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Vacuum-ultraviolet circular dichroism spectrophotometer using synchrotron radiation
Journal of Electron Spectroscopy and Related Phenomena 144–147 (2005) 1023–1025
Vacuum-ultraviolet circular dichroism spectrophotometer using synchrotron radiation K. Matsuo a, ∗ , T. Fukuyama a , R. Yonehara a , H. Namatame b , M. Taniguchi b , K. Gekko a, b a
Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan b Hiroshima Synchrotron Radiation Center, Hiroshima University, Higashi-Hiroshima 739-8526, Japan Available online 16 February 2005
Abstract We have constructed a vacuum-ultraviolet circular dichroism (VUVCD) spectrophotometer using a synchrotron radiation and an assembledtype MgF2 cell endurable under a high vacuum, to measure the CD spectra of biomaterials in aqueous solutions from 310 to 140 nm. To avoid the absorption of light by air and water vapor, all optical devices of the spectrophotometer were set up under a high vacuum (10−4 Pa). A path length of the optical cell can be adjusted by various Teflon spacers in the range from 1.3 to 50 m and its temperature can be controlled to an accuracy of ±1 ◦ C over the range from −30 to 70 ◦ C by a temperature-control unit using a Peltier thermoelectric element. The performance of the spectrophotometer and the optical cell constructed was tested by measuring the CD spectra of ammonium d-camphor-10-sulfonate, dand l-isomers of amino acids, and myoglobin in aqueous solutions. The spectra obtained demonstrate that the optical system and the sample cell constructed operate normally under a high vacuum and provide useful information on the structure of biomolecules based on the higher energy chromophores. © 2005 Elsevier B.V. All rights reserved. Keywords: Biomaterials; Circular dichroism; Synchrotron radiation; Vacuum ultraviolet
1. Introduction Circular dichroism (CD) spectroscopy is a powerful technique for analyzing the structures of optically active materials such as biomolecules. However, no commercial CD spectrophotometer is capable of measuring the CD in the vacuum ultraviolet (VUV) region below 190 nm because of technical difficulties associated with the light source, optical device, and sample cell. The extension of CD measurements into the VUV region could provide more detailed and new information on the structure of biomolecules based on the higher energy transitions of chromophores such as hydroxyl and acetal groups. Considerable effort has been made to construct the vacuum-ultraviolet circular dichroism (VUVCD) spectrophotometer using a synchrotron ra∗
Corresponding author. E-mail address: [email protected] (K. Matsuo).
0368-2048/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.elspec.2005.01.102
diation as a light source [1,2]. However, because all of these spectrophotometers operate only under a nitrogen-gas atmosphere, the short-wavelength limit is currently about 170 nm for aqueous solutions. A VUVCD spectrophotometer operating under a high vacuum would extend the lower wavelength limit. For such a VUVCD measurement, the use of a thin cell is indispensable for attaining a high transmittance in the VUV region by minimizing the absorption of solvent water. However, such a vacuum-tolerant optical cell remains undeveloped due to some technical difficulties, although some thin cells have been used for a nitrogen-gas atmosphere [3]. We constructed a VUVCD spectrophotometer using a small-scale synchrotron radiation source (HiSOR, 0.7 GeV) at the Hiroshima Synchrotron Radiation Center (HSRC) and an assembled-type optical cell with MgF2 windows that can operate under a high vacuum (10−4 Pa). The system can measure the CD spectra of biomolecules
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K. Matsuo et al. / Journal of Electron Spectroscopy and Related Phenomena 144–147 (2005) 1023–1025
in aqueous solutions from 310 to 140 nm at various temperatures.
