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The FT-IR spectrometric analysis of the changes of polyphenol oxidase II secondary structure
Journal of Molecular Structure 644 (2003) 139–144 www.elsevier.com/locate/molstruc
The FT-IR spectrometric analysis of the changes of polyphenol oxidase II secondary structure Chunhua Shia, Ya Daib, Qingliang Liua,*, Yongshu Xiea, Xiaolong Xua a
Department of Chemistry, University of Science and Technology of China, Hefei 230026, People’s Republic of China b Chongqing Tobacco Industrial Corp. Ltd, Chongqing, People’s Republic of China Received 27 June 2002; accepted 13 September 2002
Abstract Polyphenol oxidase II is a novel protein purified from tobacco, which acts as a key role in plant defense system. From the analysis of FT-IR spectrums, Fourier self-deconvolution (FSD) spectrums and second-derivative spectrums of PPO II at different pH and peroxide PPO II adduct, the secondary structure fractions are analyzed. PPO II at low pH (pH ¼ 3.0) and peroxide PPO II adduct almost keep the same secondary structure of native PPO II. The percentages of b-turn and random coil increase rapidly and the percentages of a-helix and anti-parallel b-sheet decrease rapidly at high pH (pH ¼ 10.0) comparing with that of native PPO II. All these conclusions are proved by the secondary structure calculations of circular dichroism spectrums in different states. q 2002 Elsevier Science B.V. All rights reserved. Keywords: FT-IR; Secondary structure; Polyphenol oxidase; Tobacco; CD spectrum; pH; Peroxide
1. Introduction Polyphenol oxidases (PPO) are a group of copperproteins distributed through all the phylogenetic scale widely from bacteria to mammals [1]. The common feature of this group is their capacity to catalyze the oxidation of polyphenols through molecular oxygen [2]. According to the Enzyme Commission, three types of activities are associated to PPO as followings: Catechol oxidase or o-diphenol: oxygenoxidoreductase (EC 1. 10. 3. 1); Laccase or p-diphenol: oxygen-oxidoreductase (EC 1. 10. 3. 2) and Cresolase or monophenol monooxygenase (EC 1. 18. 14. 1) [3]. They can catalyze two reactions: the hydroxylation of monophenols to o-diphenols (monophenolase activity) and the oxidation of o-diphenols to o-quinones * Corresponding author. E-mail address: [email protected] (Q. Liu).
(diphenol activity). Two PPOs, PPO I and PPO II, have been obtained from tobacco (Nicotiana Tobacum ) [4,5], while PPO II is the dominant one. PPO II has a molecular weight of 35,600 Da and contains two antiferromagentically coupled copper ions. The optimum pH and optimum temperature of PPO II are about pH ¼ 6.5 and 40 8C, respectively. PPO II does not show the activity of the catalytic oxidation to p-diphenol or m-diphenol, and only possesses lower activity to chlorgenic acid [6,7]. PPO II is activated after one azide molecule is coordinated in terminal mode with PPO II [8]. PPO II acts as a key role in plant defense system and it inhibits the culture of E. Coli in solution [4,5]. A variety of analytical tools are available to study protein conformation, such as X-Ray Diffraction, Circular Dichroism (CD), Fourier transform infrared spectrometry (FT-IR) and Raman Reson-
0022-2860/03/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 2 8 6 0 ( 0 2 ) 0 0 4 7 1 - 4
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C. Shi et al. / Journal of Molecular Structure 644 (2003) 139–144
ance. Of these methods, FT-IR has proved to be most versatile. It allows analysis of protein conformation in a diverse range of environments. The versatility of FT-IR is based on the long wavelength of the radiation, which minimizes scattering problems. In addition, a wide range of sampling methods has been developed using transmission, reflection, or emission technique [9]. Although FT-IR has become a common technique to investigate protein conformation, it is not without pitfalls. It is common to compare infrared spectra of protein recorded in different physical state or sampling technique on shape, position, and intensity of infrared absorption bands. In this work, the secondary structure changes of PPO II upon pH value and the coordination of peroxide in the active site were discussed and further experiments of CD spectrums were proved the conformation changes.
