Technical Note pubs.acs.org/ac
Assessing the Impact of Synchrotron X‑ray Irradiation on Proteinaceous Specimens at Macro and Molecular Levels Mehdi Moini,*,† Christopher M. Rollman,† and Loïc Bertrand‡,§ †
George Washington University, Department of Forensic Sciences, Washington, D.C., 20007, United States IPANEMA, CNRS, MCC, USR 3461, BP48 Saint-Aubin, F-91192 Gif-sur-Yvette, France § Synchrotron SOLEIL, BP48 Saint-Aubin, F-91192 Gif-sur-Yvette, France ‡
S Supporting Information *
ABSTRACT: Synchrotron radiation (SR) has become a preferred technique for the analysis of a wide range of archeological samples, artwork, and museum specimens. While SR is called a nondestructive technique, its effect on proteinaceous specimens has not been fully investigated at the molecular level. To investigate the molecular level effects of synchrotron X-ray on proteinaceous specimens, we propose a methodology where four variables are considered: (1) type of specimen: samples ranging from amino acids to proteinaceous objects such as silk, wool, parchment, and rabbit skin glue were irradiated; (2) synchrotron X-ray energy; (3) beam intensity; (4) irradiation time. Irradiated specimens were examined for both macroscopic and molecular effects. At macroscopic levels, color change, brittleness, and solubility enhancement were observed for several samples within 100 s of irradiation. At molecular levels, the method allowed one to quantify significant amino acid modifications. Aspartic acid (Asp), wool, parchment, and rabbit skin glue showed a significant increase in Asp racemization upon increasing irradiation time with rabbit skin glue showing the greatest increase in D-Asp formation. In contrast, Asp in silk, pure cystine (dimer of cysteine), and asparagine (Asn) did not show signs of racemization at the irradiation times studied; however, the latter two compounds showed significant signs of decomposition. Parchment and rabbit skin glue exhibited racemization of Asp, as well as racemization of isoleucine (Ile) and phenylalanine (Phe) after 100 s of irradiation with a focused beam. Under the experimental conditions and sample type and dimensions used here, more change was observed for focused and low energy (8 keV) beams than unfocused or higher energy (22 keV) beams. These results allow quantification of the change induced at the molecular level on proteinaceous specimens by synchrotron X-ray radiation and help to define accurate thresholds to minimize the probability of damage occurring to cultural heritage specimens. For most samples, damage was usually observed in the 1−10 s time scale, which is about an order of magnitude longer than SR studies of cultural heritage under X-ray fluorescence (XRF) mode; however, it is consistent with the duration of X-ray absorption spectroscopy (XAS) and microcomputed tomography (μCT) measurements.
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photometry,18 crystallography,19 and wide-angle X-ray scattering20 have been used. Raman and FT-IR analysis showed that KrF excimer laser is capable of inducing conformational changes in irradiated collagen film mainly as a result of the breaking of the hydrogen bond network and loss of water molecules which maintain the ordered structure.21 Also, fluorescence measurements showed that the UV light is capable of causing tyrosine oxidation by generating excited states or radical photoionization.22 Moreover, it has been shown that Xray free electron lasers (X-FEL) induce damage to biological specimens almost immediately after irradiation at high flux levels.22 Diffuse reflectance infrared Fourier transform (DRIFT) analysis of parchment subjected to excimer laser irradiation showed no auto oxidation below destruction
ynchrotron radiation (SR) and laser sources are today frequently used for the analysis of a wide range of precious and rare specimens, as well as biological samples.1−9A variety of X-ray modes such as fluorescence, diffraction, small-angle scattering, tomography, and X-ray absorption spectroscopy have been applied to these specimens.1−7 Depending on the type of analysis, the information sought, and the type of sample, a wide variety of energy levels and photon fluxes has been utilized.2−11 Most spectroscopic techniques that are used to study works of art using intense radiation sources are labeled as “non-destructive” based on microscopic analysis and stress/ strain testing;12−14 however, from the early 20th century, researchers realized the adverse effects of intense UV and X-ray radiation on proteinaceous samples. This early work was reviewed in a 1936 article.15 Originally, ultracentrifugation and light scattering techniques were used to detect both cleavage and aggregation of the protein molecules; however, more recently, infrared spectroscopy,16,17 ultraviolet−visible spectro© 2014 American Chemical Society