2. Design and construction of VUVCD spectrophotometer Fig. 1 shows the block diagram of our VUVCD spectrophotometer. All optical devices are located in two vacuum chambers (i.e., polarization modulation chamber and sample chamber) in order to avoid the absorption of light by air and water vapor. The incident light from monochromator is separated into two orthogonal linearly polarized light beams by a Karl Lambrecht magnesium fluoride Rochon prism. Both of these linearly polarized light beams are modulated to circularly polarized light at 50 kHz by a JASCO LiF photoelastic modulator (PEM) [4,5]. An optical servocontrol system is used to control the PEM accurately and to stabilize the lock-in amplifier under a high vacuum. This system implements achromatic modulation and compensation for the thermal drift of the PEM using a double-beam configuration [6]. The main light beam in the center of the chamber is led to the sample cell, and the CD signal is detected with a Hamamatsu R6836 photomultiplier tube. The other light beam is used as the reference signal to synchronize the polarization modulation. In order to protect the sample from damage by VUV irradiation, a shutter opens automatically only during the accumulation of signals.
3. Design and construction of the optical cell An optical cell for VUVCD measurements must meet the following requirements: (I) it must be airtight to avoid the evaporation of the sample solution under a high vacuum; (II) the path length must be adjustable and short enough to minimize light absorption by the solvent water in the VUV region; (III) the cell windows must have a high transmittance and exhibit no birefringence in the VUV region down to 140 nm;
Fig. 1. A block diagram of VUVCD spectrophotometer. A, preamplifier; ANA, analyzer; GV, gate valve; HV, high voltage supply; MR, mirror; PEM, photo-elastic modulator; PM, photomultiplier; POL, polarizer; S, shutter; TMP, turbo molecular pump.
(IV) the cell must be easily assembled and disassembled with good reproducibility, to allow cleaning the cell windows; (V) the temperature of the sample must be adjustable over a wide range. To satisfy these requirements, we designed an assembled-type cell, as shown in Fig. 2a. The cell consists of a stainless-steel container with a cylindrical screw and two MgF2 windows that are 20 mm in diameter and 1 mm thick. A c-axis-cut MgF2 disc is used to eliminate the birefringence of the windows. The optical path length can be adjusted from 1.3 m (without spacer) to 50 m using various donut-shaped Teflon spacers. The sample solution is sealed inside the cell with three fluoride-rubber O-rings. We used a Peltier thermoelectric element system to control the temperature of the sample. A heat-radiation unit of the Peltier thermoelectric element was also constructed, using liquid nitrogen or aqueous ethylene glycol as a coolant. Fig. 2b shows a block diagram of the temperature-control unit. Heat generated by the Peltier thermoelectric element is transferred to the MgF2 cell windows via a copper heat conductor. All of the cell and temperature-control units constructed are located in the vacuum sample chamber of the VUVCD spectrophotometer. The copper jacket is linked through flexible stainless-steel tubes to a liquid-nitrogen reservoir outside the vacuum chamber. The Peltier thermoelectric element and Pt thermistor are connected to a Peltier thermocontroller. The direct current to the Peltier thermoelectric element is controlled using a thermocontroller and the temperature of the heat conductor is monitored by the Pt thermistor and the thermocontroller. We estimated the temperature of the sample solution from the predetermined temperature gradient between the heat conductor and the MgF2 cell windows [7].
4. Performance of the VUVCD spectrophotometer and optical cell The performance of the VUVCD spectrophotometer and optical cell constructed were tested with ammonium dcamphor-10-sulfonate (ACS; Katayama Chemical), which is a standard material of CD spectrum. ACS was dissolved in double-distilled water. Fig. 3a shows the VUVCD spectrum for a 5% ACS aqueous solution from 310 to 140 nm at 25 ◦ C. The baseline signal for water is perfectly straight within an accuracy of ±2 mdeg, indicating the absent of birefringence of the cell windows (MgF2 ). Characteristic peaks are observed at 291 and 192 nm with an intensity ratio of 1:2, as expected for the normal operation of the instrument [8]. No change was found in the spectrum after the sample cell was maintained under a vacuum (10−4 Pa) for 10 h at room temperature, indicating no leakage of the sample solution. The CD spectrum was reproducible within 5% when the spacer and solution were exchanged. Fig. 3b shows the VUVCD spectra of d- and l-isomers of alanine and proline [9]. These two isomers exhibited the symmetrically inverted spectra with positive and negative ellipticities, consistent with the
K. Matsuo et al. / Journal of Electron Spectroscopy and Related Phenomena 144–147 (2005) 1023–1025
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Fig. 2. Block diagram of the optical cell (a) and the temperature-control unit (b).