2. Material and methods 2.1. Materials The fresh tobacco leaves (N. Tobacum ) were harvested directly from the field, washed and then kept in refrigerator below 4 8C for about 24 h. DEAESephadex A-50, CM-Sephadex C-50 and Sephadex G-75 were purchased from Pharmacia Corporation Sweden. Other chemicals were analytical reagents. 2.2. Protein preparation Polyphenol oxidase II sample was prepared using the efficient method of acetone powder, purified by 30 and 80% ammonium sulfate precipitation and then the column chromatography of DEAE-Sephadex A-50, CM-Sephadex C-50 and Sephadex G-75, respectively, according to the method of Shi et al. [4,5]. PPO II obtained in above process has been detected by PAGE, SDS-PAGE and MALDI-TOF-MS spectra (LDI 1700 Linear Scientific Inc) as a single enzyme and the molecular weight 35,600 Da. The protein concentration was determined using the method mentioned by Bradford [10].
2.3. FT-IR and CD experiments Infrared spectra were obtained at a resolution of 2 cm 21, on a Fourier-transform instrument (MAGNA-IR 750) with KBr pellets at room temperature. The samples were prepared as the following: 10 ul 20 mg/ml PPO II solutions (containing none or HCl (pH ¼ 3.0) or NaOH (pH ¼ 10.0) or 1 mM H2O2 or D2O, respectively) were dried by blowing nitrogen, then mixed with KBr pellet. The mixed was pressed into a disk. CD spectrum was measured on a Jasco720 spectropolarimeter in a cell with a 0.1 cm path length. The samples were prepared at 0.1 mg/ml PPO II containing pH ¼ 3.0, 6.0, 6.5, 8.0, 9.0 and 10.0, 0.02 mM H3PO4-NaOH buffer, respectively. 2.4. Data analysis Compsite bands of the amide I, amide II and amide III were obtained by using Fourier self-deconvolution (FSD) with an enhancement factor K ¼ 1.8 and a halfwidth of 30.2 cm21. The second-derivative spectrums of amide I, amide II or amide III were obtained by using origin 6.0. The calculations of secondary structure from CD spectrum were based on the method of Parcel et al. [24], a-helix, b-turn, antiparallel b-sheet, random coil and aromatic/disufide attribution were considered in the calculation.
3. Results and discussion A protein conformation can be defined by a set of dihedral angles Fi and Ci and by the short- or longrange interactions between different peptide groups, i.e. hydrogen bonding. The relationship between conformation and band position can be studied using following relationship [21]: " # X X 0 0 00 nðd; d Þ ¼ n0 þ Dj cosðjdÞ þ Dj cosðjd Þ i
l
where the first term n0 corresponds to the unperturbed amide frequency, d and d0 are the phase angles (0 or p ) between adjacent groups in the same chain or in the neighbouring chain connected by a hydrogen bond, Dj and Dj 0 are coupling constants corresponding to the intrachain or interchain interactions. Equation
C. Shi et al. / Journal of Molecular Structure 644 (2003) 139–144
Fig. 1. FT-IR spectrum of polyphenol oxidase II.
can describe in principle the splitting and polarization of the amide I band in different conformations. However, it does not furnish direct information concerning numerical frequency values or relative intensities. Some modes might be so weak as to be practically unobservable. Others might overlap, resulting in odd-shaped bands with apparently illdefined absorption maxim [21]. The infrared absorption spectrum of native PPO II between 4000 and 400 cm21 is shown in Fig. 1. The peak at 3307.4 cm21 is assigned as n N –H vibration of PPO II. The peak at 2962.2 cm21 is assigned as n C–H vibration. Peaks from 1750 to 1600 cm21, from 1600 to 1500 cm21 and from 1350 to 1200 cm21 are assigned
141
as amide I, amide II and amide III, respectively (see Fig. 1) [11,12]. The peak at 1402.0 cm21 is assigned as d C –H vibration. Amide I peak and amide II peak exist at 1650.8 and 1537.0 cm21, respectively, at pH ¼ 3.0. At pH ¼ 10.0 amide I peak and amide II exist at 1658.5 and 1533.2 cm21. While amide I and amide II peaks are at 1658.5 and 1540.9 cm21 in D2O solution (figures are not shown). Fig. 2 shows the FSD spectrum and secondderivative spectrum between 1750 and 1200 cm21 at neutral condition. The strong amide I band at around 1658 cm21 is definitely due to a-helix. The shoulder at around 1693, 1630 cm21 and the peaks around 1230 cm 21 is due to the anti-parallel b -sheet [9,13 – 16]. The absorption at around 1674 cm21 is assigned as b-turn. The band of random coil at around 1650 cm21 may overlap with the a-helix. The band at around 1538 and 1264 cm21 are also due to random coil [13 – 16]. There is some agreement in the literature about the origin (b-turns or high-frequency component of anti-parallel) of bands in the 1670 – 1695 cm21 region [13,17]. The band at around 1604 cm21 is assigned as the tyrosine side-chain and the band at around 1512 cm21 is assigned as the tyrosine ring vibration [11,12,18,19]. The absorption at around 1738 and 1718 cm21 are assigned as ‘free’ CyO group and hydrogen bonded CyO group, respectively (see Table 1) [20]. In the acidic condition (pH ¼ 3.0), A band at around 1725 cm21 appears clearly, which is assigned as the hydrogen bonded CyO group [20]. The band of a-helix at around 1658 cm21 weakly decreases and Table 1 Summary of the frequencies and proposed structural assignments of the bands for the decomposition of amide I, amide II and amide III
Fig. 2. Fourier self-deconvolution spectrum (upper) and secondderivative spectrum (down) of native PPO II between 1750 and 1200 cm21.