Received: July 18, 2014 Accepted: September 3, 2014 Published: September 3, 2014 9417
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Table 1. Information and Experimental Conditions for Analyzed Samples sample L-cystine L-cystine L-asparagine L-asparagine L-aspartic acid parchment parchment rabbit skin glue rabbit skin glue rabbit skin glue modern wool modern wool modern silk modern silk
energy (keV)
focused/unfocused
time (s)
8 22 8 22 8 8 22 8 8 22 8 22 8 22
unfocused focused unfocused focused unfocused focused focused unfocused focused focused focused focused focused focused
12, 120, 1200 100 12, 120, 1200 100 12, 120, 1200 1, 100 100 10, 1000 10, 100 100 1, 10, 100 100 1, 10, 100 100
D/L-Asp
(%)
1.4, 1.3, 1.3 1.4 0, 0, 0 0 0, 0.6, 5.8 6.0, 10.4 7.6 11.5, 18.3 12.7, 25.2 18.6 1.6, 2.5, 3.6 1.6 2.5, 2.2, 2.7 1.3
identified fragments deaminated, decarboxylated, cystine dioxide cystine dioxide aspartic acid, decarboxylated aspartic acid decarboxylated N/A N/A N/A N/A N/A N/A N/A N/A N/A
only a 1 × 1 mm2 section of the X-ray with uniform beam intensity was selected (using slits) for sample irradiation. For samples longer than 1 mm, a program was used to scan the specimen with the beam using the desired irradiation time. Moreover, since the thickness of the specimens was usually less than the penetration depth of the X-ray beams and the size of the sample selected for MS analysis included several X-ray spots with slightly overlapped edges to ensure complete irradiation, the data shown in this paper represent irradiation of the entire sample. A camera, positioned in the X-ray beam path behind the sample, was used to measure the flux and beam profile (Basler). The flux of the 8 keV unfocused X-ray beam was estimated at 6.3 × 1012 Ph/s (1 × 1 mm2 beam size). The flux for the 8 and 22 keV focused X-ray beams were 9.9 × 1012 Ph/s (90 × 480 μm2 beam size) and 9.4 × 1011 Ph/s (75 × 660 μm2 beam size), respectively. Sample Analysis. Table 1 provides the list of samples investigated during this study. ∼250 μg of the completely irradiated amino acid samples was removed from the tubes and dissolved in 50 μL of 0.1 N HCl for CE-MS analysis without any further sample preparation. Irradiated portions of the proteinaceous specimens (∼75 μg) were digested in 6 N HCl at 110 °C for 2 h, dried in a vacuum centrifuge, and then redissolved in ∼15 μL of 0.1 N HCl for CE-MS analysis. Analyses were performed on a capillary electrophoresis (Beckman Coulter ProteomeLab PA 800, Fullerton, CA) interfaced to a Finnigan LCQ Duo mass spectrometer (San Jose, CA) using a porous tip.35 Electrospray voltage was set at 1.1 kV, and the mass spectrometer heated capillary was 200 °C. Amino acid fragmentation analyses were performed using 0.5 M formic acid, while racemization analyses were performed using a 30 mM solution of (+)-(18-crown-6)-2,3,11,12tetracarboxylic acid (18-crown-6-TCA) in water as background electrolytes.36
thresholds, but it was noted that the age of the parchment does have an effect on the threshold.23 Another study of the effects of laser irradiation on parchment showed no FT-Raman changes; however, fluorescence changes related to photodegradation could be seen.24 Furthermore, Young concludes that, though parchment suffers a loss of thermal stability (tested using thermal microscopy) when exposed to “high flux” X-ray radiation (5 × 1012 Ph/mm2), accelerated aging studies performed after irradiation showed no significant influence in the long term.13 Previous X-ray and gamma-ray studies on the sulfur containing amino acids resulted in significant molecular change.25−28 A study of damage to proteins by synchrotron Xray (flux of 5 × 1012 Ph/s) at cryogenic temperatures reports disulfide bond breakages and decarboxylation.29 Kminek and Bada studied the effects of intense γ radiation on amino acids for the purpose of cosmogeochemistry and found that amino acids will degrade substantially unless they are shielded (i.e., about 2 m below the surface of Mars).30 Ionizing radiation is used in foods to prevent microbial growth, and a study of the effects of gamma irradiation on rice proteins showed a dosedependent increase in soluble free amino acid content.31 Furthermore, numerous studies report increased solubility of silk with exposure to gamma, electron beam, or UV radiation.32,33 Previously, we studied the effect of UV radiation on racemization of aspartic acid (Asp) in silk. It was observed that irradiation of 1920 h resulted in a 2% increase of D/L for Asp.34 In this article, we propose an approach to measure the effect of synchrotron X-ray radiation on several amino acids, a dipeptide (cystine), and proteinaceous specimens at both macro and molecular levels.
