator A mino acids are well known chiral molecules, which have handedness or chirality. Right- and left- handed amino acids are called D- and L-amino acid. Life forms on the earth use only L-type amino acids to make proteins. This is known as the homochirality of amino acids (or proteins) in the body of life forms. Although almost all amino acids found in meteorites were a mixture of 50% D-amino and 50% L-amino acid (i.e., racemic), some meteorites such as the Merchison meteorite were found recently to have enrichment of L-amino acids [1]. Among various hypotheses for this enrichment, photochemical enrichment is studied extensively by several groups with the circularly polarized light from the ultraviolet- to-vacuum ultraviolet region using the abundant data of circular dichroism (CD), which is the difference in absorption coefficient μ between left and right circularly polarized lights; CD = μ L – μ R . However, no CD data was available in the soft X-ray region, at which the energy region interaction of electromagnetic wave s and matter is violently stronger than the ultraviolet region. In this work, we attemped at the first time to detect the CD of biomolecules in the soft X-ray region [2]. The basic idea of this work is that the L- and D-amino acids should show CD spectra with the same magnitude but opposite signs and the racemic amino acid should show no CD just like the abundant data obtained in the ultraviolet region and the pioneering soft X-ray work of a metal complex by Goulon e t al. [ 3 ]. T he experiment was carried out at beamline BL23SU by mechanical switching the left and right polarized light with 0.1 Hz using a polarizing undulator. The values of μ L and μ R were determined from the sample photocurrent i divided by the incident photonumber I 0 I I estimated from the photocurrent of the focusing mirror M 3 f 3 or the left and right polarized light, respectively. Amino acid film samples were prepared o n Cu- B e substrates by the vacuum sublimation techni q ue. F ilm thickness was monitored during sublimation using a q uartz oscillator thickness monitor and confirmed after sublimation by H P LC analysis. H P LC analysis also confirmed that no thermal decomposition occurred during vacuum sublimation. F ig ure 1 shows the molecular structures of L- and D-serine, and L- and D-phenylalanine (b) (a) Fig. 1. Molecular structures of amino acids. (a) L- and D-phenylalanine, ( b) L- and D-serine. used i n this experiment. F igure 2 shows the absorption spectrum of a phenylalanine film ( F ig . 2(a)) measured by us in the K - K K edge region of nitrogen atom, CD measured in this work ( F ig. 2(b)), and result of the theoretical calculation ( F ig . 2(c)) by Li Y ang et al. [ 4 ] . As seen in the figure, the sign of CD spectrum of L-phenylalanine is opposite that of D-phenylalanine and the absolute magnitude of the CD spectrum of L-phenylalanine seems to be the same as that of D-phenylalanine. Although somewhat noisy, racemic phenylalanine seems to show no CD. F ig ure 3 shows, in the similar manner, the absorption spectra of serine film ( F ig. 3 ( a)) in the K -edge region of oxygen atom, CD measured in this work ( F ig. 3 ( b)), and result of the theoretical calculation ( F ig . 3 ( c)) by P lashkevych et al. [ 5]. As seen in the figure, the sign of CD spectrum of L- serine is opposite that of D- serine and the absolute magnitude of the CD spectrum of L- serine seems to be the same as that of D- serine. It s hould be noted that theoretical calculations by Y ang et al. [ 4 ] and P lashkevych et al. [ 5] seem to reproduce our experimental result. However, the mechanism of CD is not yet clear. B oth t heo retical 3 3 3 3 3 3 3 3 3 3 3 3 3 3 73 73 73 73 73 73 73 73 73 73 73 73 73 73 73 73 73 73 73 73 73 73 73 73 73 73 73 73 73 73 73 7 7 7 7 7 7 7 7 7 73 73 73 73 73 73 73 73 73 73 73 73 73 73 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 Kazumichi Nakagawa Faculty of Human Development, Kobe University E-mail: nakagawa@kobe-u.ac.jp References [1] J.R. Cronin and S. Pizzarello: Science 275 (1997) 951. [2] M. Tanaka, K. Nakagawa, A. Agui, K. Fujii and A. Yokoya: Physica Scripta (2004) - in press. [3] J. Goulon et al. : Jpn. J. Appl. Phys. 32 Suppl. (1993) 2 8 4. [4] L . Yang et al. : J. Synchrotron Rad. 6 (1999) 70 8 . [5] O . Plashkevych et al. : Chem. Phys. 232 (199 8 ) 49. (a) (b) (c) D-Ser L-Ser Normalized CD Intensity μ L - μ R (10 -2 ) 530 535 540 545 550 555 560 0.0 0.5 1.0 –0.4 –0.3 –0.2 –0.1 0.0 0.1 0.2 0.3 –0.002 0.000 0.002 Normalized XANES (a.u.) Photon Energy (eV) Photon Energy (eV) 530 535 540 545 550 555 560 525 Rotary Strength (10 -40 ) cgs (a) (b) (c) D-Phe rac-Phe L-Phe Normalized μ L - μ R Intensity (10 -2 ) 400 405 410 415 1.0 0.5 0.0 0.2 0.1 0.0 –0.1 –0.2 0.000 0.005 0.010 Normalized XANES (a.u.) ∆σ (Mb) Photon Energy (eV) Fig. 3. (a) Absorption spectrum of a serine film. (b) CD spectra measured in this work. (c) Result of the theoretical calculation by Plashkevych et al. [5]. Fig. 2. (a) Absorption spectrum of a phenylalanine film. (b) CD spectra measured in this work. (c) Result of the theoretical calculation by Yang et al. [4]. calculations were carried out on the basis of the interference between the electric dipole (E1) and the magnetic dipole (M1) transitions. O n the other hand, the ESRF group headed by Goulon ascribed many CD transitions in the hard X -ray region to be the E1- E2 interference mechanism. Moreover, in the U V -to- V U V region, the mechanism of CD is believed to be the E1-M1 mechanism. B ecause the soft X -ray region is the bridge energy region between U V and hard X -ray, the mechanism from which CD arise s is a challenging subject for e x perimental and theoretical physicists and chemists. 74
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