Frequency (cm21) Assignment
References
1738 1719 1693 1674 1658 1630 1604 1538 1512 1264 1230
[20] [20] [9,13–16] [9,13–16] [21] [9,13–16] [16] [9] [11,12,18,19] [9] [13–16]
‘Free’ CyO Group Hydrogen bonded CyO Group Anti-parallel b-sheet b-turn a-helix and random coil anti-parallel b-sheet Side chain Random coil Tyrosine ring vibration Random coil Anti-parallel b-sheet
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Fig. 3. Fourier self-deconvolution spectrum (A) and second-derivative spectrum (B) of PPO II at different pH (3.0, neutral and 10.0) and peroxide PPO II adduct in amide I band; Fourier self-deconvolution spectrum (C) and second-derivative spectrum (D) in amide II band; Fourier self-deconvolution spectrum (E) and second-derivative spectrum (F) in amide III band.
C. Shi et al. / Journal of Molecular Structure 644 (2003) 139–144
the absorption at around 1650 cm21 increases a little comparing with the absorption at neutral condition, which suggest that the concentration of a-helix decreases weakly and the random coil increases a little at low pH (see Fig. 3A,B). The absorption at around 1538 cm21 in amide II band (see Fig. 3C, D) and at around 1264 cm21 (see Fig. 3E, F) increase too at low pH, which proves again that percentage of random coil at low pH increase weakly. At high pH (pH ¼ 10.0), The absorption at around 1658 cm21 almost disappear (see Fig. 3A, B), the optimal absorption red-shifts to around 1650 cm21 (in other words, the absorption at around 1650 cm21 increases rapidly) and the band at around 1264 cm21 increase too, which indicates the rapid increase of random coil and the rapid decrease of a-helix. The absorption at around 1630 cm21 decreases and the absorption at 1674 cm21 increases at high pH, which suggest the increase of b-turn and the decrease of anti-parallel bsheet. The band at around 1658 cm21 increases after D2O exchange, and it shows the percentage of a-helix increases. The shoulder at around 1688 cm21 is due to the red-shift of anti-parallel after the exchange of D2O [21], which proves the band at around 1693 cm21 is attributed to anti-parallel b-sheet (see Fig. 2). Peroxide can coordinate in the active site of PPO II and form peroxide PPO II adduct [22,23]. The secondary structure of peroxide PPO II adduct does not change clearly comparing with native PPO II. The difference can be found in the absorption shoulder at around 1678 cm21 and the red-shift of secondderivative at around 1678 cm21 (see Fig. 3), which suggests that the percentage of b-turn in peroxide PPO II adduct increases weakly. Using CD measurements, the secondary structure of PPO II in different state (different pH and peroxide adduct) is studied. Fig. 4 shows the CD spectrums of PPO II at different pH and peroxide adduct. It exhibits
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Fig. 4. CD spectrum of PPO II at different pH (3.0, 6.0, 6.5, 8.0, 9.0 and 10.0) and peroxide PPO II adduct between 190 and 250 nm.