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EXPERIMENTAL SECTION Sample Irradiation. All sample irradiations took place at the SOLEIL synchrotron source in Gif-sur-Yvette, France at the CRISTAL beamline. Detailed experimental conditions are documented in the Supporting Information. Solid samples (Lcystine, L-Asn (asparagine), L-Asp) were placed in glass X-ray capillaries (O.D. 0.9 or 1.0 mm and wall thickness of 1/100 mm). Parchment samples were mounted using a microvice, and fiber samples were taped to a washer to hold the sample in the middle of the washer. Samples were irradiated at two energy levels (8 and 22 keV) using either an unfocused or focused Xray beam for various lengths of time (1−1200 s). The size of the unfocused beam was approximately 1 × 3 mm2; however, since most samples were not thicker than 1 mm in diameter,
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RESULTS AND DISCUSSION Our approach allows one to study of the effect of synchrotron X-ray radiation on proteinaceous specimens, through three factors: (1) visible change (For this study, the specimens were monitored by the naked eye and by taking microscopy images.); (2) molecular fragmentation (Amino acids were analyzed by CE-MS using 0.5 M formic acid in water to separate and detect molecular fragmentation.); (3) amino acid racemization (Amino acids and the HCl digests of proteins and proteinaceous specimens were analyzed by CE-MS using 9418
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Figure 1. Light microscopy images of L-cystine, wool, and rabbit skin glue (top to bottom) showing visible damage as a result of increasing radiation duration. Cystine was irradiated with 8 keV unfocused X-ray, and the wool and rabbit skin glue were irradiated with 8 keV focused X-ray.
(+)18-crown-6-TCA as the BGE to investigate the formation of D-amino acid resulting from radiation.). Furthermore, the effect of the sample type, beam energy, beam intensity, and irradiation time were investigated. Visible Change. Figure 1 shows pictures of cystine, rabbit skin glue, and wool before and after irradiation for various times. As is evident in Figure 1, these samples showed dramatic visible change (damage) with increased irradiation time. After 1200 s of unfocused, 8 keV irradiation, cystine went from white to green and yellow in color. Using focused 8 keV radiation, the wool sample became faint yellow at 10 s and darker yellow at 100 s, while rabbit skin glue became opaque within 100 s. Photoyellowing of wool induced by UV radiation has been reported previously.16,37 In addition to color change, significant mechanical and solubility changes were also observed. The wool that was irradiated for 100 s became very brittle and fell apart easily. While unirradiated silk and parchment did not dissolve in methanol, upon irradiation with an 8 keV focused beam for 10 s, they dissolved in methanol with ease, which is consistent with previous studies.32,33 Molecular Fragmentation. The effect of synchrotron Xray radiation was studied on two amino acids (Asn, Asp; Figure 2A,B) and one dipeptide (cystine; Figure 2C) at two irradiation energies and several irradiation times. As shown, all three compounds showed fragmentation and other molecular level changes upon irradiation. Figure 2A1−A3 shows increased fragmentation of asparagine and its conversion to aspartic acid upon increased irradiation time. With 100 s of 22 keV focused irradiation, no fragmentation was observed for Asn (data not shown). Aspartic acid fragmentation included decarboxylation of both carboxylic acids. Increasing irradiation time to 1200 s significantly increased the intensities of the decarboxylated peaks (Figure 2B1−B3). Cystine showed the greatest