Fig. 3. (a) VUVCD spectra of 5% ACS aqueous solutions at 25 ◦ C. (b) VUVCD spectra of l-alanine (10%), d-alanine (10%), l-proline (4%) and d-proline (4%) in aqueous solutions at 25 ◦ C. (c) VUVCD spectra of myoglobin (pH 3.7) at −7, 30, and 68 ◦ C. All spectra were recorded with a 1.0 mm slit, a 16 s time constant, a 4 nm/min scan speed, and 4–16 accumulations. Open circles indicate the CD spectra measured by a JASCO J-720W spectropolarimeter.
theoretical expectation for the optical isomers. The spectra of ACS, l-alanine, and l-proline obtained using the VUVCD spectrophotometer completely superposed those obtained using a commercial JASCO J-720W spectropolarimeter. These results confirm a good performance of the present instrument. The performance of the temperature-control unit was tested by monitoring the VUVCD spectra of a model protein, myoglobin (Sigma), at several temperatures. Myoglobin was dialyzed against double-distilled water. The concentration of the protein was determined by an absorption measurement with a molar extinction coefficient of 1.43 M−1 cm−1 at 280 nm. The sample solution was adjusted to pH 3.7 with acetic acid. This protein is known to unfold at both low and high temperatures due to cold and heat denturation, respectively [10]. Fig. 3c shows the VUVCD spectra of myoglobin (pH 3.7) at three temperatures (−7, 30, and 68 ◦ C). The short-wavelength limit of the spectra was 160 nm because of the large absorption by the solvent (acetic acid). It is evident that the spectrum of colddenatured protein at −7 ◦ C differs significantly from that of the heat-denatured one at 68 ◦ C. These results demonstrate that the constructed temperature-control unit operates normally under a high vacuum and that it is useful for studying the effects of temperature on the conformation of biomolecules.
The present study successfully constructed a VUVCD spectrophotometer using synchrotron radiation as a light source and an assembled-type optical cell, which are capable of measuring CD spectra down to 140 nm in aqueous solutions by keeping all the optical devices under a high vacuum. This instrument represents a breakthrough for VUVCD measurements of optically active materials, which is not currently possible using commercial CD spectropolarimeters.
References [1] J.C. Sutherland, E.J. Desmond, P.Z. Takacs, Nucl. Instrum. Meth. 172 (1980) 195. [2] B.A. Wallace, Nat. Struct. Biol. 7 (2000) 708. [3] D.M. Gray, D. Lang, E. Kuner, M. Vaughan, J.C. Sutherland, J. Anal. Biochem. 136 (1984) 247. [4] N. Ojima, K. Sakai, T. Fukazawa, K. Gekko, Chem. Lett. (2000) 832. [5] N. Ojima, K. Sakai, K. Matsuo, T. Matsui, T. Fukazawa, H. Namatame, M. Taniguchi, K. Gekko, Chem. Lett. (2001) 522. [6] T. Fukazawa, Y. Fujita, Rev. Sci. Instrum. 67 (1996) 1951. [7] K. Matsuo, K. Sakai, Y. Matsushima, T. Fukuyama, K. Gekko, Anal. Sci. 19 (2003) 129. [8] T. Takakuwa, T. Konno, H. Meguro, Anal. Sci. 1 (1985) 215. [9] P.L. Privalov, Yu.V. Griko, S.Yu. Venyaminov, V.P. Kutyshenko, J. Mol. Biol. 190 (1986) 487. [10] K. Matsuo, Y. Matsushima, T. Fukuyama, S. Senba, K. Gekko, Chem. Lett. (2002) 826.