a clear negative band at approximately 208 nm and other prominent negative bands at 221 nm. The CD spectrums at pH 3.0, 6.0, 6.5, 8.0, 9.0 and peroxide adduct indicates that PPO II in the above state have a relative average fraction of a-helix, anti-parallel b-sheet, b-turn and random coil. The fractions at pH 6.5 are calculated as 26.3%, 17.0% b-turn, 25.9% anti-parallel b-sheet, 29% random coil and 1.7% aromatic/disulfide contributions using the method of Parcel et al. [24]. At pH 6.0, the fractions are almost as same as that at pH 6.5 and they are 25.2, 17.1, 25.1, 31.2 and 1.5%, respectively. At low pH (pH ¼ 3.0), the fraction of a-helix is decreased to 22.4%, the fractions of b-turn and anti-parallel b-sheet almost do not change (17.4 and 25.5%, respectively,) and the fraction of random coil increases to 32.1%. The pH value does not affect the secondary structure of PPO II
Table 2 The percentage of secondary structure of PPO II at different state calculated from CD spectrums Sample
a-helix
b-turn
Anti-parallel b-sheet
Random coil
Aromatic/disulfide contribution
pH ¼ 3.0 pH ¼ 6.0 pH ¼ 6.5 pH ¼ 10.0 Peroxide adduct
22.4% 25.2% 26.3% 0.1% 24.8%
17.4% 17.1% 17.0% 43.2% 20.1%
25.5% 25.1% 25.9% 14.0% 22.5%
32.1% 31.2% 29.0% 42.6% 32.6%
2.6% 1.5% 1.7% 0.2% 0%
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clearly from the CD spectrums at pH 8 and 9.0. At high pH (pH ¼ 10.0), CD spectrum changes completely (see Fig. 4). The fractions of random coil and bturn are increased to 42.6 and 43.2%, respectively, the fraction of a-helix almost disappears (0.1%), and the fraction of anti-parallel b-sheet is decreased to 14.0%. The fractions of peroxide adduct are 24.8% (a-helix), 20.1% (b-turn), 22.5% (anti-parallel b-sheet), 32.6% (random coil), respectively, (see Table 2). The secondary structure fractions of CD spectrum’s calculations well fit the analysis of that from FT-IR spectrum.
4. Conclusion From the analysis of FT-IR spectrums, FSD spectrums and second derivative spectrums of PPO II at different pH and peroxide PPO II adduct, the secondary structure fractions are analyzed. The percentage of a-helix decreases a little and the percentage of random coil increases a little at low pH (pH ¼ 3.0), and the percentages of b-turn and random coil increase rapidly and the percentages of a-helix and anti-parallel b-sheet decreased rapidly at high pH (pH ¼ 10.0) comparing with that of native PPO II. The percentages of b-turn and random coil increase a little, and the percentage of a-helix and anti-parallel b-sheet decrease a little in peroxide PPO II adduct. All these conclusions are proved by the secondary structure calculations of CD spectrums in different states.
Acknowledgements This research was financially supported by National foundation of Science (No. 30270321) and the National Tobacco Monopolization Bureau (NO. 110200001027) and foundation of University of Science and Technology of China.
References [1] D.A. Robb, in: R. Lontie (Ed.), Copper Proteins and Copper Enzymes, vol. 207–241, CRC Press, Boca Raton, FL, 1984, pp. 207–241. [2] B.G. Malmstro¨m, L.E. Andreasson, B. Reinhammar, in: P.D. Boyer (Ed.), Enzymes, vol. 507 –579, Academic Press, NY, 1975, pp. 507 –579. [3] A.M. Mayer, Phytochemistry 26 (1987) 11–20. [4] C.H. Shi, Y. Dai, X.L. Xu, Y.S. Xie, Q.L. Liu, Protein Expr. Purif. 24 (2002) 51– 55. [5] C.H. Shi, Y. Dai, B.L. Xia, X.L. Xu, Y.S. Xie, Q.L. Liu, et al., Plant Mol. Biol. Reporter 19 (2001) 381–382. [6] C.H. Shi, Y. Dai, X.L. Xu, Y.S. Xie, Q.L. Liu, Spectrosc. Lett. 34 (6) (2001) 675 –683. [7] C.H. Shi, Y. Dai, X.L. Xu, Y.S. Xie, Q.L. Liu, Spectrosc. Lett. 35 (2) (2002) 199 –206. [8] C.H. Shi, Y. Dai, X.L. Xu, Y.S. Xie, H.L. Han, Q.L. Liu, J. Protein Chem. 20 (6) (2001) 463–468. [9] M. Weert, P.I. Haris, W.E. Hennink, D.J.A. Crommelin, Anal. Biochem. 