Raman spectroscopic study of photosensitive damage to ... - Hindawi

Spectroscopy 20 (2006) 269–273 IOS Press

269

Raman spectroscopic study of photosensitive damage to lysozyme structure sensitized by hypocrellin A J.H. Zhou a,∗ , X.H. Wu a , S.H. Wei a , X.T. Gu a , Y.Y. Feng a , X.S. Wang b and B.W. Zhang b a

Analysis & Testing Center, Nanjing Normal University, 210097 Nanjing, P.R. China Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100101, P.R. China

b

Abstract. Laser Raman spectroscopy was used to detect the nature of the structural change in lysozyme sensitized by hypocrellin A (HA) at the molecular level. The results indicated that the orderly structure of lysozyme has been damaged by the active oxygen (1 O2 , O−· 2 and ·OH, etc.) generated by HA, and cause the changes in H-bonds system of the main chain and the side chain of lysozyme. Keywords: Hypocrellin A, lysozyme, photosensitive damage, Raman spectroscopy, protein conformation

1. Introduction Hypocrellin A (4,9-dihydroxyperylene-3-10-quinone, HA), which can be extracted from Hypocreila bambuase, a parasitic fungus of Siramudinaria, is structurally related to perihydroxylated polycyclic quinines, such as hypericin [1]. It has been used as a phototherapeutic agent to cure various skin diseases, and has been taken orally as a folk medicine for several centuries in China [1,2]. Recent studies show that this natural perylenequinonoid compound also displays anti-tumor and antiviral activities against several types of viruses, including human immunodeficiency virus [3]. It exhibits several advantages over the presently used hematoporphyrin derivatives (HPD), i.e., ready preparation and easy purification relative to HPD, small aggregation tendency, strong red light absorptivity and high quantum yields of singlet oxygen [4,5]. Photophysical and photochemical properties, photobiological activities and biomedical applications of hypocrellin A have been intensively investigated by a broad spectrum of researchers over the past decade. The results indicated that HA could photosensitize damage compositions of cellular membranes, especially membrane proteins and membrane lipids, then induce tumor cells to death. In order to reveal the detail photosensitive damage mechanism of HA at molecular level, the photodamage activity of HA on a protein with well-known structure need to be further studied. According to our knowledge, this has not been previously investigated. In this study, laser Raman spectroscopy has been used to gain detailed information on structural changes in a protein, specifically investigating the photosensitive damage of lysozyme sensitized by *

Corresponding author: Jiahong Zhou, Associate Professor, Analysis & Detecting Center, Nanjing Normal University, 122 Ninghai Road, Nanjing, Jiangsu 210097, P.R. China. Fax: +86 25 8359 8359; E-mail: [email protected]. 0712-4813/06/$17.00  2006 – IOS Press and the authors. All rights reserved

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J.H. Zhou et al. / Raman spectroscopic study of photosensitive damage to lysozyme structure