Vacuum-ultraviolet circular dichroism spectrophotometer using synchrotron radiation K. Matsuo a, ∗ , T. Fukuyama a , R. Yonehara a , H. Namatame b , M. Taniguchi b , K. Gekko a, b a
Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan b Hiroshima Synchrotron Radiation Center, Hiroshima University, Higashi-Hiroshima 739-8526, Japan Available online 16 February 2005
Abstract We have constructed a vacuum-ultraviolet circular dichroism (VUVCD) spectrophotometer using a synchrotron radiation and an assembledtype MgF2 cell endurable under a high vacuum, to measure the CD spectra of biomaterials in aqueous solutions from 310 to 140 nm. To avoid the absorption of light by air and water vapor, all optical devices of the spectrophotometer were set up under a high vacuum (10−4 Pa). A path length of the optical cell can be adjusted by various Teflon spacers in the range from 1.3 to 50 m and its temperature can be controlled to an accuracy of ±1 ◦ C over the range from −30 to 70 ◦ C by a temperature-control unit using a Peltier thermoelectric element. The performance of the spectrophotometer and the optical cell constructed was tested by measuring the CD spectra of ammonium d-camphor-10-sulfonate, dand l-isomers of amino acids, and myoglobin in aqueous solutions. The spectra obtained demonstrate that the optical system and the sample cell constructed operate normally under a high vacuum and provide useful information on the structure of biomolecules based on the higher energy chromophores. © 2005 Elsevier B.V. All rights reserved. Keywords: Biomaterials; Circular dichroism; Synchrotron radiation; Vacuum ultraviolet
1. Introduction Circular dichroism (CD) spectroscopy is a powerful technique for analyzing the structures of optically active materials such as biomolecules. However, no commercial CD spectrophotometer is capable of measuring the CD in the vacuum ultraviolet (VUV) region below 190 nm because of technical difficulties associated with the light source, optical device, and sample cell. The extension of CD measurements into the VUV region could provide more detailed and new information on the structure of biomolecules based on the higher energy transitions of chromophores such as hydroxyl and acetal groups. Considerable effort has been made to construct the vacuum-ultraviolet circular dichroism (VUVCD) spectrophotometer using a synchrotron ra∗
Corresponding author. E-mail address: [email protected] (K. Matsuo).
0368-2048/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.elspec.2005.01.102
diation as a light source [1,2]. However, because all of these spectrophotometers operate only under a nitrogen-gas atmosphere, the short-wavelength limit is currently about 170 nm for aqueous solutions. A VUVCD spectrophotometer operating under a high vacuum would extend the lower wavelength limit. For such a VUVCD measurement, the use of a thin cell is indispensable for attaining a high transmittance in the VUV region by minimizing the absorption of solvent water. However, such a vacuum-tolerant optical cell remains undeveloped due to some technical difficulties, although some thin cells have been used for a nitrogen-gas atmosphere [3]. We constructed a VUVCD spectrophotometer using a small-scale synchrotron radiation source (HiSOR, 0.7 GeV) at the Hiroshima Synchrotron Radiation Center (HSRC) and an assembled-type optical cell with MgF2 windows that can operate under a high vacuum (10−4 Pa). The system can measure the CD spectra of biomolecules
1024
K. Matsuo et al. / Journal of Electron Spectroscopy and Related Phenomena 144–147 (2005) 1023–1025
in aqueous solutions from 310 to 140 nm at various temperatures.