fragmentation or molecular changes which included major decarboxylation, deamination, and formation of cystine dioxide (Figure 2C1−C3). Using the 8 keV unfocused beam, asparagine appeared to be least affected by irradiation, while cystine was the most affected. Modifications of cystine included decarboxylation, deamination, and oxidation, as well as numerous other unidentified modifications. However, using the 22 keV focused beam, the only molecular change observed in cystine was the formation of cystine dioxide (data not shown). Amino Acid Racemization. Using unfocused 8 keV beam and irradiation times of up to 1200 s, no racemization was observed for L-cystine and L-Asn. As shown in Figure 2B, increased irradiation time increased the formation of Asp from Asn; however, the amount of aspartic acid formed by irradiation of asparagine was too low to quantitate its racemization. Also, no racemization was observed for Asn and cystine with irradiation by the focused, 22 keV X-ray for 100 s. However, racemization of Asp increased by 5.8% using the unfocused 8 keV beam for 1200 s (see Figure 3A). In terms of racemization, Asp appears to be the most susceptible free amino acid to X-ray irradiation. Effect of Synchrotron X-ray Radiation on Silk, Wool, Parchment, and Rabbit Skin Glue. A modern, degummed, and undyed silk fiber showed no increase in D-aspartic acid with an irradiation of up to 100 s using the focused 8 keV beam; however, under the same conditions, a modern, undyed wool fiber showed an increase of ∼2% D-Asp compared with the unirradiated fibers (Figure 3B). Under the same irradiation time but using the higher energy X-ray (22 keV), no increase in D-Asp was observed for silk and wool fibers compared to the unirradiated ones. Irradiation of an ∼500 year old parchment specimen with a focused 8 keV beam for 100 s produced a D/L9419
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Figure 2. Electropherograms showing fragmentation and modifications of Asn (A1−A3), Asp (B1−B3), and cystine (C1−C3) with increasing irradiation time using an 8 keV unfocused X-ray. Unlabeled peaks have not been identified.
Overnight irradiation of ∼10 000 year old bone (Horn Shelter, TX) with 8 keV unfocused beam produced no noticeable change in racemization of Asp in bone proteins.38 As shown in Figure 3, under the experimental conditions used in this study including beam flux, irradiation time, and sample dimensions, D/L appears to increase linearly by time; however, more experiments will be conducted in the future to thoroughly investigate this trend. Racemization of Amino Acids Other than Asp in Proteinaceous Specimens. The racemization of other amino acids (in addition to Asp) in the various samples was
Asp increase of 4.7% (Figure 3C), while irradiating the parchment specimen at higher energy (22 keV) for 100 s resulted in a D/L-Asp increase of only 1.9%. The lower energy beam produced more molecular level change due to its higher absorption cross section. Rabbit skin glue is commonly used in museum specimens as an adhesive and sealant. When solid rabbit skin glue was irradiated with a focused 8 keV X-ray for 100 s, the D/L-Asp more than doubled from 12.1% to 25.2% (Figure 3D). At higher energy X-ray (22 keV), however, the D/L-Asp increased to 18.6%. Again, higher energy X-ray caused less damage. 9420
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Figure 3. Graphs showing effect of radiation on the D/L-Asp of aspartic acid irradiated with 8 keV unfocused (A), wool fiber irradiated with 8 keV focused (B), parchment irradiated with 8 keV focused (C), and rabbit skin glue irradiated with 8 keV focused (D) samples with insets showing ion electropherograms of D- and L-isomers of Asp.
variables. First, the type of specimen: of the specimens studied here, rabbit skin glue showed the greatest increase in D-Asp upon irradiation. Parchment was the second most affected, and the fiber samples (silk and wool) were the least susceptible to D-Asp formation. Second, the energy of the X-ray: generally, the higher the energy, the lower is the extent of racemization. For example, the rabbit skin glue and parchment samples showed increased D-Asp formation at 22 keV; however, the increase was less than that of the same irradiation time at 8 keV. This is because higher energy X-ray is more penetrating, resulting in less damage in thin specimens. Third, beam intensity: in general, the focused beam produced more damage than the unfocused beam. Finally, irradiation time: the extent of molecular level effects was directly related to irradiation time, and as expected, longer irradiation times produced more change. For most samples, damage was usually observed in the 1−10 s time scale, which is about an order of magnitude longer than SR studies of cultural heritage under XRF mode; however, it is consistent with the duration of XAS and μCT measurements.