297 (2001) 160– 169. [10] M.M. Bradford, Anal. Biochem. 72 (1976) 248–254. [11] X. Ruan, J. Wei, Q. Xu, J.S. Wang, Y.D. Gong, et al., J. Mol. Struct. 525 (2000) 97–106. [12] A.E. Adreeva, I.R. Karamancheva, J. Mol. Struct. 565–566 (2001) 177 –182. [13] V.A. Iconomidou, G.D. Chryssikos, V. Gionis, J.H. Willis, S. Hamodrakas, Insect Biochem. Mol. Biol. 31 (2001) 877–885. [14] S. Cai, B.R. Singh, Biophys. Chem. 80 (1999) 7–20. [15] M. Jackson, H.H. Mantsch, Crit. Rev. Biochem. Mol. Biol. 30 (2) (1995) 95–120. [16] H. Shi, L. Xiong, K.Y. Yang, C.Q. Tang, T.Y. Kuang, N.M. Zhao, J. Mol. Struct. 446 (1998) 137 –147. [17] P.L. Haris, D. Chapman, Biopolymers 37 (1995) 251 –263. [18] W.Z. He, W.R. Newell, P.I. Haris, D. Chapman, J. Barber, Biochemistry 30 (1991) 4552–4559. [19] G.M. Macdonald, B.A. Barry, Biochemistry 31 (1992) 9848–9853. [20] A. Blume, W. Hubner, G. Messner, Biochemistry 27 (1988) 8239–8249. [21] J.L.R. Arrondo, F.M. Gon˜i, Prog. Biophys. Mol. Biol. 72 (1999) 367 –405. [22] E.I. Solomon, U.M. Sundaram, T.E. Mochonkin, Chem. Rev. 7 (1996) 2563–2605. [23] C. Eichen, B. Krebs, J.C. Saccbettini, Curr. Opin. Struct. Biol. 9 (1999) 677 –683. [24] A. Perczel, K. Park, G.D. Fasman, Anal. Biochem. 203 (1992) 83– 93.
The FT-IR spectrometric analysis of the changes of polyphenol oxidase II secondary structure Chunhua Shia, Ya Daib, Qingliang Liua,*, Yongshu Xiea, Xiaolong Xua a
Department of Chemistry, University of Science and Technology of China, Hefei 230026, People’s Republic of China b Chongqing Tobacco Industrial Corp. Ltd, Chongqing, People’s Republic of China Received 27 June 2002; accepted 13 September 2002
Abstract Polyphenol oxidase II is a novel protein purified from tobacco, which acts as a key role in plant defense system. From the analysis of FT-IR spectrums, Fourier self-deconvolution (FSD) spectrums and second-derivative spectrums of PPO II at different pH and peroxide PPO II adduct, the secondary structure fractions are analyzed. PPO II at low pH (pH ¼ 3.0) and peroxide PPO II adduct almost keep the same secondary structure of native PPO II. The percentages of b-turn and random coil increase rapidly and the percentages of a-helix and anti-parallel b-sheet decrease rapidly at high pH (pH ¼ 10.0) comparing with that of native PPO II. All these conclusions are proved by the secondary structure calculations of circular dichroism spectrums in different states. q 2002 Elsevier Science B.V. All rights reserved. Keywords: FT-IR; Secondary structure; Polyphenol oxidase; Tobacco; CD spectrum; pH; Peroxide
1. Introduction Polyphenol oxidases (PPO) are a group of copperproteins distributed through all the phylogenetic scale widely from bacteria to mammals [1]. The common feature of this group is their capacity to catalyze the oxidation of polyphenols through molecular oxygen [2]. According to the Enzyme Commission, three types of activities are associated to PPO as followings: Catechol oxidase or o-diphenol: oxygenoxidoreductase (EC 1. 10. 3. 1); Laccase or p-diphenol: oxygen-oxidoreductase (EC 1. 10. 3. 2) and Cresolase or monophenol monooxygenase (EC 1. 18. 14. 1) [3]. They can catalyze two reactions: the hydroxylation of monophenols to o-diphenols (monophenolase activity) and the oxidation of o-diphenols to o-quinones * Corresponding author. E-mail address: [email protected] (Q. Liu).