2. Design and construction of VUVCD spectrophotometer Fig. 1 shows the block diagram of our VUVCD spectrophotometer. All optical devices are located in two vacuum chambers (i.e., polarization modulation chamber and sample chamber) in order to avoid the absorption of light by air and water vapor. The incident light from monochromator is separated into two orthogonal linearly polarized light beams by a Karl Lambrecht magnesium fluoride Rochon prism. Both of these linearly polarized light beams are modulated to circularly polarized light at 50 kHz by a JASCO LiF photoelastic modulator (PEM) [4,5]. An optical servocontrol system is used to control the PEM accurately and to stabilize the lock-in amplifier under a high vacuum. This system implements achromatic modulation and compensation for the thermal drift of the PEM using a double-beam configuration [6]. The main light beam in the center of the chamber is led to the sample cell, and the CD signal is detected with a Hamamatsu R6836 photomultiplier tube. The other light beam is used as the reference signal to synchronize the polarization modulation. In order to protect the sample from damage by VUV irradiation, a shutter opens automatically only during the accumulation of signals.
3. Design and construction of the optical cell An optical cell for VUVCD measurements must meet the following requirements: (I) it must be airtight to avoid the evaporation of the sample solution under a high vacuum; (II) the path length must be adjustable and short enough to minimize light absorption by the solvent water in the VUV region; (III) the cell windows must have a high transmittance and exhibit no birefringence in the VUV region down to 140 nm;
Fig. 1. A block diagram of VUVCD spectrophotometer. A, preamplifier; ANA, analyzer; GV, gate valve; HV, high voltage supply; MR, mirror; PEM, photo-elastic modulator; PM, photomultiplier; POL, polarizer; S, shutter; TMP, turbo molecular pump.
(IV) the cell must be easily assembled and disassembled with good reproducibility, to allow cleaning the cell windows; (V) the temperature of the sample must be adjustable over a wide range. To satisfy these requirements, we designed an assembled-type cell, as shown in Fig. 2a. The cell consists of a stainless-steel container with a cylindrical screw and two MgF2 windows that are 20 mm in diameter and 1 mm thick. A c-axis-cut MgF2 disc is used to eliminate the birefringence of the windows. The optical path length can be adjusted from 1.3 m (without spacer) to 50 m using various donut-shaped Teflon spacers. The sample solution is sealed inside the cell with three fluoride-rubber O-rings. We used a Peltier thermoelectric element system to control the temperature of the sample. A heat-radiation unit of the Peltier thermoelectric element was also constructed, using liquid nitrogen or aqueous ethylene glycol as a coolant. Fig. 2b shows a block diagram of the temperature-control unit. Heat generated by the Peltier thermoelectric element is transferred to the MgF2 cell windows via a copper heat conductor. All of the cell and temperature-control units constructed are located in the vacuum sample chamber of the VUVCD spectrophotometer. The copper jacket is linked through flexible stainless-steel tubes to a liquid-nitrogen reservoir outside the vacuum chamber. The Peltier thermoelectric element and Pt thermistor are connected to a Peltier thermocontroller. The direct current to the Peltier thermoelectric element is controlled using a thermocontroller and the temperature of the heat conductor is monitored by the Pt thermistor and the thermocontroller. We estimated the temperature of the sample solution from the predetermined temperature gradient between the heat conductor and the MgF2 cell windows [7].
4. Performance of the VUVCD spectrophotometer and optical cell The performance of the VUVCD spectrophotometer and optical cell constructed were tested with ammonium dcamphor-10-sulfonate (ACS; Katayama Chemical), which is a standard material of CD spectrum. ACS was dissolved in double-distilled water. Fig. 3a shows the VUVCD spectrum for a 5% ACS aqueous solution from 310 to 140 nm at 25 ◦ C. The baseline signal for water is perfectly straight within an accuracy of ±2 mdeg, indicating the absent of birefringence of the cell windows (MgF2 ). Characteristic peaks are observed at 291 and 192 nm with an intensity ratio of 1:2, as expected for the normal operation of the instrument [8]. No change was found in the spectrum after the sample cell was maintained under a vacuum (10−4 Pa) for 10 h at room temperature, indicating no leakage of the sample solution. The CD spectrum was reproducible within 5% when the spacer and solution were exchanged. Fig. 3b shows the VUVCD spectra of d- and l-isomers of alanine and proline [9]. These two isomers exhibited the symmetrically inverted spectra with positive and negative ellipticities, consistent with the
K. Matsuo et al. / Journal of Electron Spectroscopy and Related Phenomena 144–147 (2005) 1023–1025
1025
Fig. 2. Block diagram of the optical cell (a) and the temperature-control unit (b).