investigated. In the parchment sample, there was also an increase in the formation of D-Ile (isoleucine) and D-Phe (phenylalanine) with increasing irradiation time. For the parchment sample irradiated with 8 keV focused X-ray, the D/L of Ile increased from 0.6% (unirradiated) to 4% (100 s irradiation). Also, D/L of Phe increased from 2.2% (unirradiated) to 3.9% (100 s irradiation). In the rabbit skin glue sample, irradiation with an 8 keV unfocused X-ray resulted in a D-Ile increase of ∼2% and D-Phe increase of ∼1%, both with a 1000 s irradiation time. Under 8 keV focused X-ray and 100 s of irradiation time, the rabbit skin glue had a D/L-Ile increase of ∼5% and D/L-Phe increase of ∼2%. As expected, this data suggests that focused beam (higher flux) leads to increased racemization in biological molecules at the same energy level. As shown in Figure 3, for most samples, damage was usually observed in the 1−10 s time scale, which is about an order of magnitude longer than SR studies of cultural heritage under XRF or XRD modes; however, it is in the typical irradiation time range of XAS or μCT where irradiation can be on the order of minutes to ∼an hour.8,9,39 In these circumstances, partial molecular level modification may have occurred before reaching any XAS characteristic edge feature.
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ASSOCIATED CONTENT
S Supporting Information *
CONCLUSIONS The proposed method allowed one to measure the significant degradation undergone by free amino acids upon irradiation with synchrotron X-ray including fragmentation, modification (Asn, Asp), and racemization (Asp). Cystine shows color changes, oxidation, and fragmentation. Furthermore, proteinaceous specimens subjected to synchrotron X-ray exhibited color change (wool and rabbit skin glue) and racemization of several of their constituent amino acids (Asp, Ile, Phe). The extent of racemization induced by X-ray irradiation depends on several
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest. 9421
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Technical Note
(27) Markakis, P.; Tappel, A. L. J. Am. Chem. Soc. 1960, 82, 1613− 1617. (28) Owen, T. C.; Rodriguez, M.; Johnson, B. G.; Roach, J. A. G. J. Am. Chem. Soc. 1968, 90, 196−200. (29) Weik, M.; Ravelli, R. B. G.; Kryger, G.; McSweeney, S.; Raves, M. L.; Harel, M.; Gros, P.; Silman, I.; Kroon, J.; Sussman, J. L. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 623−628. (30) Kminek, G.; Bada, J. L. Earth Planet. Sci. Lett. 2006, 245, 1−5. (31) Maity, J. P.; Chakraborty, S.; Kar, S.; Panja, S.; Jean, J.-S.; Samal, A. C.; Chakraborty, A.; Santra, S. C. Food Chem. 2009, 114, 1237− 1244. (32) Kojthung, A.; Meesilpa, P.; Sudatis, B.; Treeratanapiboon, L.; Udomsangpetch, R.; Oonkhanond, B. International Biodeterioration 2008, 62, 487−490. (33) Ishida, K.; Takeshita, H.; Kamiishi, Y.; Yoshii, F. Radiation Degradation of Silk. In Proceedings of the Takasaki Symposium on Radiation Processing of Natural Polymers, Takasaki, Japan; Japan Atomic Energy Research Institute: Tokai-mura, Japan, 2001; pp 130−138. (34) Moini, M.; Klauenberg, K.; Ballard, M. Anal. Chem. 2011, 83, 7577−7581. (35) Moini, M. Anal. Chem. 2007, 79, 4241−4246. (36) Schultz, C. L.; Moini, M. Anal. Chem. 2003, 75, 1508−1513. (37) Zhang, H.; Cookson, P.; Wang, X. Text. Res. J. 2008, 78, 1004− 1010. (38) Moini, M.; Rollman, C. M.; France, C. M. F. Anal. Chem. 2013, 85, 11211−11215. (39) Smith, E.; Kempson, I.; Juhasz, A. L.; Weber, J.; Skinner, W. M.; Grafe, M. Chemosphere 2009, 76, 529−535.
ACKNOWLEDGMENTS We thank Dr. Sylvain Ravy (Synchrotron SOLEIL), Dr. Felisa Berenguer, and Ms. Alessandra Vichi (IPANEMA) for their experimental help and Dr. Matija Strlic (University College London) for providing the parchment sample. Beam time was funded by Synchrotron SOLEIL under project no. 20120976. This material is based upon work supported by the National Science Foundation under grant numbers CHE 1241672 and CHE 1440849. The IPANEMA platform (CNRS, MCC) is supported by a CPER grant (MENESR, Région Il̂ e-de-France).