HA at a molecular level. The results of the spectroscopic analysis is used to understand the mechanism of photosensitive damage by HA. 2. Material and methods 2.1. Material Lysozyme from hen egg white produced by American Sigma Chemical Co. was dissolved in the buffer solution (30 mM Na2 SO4 , pH 7.0). The concentration of lysozyme was 5% (V/V). HA was obtained according to the procedure described in literature, and the purity was confirmed as >97% by HPLC. 1 mmol/L HA DMSO solution was put into 5% (V/V) lysozyme, and the final concentration of HA was 1 µmol/L. After mixing, the sample was injected into 1 mm (inside diameter) quartz capillaries for laser Raman spectroscopy analyses. 2.2. Spectroscopic method Each Raman spectrum was obtained on Labram HR800 Raman spectrophotometer (French Jobin Yvon Company), which was fitted with argon-ion laser (Innova 70, Coherent, USA). The 514.5 nm line was used as irradiation for the excitation of the samples. The irradiation for photosensitization was from the INNOVA70 laser made in Coherent Company, and its power was 150 mW. The experimental conditions were as follows: the exciting line 514.5 nm and power 10 mW; spectral resolution 1 cm−1 ; scanning range 400–1700 cm−1 ; room temperature (20 ± 2◦ C). 3. Result and discussion To study structural changes of lysozyme before and after photosensitive damage, we observed and calculated the changes of the important Raman bands between 400–1700 cm−1 . The results of the Raman spectroscopic analysis are shown in Fig. 1. There are no bands of HA in these spectra. The internal standard in the spectra is the strong band at 983 cm−1 of sodium sulfate, and the intensity of other bands in text was determined relative to it. Amide III is the most sensitive band in the laser Raman spectroscopy which could reflect the conformational change of main-chain in protein, such as α-helix, β-sheet, random coil, β-turn, etc., which are called the secondary structure of proteins [6,7]. From the table we know that the Amide III of original lysozyme is composed of 1303 cm−1 (β-turn), 1273, 1288 cm−1 (α-helix), 1252 cm−1 (random coil) [8]. After lysozyme was photodamaged for 15 min, the Amide III bands changed as follows: The band belonging to β-turn at 1303 cm−1 shifted to 1305 cm−1 . The bands assigned to α-helix was only left at 1288 cm−1 . The band assigned to random coil shifted to 1257 cm−1 , and the intensity increased. After photodamage for 30 min, the Amide III bands changed further as follows: The bands assigned to α-helix shifted to 1269 cm−1 . A new band appeared at 1237 cm−1 which belongs to β-sheet. There was only one 1237 cm−1 which belongs to β-sheet. Further irradiation for 75 min, there was only one strong band which belongs to random coil was left at 1257 cm−1 . The Amide I band of native lysozyme at 1657 cm−1 , which can be assigned to α-helix, shifted to 1661 cm−1 [8]. The results suggested that the conformation of lysozyme has been damaged severely when irradiated for 75 min, and the ordered conformation decreased significantly while the random coil increased.


271

Fig. 1. Raman spectroscopy of lysozyme (a: the native (control) lysozyme; b: the lysozyme photodamaged by HA for 15 min; c: the lysozyme photodamaged by HA for 30 min; d: the lysozyme photodamaged by HA for 75 min).

There are six tryptophan residues in lysozyme, four outside and two inside [9]. In the laser Raman spectroscopy, nine bands belong to the indole ring of tryptophan. When photodamaged by HA, the band intensity of 760 cm−1 decreased by 10% after irradiating for 75 min, indicating that the tryptophan residues were damaged. The 1360 cm−1 is conformational-sensitive [10], and its existence denotes that the tryptophan is “buried” [8]. When irradiated for 75 min the band became broader, meaning that part of tryptophan outside the lysozyme change to be “exposed”. It is well known that the disulfide bond can make a protein stable. There is only one kind of disulfide bond (gauche-gauche-gauche, g-g-g) in native lysozyme, which appeared at 511 cm−1 , as one of the stable conformations [7,8]. After irradiating for 75 min, the band at 511 cm−1 shifted to 508 cm−1 , and a new band appeared at 536 cm−1 which denotes to the conformation (gauche-gauche-trans, g-g-t), indicating that the conformation of the disulfide bond changed to g-g-g and t-g-t, so the stabilization of lysozyme has been decreased. The bands at 670, 699 cm−1 belongs to the stretching vibrations of the C–S bond of cystine and methionine, respectively [11], after irradiation, the two bands disappeared and a new band at 679 cm−1 appeared. The origin of this band is not clear, but it may be indicative of a break-up of the C–S bond. It is well known that the amino groups containing aromatic ring, imidazole ring and mercapto group, etc., are the direct targets of active oxygen produced by HA [12]. Therefore, the reasons that caused the structural change of lysozyme may be explained as follows: Firstly, HA was excited to generate the active oxygen species (1 O2 , O−· 2 and ·OH) and semiquinone radical anion [13], which damaged the H-bonds, disulfide bond and carbon–sulfur bond that keep the space structure of lysozyme, and changed the system of H-bonds. Secondly, the amino groups in side chains of lysozyme are sensitive to photooxidization, and some amino acid residues in α-helix and β-sheet conformations may be photodamaged resulting in re-arrangement and break-up of H-bonds. In addition, the change of the micro-environment of tryptophan residues in lysozyme shows that the conformation of the main chain is altered. In summary, the results of Laser Raman spectroscopy indicated that active oxygen species (1 O2 , O−· 2 and ·OH, etc.) generated by HA can photodamage the structural of lysozyme, including the main-chain,