Fig. 3. (a) VUVCD spectra of 5% ACS aqueous solutions at 25 ◦ C. (b) VUVCD spectra of l-alanine (10%), d-alanine (10%), l-proline (4%) and d-proline (4%) in aqueous solutions at 25 ◦ C. (c) VUVCD spectra of myoglobin (pH 3.7) at −7, 30, and 68 ◦ C. All spectra were recorded with a 1.0 mm slit, a 16 s time constant, a 4 nm/min scan speed, and 4–16 accumulations. Open circles indicate the CD spectra measured by a JASCO J-720W spectropolarimeter.
theoretical expectation for the optical isomers. The spectra of ACS, l-alanine, and l-proline obtained using the VUVCD spectrophotometer completely superposed those obtained using a commercial JASCO J-720W spectropolarimeter. These results confirm a good performance of the present instrument. The performance of the temperature-control unit was tested by monitoring the VUVCD spectra of a model protein, myoglobin (Sigma), at several temperatures. Myoglobin was dialyzed against double-distilled water. The concentration of the protein was determined by an absorption measurement with a molar extinction coefficient of 1.43 M−1 cm−1 at 280 nm. The sample solution was adjusted to pH 3.7 with acetic acid. This protein is known to unfold at both low and high temperatures due to cold and heat denturation, respectively [10]. Fig. 3c shows the VUVCD spectra of myoglobin (pH 3.7) at three temperatures (−7, 30, and 68 ◦ C). The short-wavelength limit of the spectra was 160 nm because of the large absorption by the solvent (acetic acid). It is evident that the spectrum of colddenatured protein at −7 ◦ C differs significantly from that of the heat-denatured one at 68 ◦ C. These results demonstrate that the constructed temperature-control unit operates normally under a high vacuum and that it is useful for studying the effects of temperature on the conformation of biomolecules.
The present study successfully constructed a VUVCD spectrophotometer using synchrotron radiation as a light source and an assembled-type optical cell, which are capable of measuring CD spectra down to 140 nm in aqueous solutions by keeping all the optical devices under a high vacuum. This instrument represents a breakthrough for VUVCD measurements of optically active materials, which is not currently possible using commercial CD spectropolarimeters.
References [1] J.C. Sutherland, E.J. Desmond, P.Z. Takacs, Nucl. Instrum. Meth. 172 (1980) 195. [2] B.A. Wallace, Nat. Struct. Biol. 7 (2000) 708. [3] D.M. Gray, D. Lang, E. Kuner, M. Vaughan, J.C. Sutherland, J. Anal. Biochem. 136 (1984) 247. [4] N. Ojima, K. Sakai, T. Fukazawa, K. Gekko, Chem. Lett. (2000) 832. [5] N. Ojima, K. Sakai, K. Matsuo, T. Matsui, T. Fukazawa, H. Namatame, M. Taniguchi, K. Gekko, Chem. Lett. (2001) 522. [6] T. Fukazawa, Y. Fujita, Rev. Sci. Instrum. 67 (1996) 1951. [7] K. Matsuo, K. Sakai, Y. Matsushima, T. Fukuyama, K. Gekko, Anal. Sci. 19 (2003) 129. [8] T. Takakuwa, T. Konno, H. Meguro, Anal. Sci. 1 (1985) 215. [9] P.L. Privalov, Yu.V. Griko, S.Yu. Venyaminov, V.P. Kutyshenko, J. Mol. Biol. 190 (1986) 487. [10] K. Matsuo, Y. Matsushima, T. Fukuyama, S. Senba, K. Gekko, Chem. Lett. (2002) 826.