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REFERENCES
(1) Lang, J.; Middleton, A., Eds. Radiography of Cultural Material; Elsevier: Burlington, MA, 2005. (2) Cotte, M.; Susini, J.; Dik, J.; Janssens, K. Acc. Chem. Res. 2010, 43, 705−714. (3) Howard, D. L.; de Jonge, M. D.; Lau, D.; Hay, D.; Varcoe-Cocks, M.; Ryan, C. G.; Kirkham, R.; Moorhead, G.; Paterson, D.; Thurrowgood, D. Anal. Chem. 2012, 84, 3278−3286. (4) Dumont, M.; Zoeger, N.; Streli, C.; Wobrauschek, P.; Falkenberg, G.; Sander, P. M.; Pyzalla, A. R. Powder Diffr. 2009, 24, 130−134. (5) Kennedy, C. J.; Wess, T. J. The Use of X-ray Scattering to Analyse Parchment Structure and Degradation. In Physical Techniques in the Study of Art, Archaeology and Cultural Heritage; Bradley, D.; Creagh, D., Eds.; Elsevier B. V.: Burlington, MA, 2006; pp 151−172. (6) Bertrand, L.; Cotte, M.; Stampanoni, M.; Thoury, M.; Marone, F.; Schöder, S. Phys. Rep. 2012, 519, 51−96. (7) Bertrand, L.; Robinet, L.; Thoury, M.; Janssens, K.; Cohen, S. X.; Schöder, S. Appl. Phys. A: Mater. Sci. Process. 2012, 106, 377−396. (8) Hummer, A. A.; Rompel, A. Metallomics 2013, 5, 597−614. (9) Lombi, E.; Scheckel, K. G.; Kempson, I. M. Environ. Exp. Bot. 2011, 72, 3−17. (10) Garside, P.; O’Connor, S. e-Preservation Sci. 2007, 4, 1−7. (11) Van der Snickt, G.; Janssens, K.; Dik, J.; De Nolf, W.; Vanmeert, F.; Jaroszewicz, J.; Cotte, M.; Falkenberg, G.; Van der Loeff, L. Anal. Chem. 2012, 84, 10221−10228. (12) Machnowski, W.; Gutarowska, B.; Perkowski, J.; Wrzosek, H. Text. Res. J. 2013, 83, 44−55. (13) Young, G. Effect of High Flux X-radiation on Parchment; Canadian Conservation Institute: Ottawa, Ontario, 2005. (14) Mantler, M.; Klikovits, J. Adv. X-Ray Anal. 2004, 47, 42−46. (15) Arnow, L. E. Physiol. Rev. 1936, 16, 671−685. (16) Millington, K. R.; Church, J. S. J. Photochem. Photobiol. B: Biol. 1997, 39, 204−212. (17) Osman, E. M.; Michael, M. N.; Gohar, H. Int. J. Chem. 2010, 2, 28−39. (18) Nicholls, C. H.; Pailthorpe, M. T. J. Text. Inst. 1976, 67, 397− 403. (19) Ravelli, R. B. G.; McSweeney, S. M. Structure 2000, 8, 315−328. (20) Sangappa; Asha, S.; Parameswara, P.; Somashekar, R. Bull. Mater. Sci. 2011, 34, 1583−1590. (21) Wisniewski, M.; Sionkowska, A.; Kaczmarek, H.; Lazare, S.; Tokarev, V.; Belin, C. J. Photochem. Photobiol. A: Chem. 2007, 188, 192−199. (22) Wilmanns, M. J. Synchrotron Radiat. 2000, 7, 41−46. (23) Kautek, W.; Pentzien, S.; Conradi, A.; Leichtfried, D.; Puchinger, L. J. Cult. Heritage 2003, 4, 179s−184s. (24) Walczak, M.; Oujja, M.; Crespo-Arca, L.; Garcia, A.; Mendez, C.; Moreno, P.; Domingo, C.; Castillejo, M. Appl. Surf. Sci. 2008, 255, 3179−3183. (25) Kobayashi, K.; Yokoya, A.; Usami, N.; Ishizaka, S. Fragmentation of Amino Acids due to Inner Shell X-ray Absorption in Sulfur Atoms. In Biophysical Aspects of Auger Processes, AAPM Symposium Series; Howell, R. W., Narra, V. R., Sastry, K. S. R., Rao, D. V., Eds.; American Institute of Physics, Inc.: Woodbury, NY., 1992; pp 014−023. (26) Dale, W. M.; Davies, J. V. Biochemistry 1951, 48, 129−132. 9422
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