(diphenol activity). Two PPOs, PPO I and PPO II, have been obtained from tobacco (Nicotiana Tobacum ) [4,5], while PPO II is the dominant one. PPO II has a molecular weight of 35,600 Da and contains two antiferromagentically coupled copper ions. The optimum pH and optimum temperature of PPO II are about pH ¼ 6.5 and 40 8C, respectively. PPO II does not show the activity of the catalytic oxidation to p-diphenol or m-diphenol, and only possesses lower activity to chlorgenic acid [6,7]. PPO II is activated after one azide molecule is coordinated in terminal mode with PPO II [8]. PPO II acts as a key role in plant defense system and it inhibits the culture of E. Coli in solution [4,5]. A variety of analytical tools are available to study protein conformation, such as X-Ray Diffraction, Circular Dichroism (CD), Fourier transform infrared spectrometry (FT-IR) and Raman Reson-
0022-2860/03/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 2 8 6 0 ( 0 2 ) 0 0 4 7 1 - 4
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C. Shi et al. / Journal of Molecular Structure 644 (2003) 139–144
ance. Of these methods, FT-IR has proved to be most versatile. It allows analysis of protein conformation in a diverse range of environments. The versatility of FT-IR is based on the long wavelength of the radiation, which minimizes scattering problems. In addition, a wide range of sampling methods has been developed using transmission, reflection, or emission technique [9]. Although FT-IR has become a common technique to investigate protein conformation, it is not without pitfalls. It is common to compare infrared spectra of protein recorded in different physical state or sampling technique on shape, position, and intensity of infrared absorption bands. In this work, the secondary structure changes of PPO II upon pH value and the coordination of peroxide in the active site were discussed and further experiments of CD spectrums were proved the conformation changes.
2. Material and methods 2.1. Materials The fresh tobacco leaves (N. Tobacum ) were harvested directly from the field, washed and then kept in refrigerator below 4 8C for about 24 h. DEAESephadex A-50, CM-Sephadex C-50 and Sephadex G-75 were purchased from Pharmacia Corporation Sweden. Other chemicals were analytical reagents. 2.2. Protein preparation Polyphenol oxidase II sample was prepared using the efficient method of acetone powder, purified by 30 and 80% ammonium sulfate precipitation and then the column chromatography of DEAE-Sephadex A-50, CM-Sephadex C-50 and Sephadex G-75, respectively, according to the method of Shi et al. [4,5]. PPO II obtained in above process has been detected by PAGE, SDS-PAGE and MALDI-TOF-MS spectra (LDI 1700 Linear Scientific Inc) as a single enzyme and the molecular weight 35,600 Da. The protein concentration was determined using the method mentioned by Bradford [10].
2.3. FT-IR and CD experiments Infrared spectra were obtained at a resolution of 2 cm 21, on a Fourier-transform instrument (MAGNA-IR 750) with KBr pellets at room temperature. The samples were prepared as the following: 10 ul 20 mg/ml PPO II solutions (containing none or HCl (pH ¼ 3.0) or NaOH (pH ¼ 10.0) or 1 mM H2O2 or D2O, respectively) were dried by blowing nitrogen, then mixed with KBr pellet. The mixed was pressed into a disk. CD spectrum was measured on a Jasco720 spectropolarimeter in a cell with a 0.1 cm path length. The samples were prepared at 0.1 mg/ml PPO II containing pH ¼ 3.0, 6.0, 6.5, 8.0, 9.0 and 10.0, 0.02 mM H3PO4-NaOH buffer, respectively. 2.4. Data analysis Compsite bands of the amide I, amide II and amide III were obtained by using Fourier self-deconvolution (FSD) with an enhancement factor K ¼ 1.8 and a halfwidth of 30.2 cm21. The second-derivative spectrums of amide I, amide II or amide III were obtained by using origin 6.0. The calculations of secondary structure from CD spectrum were based on the method of Parcel et al. [24], a-helix, b-turn, antiparallel b-sheet, random coil and aromatic/disufide attribution were considered in the calculation.
3. Results and discussion A protein conformation can be defined by a set of dihedral angles Fi and Ci and by the short- or longrange interactions between different peptide groups, i.e. hydrogen bonding. The relationship between conformation and band position can be studied using following relationship [21]: " # X X 0 0 00 nðd; d Þ ¼ n0 þ Dj cosðjdÞ þ Dj cosðjd Þ i
l
where the first term n0 corresponds to the unperturbed amide frequency, d and d0 are the phase angles (0 or p ) between adjacent groups in the same chain or in the neighbouring chain connected by a hydrogen bond, Dj and Dj 0 are coupling constants corresponding to the intrachain or interchain interactions. Equation
C. Shi et al. / Journal of Molecular Structure 644 (2003) 139–144
Fig. 1. FT-IR spectrum of polyphenol oxidase II.