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J.H. Zhou et al. / Raman spectroscopic study of photosensitive damage to lysozyme structure Table 1 The Raman spectroscopy of lysozyme photodamaged by HA

L0 1657 — 1583 1553 1526 1484 1455 — 1362 1340 1303 1288 1273 1252 — — 1198 1150 1129 1107 1090 —

Frequency (cm−1 ) L1 L2 1656 1657 1626 — 1588 1589 1553 1555 1514 1521 1494 1486 1450 1457 1425 — 1364 1364 1333 1338 1305 — 1288 — — 1269 1257 1254 — 1237 1205 1216 1193 1190 1160 1164 1129 1116 1104 1100 1078 1074 1046 1044

Ascription L3 1661 — 1581 1555 1503 1484 1447 — — 1340 — — — 1257 — 1207 1186 1164 1130 1113 1076 1047

Amide I Trp, Tyr, Phe Trp Trp His δ (CH2 ) νs (COO− ) Trp Trp

   Amide III

 

Tyr, Phe Tyr, Phe Tyr

ν (C–N)

L0 1012 984 935 903 878 — 838 807 762 723 699 — 670 633 614 566 543 — 511 483

Frequency (cm−1 ) L1 L2 1008 1014 982 982 935 933 908 903 883 881 849 — 833 831 800 807 760 763 725 719 — — 679 681 — — 649 633 612 603 569 583 545 556 — 525 508 508 474 461

Ascription L3 1012 984 935 899 874 — 833 762 716 — 679 — 629 — 586 564 536 508 485

Phe SO2− 4 ν (C–C) ν (C–C) Trp Tyr Tyr Trp ν (C–S)Met

ν (C–S)Cys

Phe Trp Trp ν (S–S) ν (S–S)

Explanation: L0, L1, L2, L3 stand for, respectively, native lysozyme and HA-lysozyme. L0, L1, L2, L3-photodamaged for 0 min, 15 min, 30 min, 75 min, respectively. Sign: ν – stretching vibration; δ – bending vibration; s – symmetry; Cys, Trp, Tyr, Phe, Met and His are abbreviations of cystine, tryptophan, tyrosine, phenylalanine, methionine and histidine, respectively.

the side-chain, tryptophan residues, etc., and these results are helpful to understand the photodamage mechanism of HA at molecular level. Acknowledgements The authors are grateful to the National Natural Science Foundation of China (20603018) and the Natural Science Foundation of Jiangsu Education Department (04KJB150068) for their support. References [1] [2] [3] [4] [5] [6]

L. J. Jiang, Chin. Sci. Bull. 21 (1990), 1608. Z.J. Diwu and J.W. Lown, Photochem. Photobiol. 52 (1990), 609. S.J. Xu, X.X. Zhang, S. Chen et al., Chin. Sci. Bull. 48(10) (2003), 1007. Z.J. Diwu and J.W. Lown, J. Photochem. Photobiol. A: Chem. 64 (1992), 273. G.G. Miller, K. Brown, A.M. Ballangrud et al., Photochem. Photobiol. 65 (1997), 714. A.T. Tu (eds), Raman Spectroscopy in Biology Principle and Applications, Ind. ed. U.S.A. John Wiley and Sons, Inc., 1982, pp. 65–99.


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[7] Y.M. Xu, Y.Z. Yang and Z.Y. Zhang, Acta Biophysica Sinica 12(4) (1996), 724. [8] Z.Y. Zhu, R.A. Gu and T.H. Lu (eds.), Chemical Application of Raman Spectroscopy, Northeast University Press, Shenyang, 1998, pp. 209–224. [9] B. Wang, J. Wang, J. Yu and H.Z. Liu, Spectroscopy and Spectral Analysis 19(4) (1999), 535. [10] Z.Y. Zhang and Y.M. Xu, Sicence in China (Series B) 35(4) (1991), 437. [11] Y.M. Xu, J. Light Scattering 16(3) (2004), 189. [12] Z.Y. Zhang, N.H. Wang, Q. Wan et al., Free Radicals Biology & Medicine 14 (1993), 1. [13] Y.Z. Hu, J.Y. An, L.J. Jiang and D.W. Chen, J. Photochem. Photobiol. A: Chem. 89 (1995), 45.

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