can describe in principle the splitting and polarization of the amide I band in different conformations. However, it does not furnish direct information concerning numerical frequency values or relative intensities. Some modes might be so weak as to be practically unobservable. Others might overlap, resulting in odd-shaped bands with apparently illdefined absorption maxim [21]. The infrared absorption spectrum of native PPO II between 4000 and 400 cm21 is shown in Fig. 1. The peak at 3307.4 cm21 is assigned as n N –H vibration of PPO II. The peak at 2962.2 cm21 is assigned as n C–H vibration. Peaks from 1750 to 1600 cm21, from 1600 to 1500 cm21 and from 1350 to 1200 cm21 are assigned
141
as amide I, amide II and amide III, respectively (see Fig. 1) [11,12]. The peak at 1402.0 cm21 is assigned as d C –H vibration. Amide I peak and amide II peak exist at 1650.8 and 1537.0 cm21, respectively, at pH ¼ 3.0. At pH ¼ 10.0 amide I peak and amide II exist at 1658.5 and 1533.2 cm21. While amide I and amide II peaks are at 1658.5 and 1540.9 cm21 in D2O solution (figures are not shown). Fig. 2 shows the FSD spectrum and secondderivative spectrum between 1750 and 1200 cm21 at neutral condition. The strong amide I band at around 1658 cm21 is definitely due to a-helix. The shoulder at around 1693, 1630 cm21 and the peaks around 1230 cm 21 is due to the anti-parallel b -sheet [9,13 – 16]. The absorption at around 1674 cm21 is assigned as b-turn. The band of random coil at around 1650 cm21 may overlap with the a-helix. The band at around 1538 and 1264 cm21 are also due to random coil [13 – 16]. There is some agreement in the literature about the origin (b-turns or high-frequency component of anti-parallel) of bands in the 1670 – 1695 cm21 region [13,17]. The band at around 1604 cm21 is assigned as the tyrosine side-chain and the band at around 1512 cm21 is assigned as the tyrosine ring vibration [11,12,18,19]. The absorption at around 1738 and 1718 cm21 are assigned as ‘free’ CyO group and hydrogen bonded CyO group, respectively (see Table 1) [20]. In the acidic condition (pH ¼ 3.0), A band at around 1725 cm21 appears clearly, which is assigned as the hydrogen bonded CyO group [20]. The band of a-helix at around 1658 cm21 weakly decreases and Table 1 Summary of the frequencies and proposed structural assignments of the bands for the decomposition of amide I, amide II and amide III
Fig. 2. Fourier self-deconvolution spectrum (upper) and secondderivative spectrum (down) of native PPO II between 1750 and 1200 cm21.
Frequency (cm21) Assignment
References
1738 1719 1693 1674 1658 1630 1604 1538 1512 1264 1230
[20] [20] [9,13–16] [9,13–16] [21] [9,13–16] [16] [9] [11,12,18,19] [9] [13–16]
‘Free’ CyO Group Hydrogen bonded CyO Group Anti-parallel b-sheet b-turn a-helix and random coil anti-parallel b-sheet Side chain Random coil Tyrosine ring vibration Random coil Anti-parallel b-sheet
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Fig. 3. Fourier self-deconvolution spectrum (A) and second-derivative spectrum (B) of PPO II at different pH (3.0, neutral and 10.0) and peroxide PPO II adduct in amide I band; Fourier self-deconvolution spectrum (C) and second-derivative spectrum (D) in amide II band; Fourier self-deconvolution spectrum (E) and second-derivative spectrum (F) in amide III band.
C. Shi et al. / Journal of Molecular Structure 644 (2003) 139–144
the absorption at around 1650 cm21 increases a little comparing with the absorption at neutral condition, which suggest that the concentration of a-helix decreases weakly and the random coil increases a little at low pH (see Fig. 3A,B). The absorption at around 1538 cm21 in amide II band (see Fig. 3C, D) and at around 1264 cm21 (see Fig. 3E, F) increase too at low pH, which proves again that percentage of random coil at low pH increase weakly. At high pH (pH ¼ 10.0), The absorption at around 1658 cm21 almost disappear (see Fig. 3A, B), the optimal absorption red-shifts to around 1650 cm21 (in other words, the absorption at around 1650 cm21 increases rapidly) and the band at around 1264 cm21 increase too, which indicates the rapid increase of random coil and the rapid decrease of a-helix. The absorption at around 1630 cm21 decreases and the absorption at 1674 cm21 increases at high pH, which suggest the increase of b-turn and the decrease of anti-parallel bsheet. The band at around 1658 cm21 increases after D2O exchange, and it shows the percentage of a-helix increases. The shoulder at around 1688 cm21 is due to the red-shift of anti-parallel after the exchange of D2O [21], which proves the band at around 1693 cm21 is attributed to anti-parallel b-sheet (see Fig. 2). Peroxide can coordinate in the active site of PPO II and form peroxide PPO II adduct [22,23]. The secondary structure of peroxide PPO II adduct does not change clearly comparing with native PPO II. The difference can be found in the absorption shoulder at around 1678 cm21 and the red-shift of secondderivative at around 1678 cm21 (see Fig. 3), which suggests that the percentage of b-turn in peroxide PPO II adduct increases weakly. Using CD measurements, the secondary structure of PPO II in different state (different pH and peroxide adduct) is studied. Fig. 4 shows the CD spectrums of PPO II at different pH and peroxide adduct. It exhibits
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Fig. 4. CD spectrum of PPO II at different pH (3.0, 6.0, 6.5, 8.0, 9.0 and 10.0) and peroxide PPO II adduct between 190 and 250 nm.
a clear negative band at approximately 208 nm and other prominent negative bands at 221 nm. The CD spectrums at pH 3.0, 6.0, 6.5, 8.0, 9.0 and peroxide adduct indicates that PPO II in the above state have a relative average fraction of a-helix, anti-parallel b-sheet, b-turn and random coil. The fractions at pH 6.5 are calculated as 26.3%, 17.0% b-turn, 25.9% anti-parallel b-sheet, 29% random coil and 1.7% aromatic/disulfide contributions using the method of Parcel et al. [24]. At pH 6.0, the fractions are almost as same as that at pH 6.5 and they are 25.2, 17.1, 25.1, 31.2 and 1.5%, respectively. At low pH (pH ¼ 3.0), the fraction of a-helix is decreased to 22.4%, the fractions of b-turn and anti-parallel b-sheet almost do not change (17.4 and 25.5%, respectively,) and the fraction of random coil increases to 32.1%. The pH value does not affect the secondary structure of PPO II
Table 2 The percentage of secondary structure of PPO II at different state calculated from CD spectrums Sample
a-helix
b-turn
Anti-parallel b-sheet
Random coil
Aromatic/disulfide contribution
pH ¼ 3.0 pH ¼ 6.0 pH ¼ 6.5 pH ¼ 10.0 Peroxide adduct
22.4% 25.2% 26.3% 0.1% 24.8%
17.4% 17.1% 17.0% 43.2% 20.1%
25.5% 25.1% 25.9% 14.0% 22.5%
32.1% 31.2% 29.0% 42.6% 32.6%
2.6% 1.5% 1.7% 0.2% 0%
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clearly from the CD spectrums at pH 8 and 9.0. At high pH (pH ¼ 10.0), CD spectrum changes completely (see Fig. 4). The fractions of random coil and bturn are increased to 42.6 and 43.2%, respectively, the fraction of a-helix almost disappears (0.1%), and the fraction of anti-parallel b-sheet is decreased to 14.0%. The fractions of peroxide adduct are 24.8% (a-helix), 20.1% (b-turn), 22.5% (anti-parallel b-sheet), 32.6% (random coil), respectively, (see Table 2). The secondary structure fractions of CD spectrum’s calculations well fit the analysis of that from FT-IR spectrum.
4. Conclusion From the analysis of FT-IR spectrums, FSD spectrums and second derivative spectrums of PPO II at different pH and peroxide PPO II adduct, the secondary structure fractions are analyzed. The percentage of a-helix decreases a little and the percentage of random coil increases a little at low pH (pH ¼ 3.0), and the percentages of b-turn and random coil increase rapidly and the percentages of a-helix and anti-parallel b-sheet decreased rapidly at high pH (pH ¼ 10.0) comparing with that of native PPO II. The percentages of b-turn and random coil increase a little, and the percentage of a-helix and anti-parallel b-sheet decrease a little in peroxide PPO II adduct. All these conclusions are proved by the secondary structure calculations of CD spectrums in different states.
Acknowledgements This research was financially supported by National foundation of Science (No. 30270321) and the National Tobacco Monopolization Bureau (NO. 110200001027) and foundation of University of Science and Technology of China.
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