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Effect of thermal treatment on secondary structure and conformational change of mushroom polyphenol oxidase (PPO) as food quality related enzyme: A FTIR study
Food Chemistry 187 (2015) 263–269
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Effect of thermal treatment on secondary structure and conformational change of mushroom polyphenol oxidase (PPO) as food quality related enzyme: A FTIR study Hande Baltacıog˘lu a,b, Alev Bayındırlı a, Mete Severcan c, Feride Severcan d,⇑ a
Department of Food Engineering, Middle East Technical University, 06800 Ankara, Turkey Department of Food Engineering, Ondokuz Mayis University, 55139 Samsun, Turkey Department of Electrical and Electronics Engineering, Middle East Technical University, 06800 Ankara, Turkey d Department of Biological Sciences, Middle East Technical University, 06800 Ankara, Turkey b c
a r t i c l e
i n f o
Article history: Received 24 November 2014 Received in revised form 25 March 2015 Accepted 21 April 2015 Available online 23 April 2015 Keywords: PPO Heat inactivation FTIR Spectroscopy Protein secondary structure
a b s t r a c t In order to understand the conformational changes of polyphenol oxidase (PPO), which is a food quality related enzyme, after thermal treatment, secondary structure changes of the enzyme were analyzed by using Fourier Transform Infrared (FTIR) spectroscopy and compared with the change in enzyme activity in the temperature range of 25–80 °C. Fourier self-deconvolution, neural network (NN) and curve-fitting analysis were applied to the amide I band of FTIR spectra for detail analysis of secondary structure elements. FTIR analysis indicated that PPO is an a-helix dominating enzyme. Detail analysis revealed that, as temperature increased, a-helix and b-sheet decreased, but aggregated b-sheet, turns and random coil increased. The marked changes were noted at 40 °C with the occurrence of new bands due to aggregated b-sheet structures, all of which indicate protein denaturation. These aggregation bands were still observed when the temperature was reduced back to 25 °C, from 70 °C, demonstrating an irreversible change in the structure. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Polyphenol oxidase (PPO, E.C 1.14.18.1) is a copper-containing enzyme that causes enzymatic browning in fresh fruit, vegetable products and also mushrooms. PPO from all of these species has six histidine residues that ligats the two copper ions of the active site (Yi et al., 2012). The mushroom PPO enzyme is commonly found as a tetrameric protein with a molecular mass of 120 kDa, composed of two subunits of 43 kDa (H subunit) and two subunits of 14 kDa (L subunit). The enzyme is a-helix dominating enzyme with the helices found in the H subunit. The H subunits contain a binuclear copper-binding site with each copper ion coordinated by three histidine residues. Binuclear copper-binding site locates in the center of the domain, between a bundle of four helices (a3, a4, a10 and a11). The L subunit consists of 12 antiparallel b-strands assembled in a cylindrical barrel of six 2-stranded sheets (Ismaya et al., 2011). Enzymatic browning is the result of the oxidation of o-diphenols into unstable quinones by PPO in the presence of molecular oxygen. o-Quinones are highly reactive ⇑ Corresponding author. E-mail address: [email protected] (F. Severcan). http://dx.doi.org/10.1016/j.foodchem.2015.04.097 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
compounds and react with other phenols and non-phenolic compounds to give brown pigments. This enzymatic browning affects color, flavor and nutritional quality of foods. Therefore, this enzyme is inactivated during food processing in order to prevent browning (Ludikhuyze, Van Loey, Smout, & Hendrickx, 2003). Thermal treatment is the most common and widely employed technique for the inactivation of food quality related enzymes in the food industry. The inactivation of enzymes during thermal treatment has been extensively studied over the past years and this process has been generally represented with simple kinetic approaches in the literature. Inactivation kinetics of PPO in apple (Yemeniciog˘lu, Özkan, & Cemerog˘lu, 1997), carrots and potatoes (Anthon & Barrett, 2002), table grape (Fortea, López-Miranda, Serrano-Martínez, Carreño, & Núñez-Delicado, 2009) and mushroom (Agaricus bisporus) (Gouzi, Depagne, & Coradin, 2012; Ionita, Aprodu, Stanciuc, Rapeanu, & Bahrim, 2014) were previously reported. However, less attention has been given to investigate the conformation changes of food enzymes during thermal treatment. There are different analytical tools to study protein conformation. Among these techniques FTIR spectroscopy has several advantages for determination of secondary structure of proteins
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in solution (Severcan, Haris, & Severcan, 2004; Goormaghtigh, Gasper, Bénard, Goldsztein, & Raussens, 2009; Yang, Yang, Kong, Dong, & Yu, 2015) and in biological systems (Cakmak, Zorlu, Severcan, & Severcan, 2011; Garip, Yapici, Simsek Ozek, Severcan, & Severcan, 2010). It provides high-quality spectra with very small amounts of protein whose size is not important. It is a rapid technique with relatively low costs. Characteristic bands in an IR spectrum of a protein are very sensitive to conformational changes in the protein (Haris & Severcan, 1999). The amide I region is generally used for the analysis of secondary structures of proteins from FTIR spectra. Neural networks (NNs) are reported to be effective ways to predict the secondary structure of proteins from FTIR spectra (Severcan, Severcan, & Haris, 2001; Severcan et al., 2004). There are limited numbers of studies about the conformational changes of PPO. Among these, FTIR spectroscopic analysis of mushroom PPO was investigated in the temperature range of 25–45 °C. It was reported that tyrosinase was predominately a-helix in nature and above 40 °C the secondary structure of the enzyme started to change (Tse, Kermasha, & Ismail, 1997). Weemaes et al. (1997) determined the thermal stability of mushroom PPO by using FTIR spectroscopy. A temperature titration curve constructed in order to determine whether thermal inactivation was due to a small change in the active site or to a global conformation change of the enzyme was used in this study. However, detailed secondary structural analysis during thermal treatment was not reported in these studies. Ionita et al. (2014) studied the heat induced changes of tyrosinase from A. bisporus by using the combination of fluorescence spectroscopic measurements, inactivation kinetics, and in silico prediction. Changes in the enzyme during thermal treatment at different temperature values were studied. The thermal induced behavior of tyrosinase at single molecule level was investigated by means of numerical approach in this study and atomic details were provided after performing molecular dynamics simulations at different temperatures on the enzyme crystal structure. It was reported that significant changes in secondary structure of tyrosinase above 60 °C might lead to enzyme inactivation according to the molecular dynamics simulations. In the present study, in addition to the temperature induced changes in the activity of PPO, in order to understand the conformational changes of PPO after thermal inactivation, secondary structural content of the enzyme in solution was determined precisely for the first time, using FTIR spectroscopy. To achieve this, neural network (NN) and curve-fitting analysis based on amide I band (1700–1600 cm 1) were used. It is well known that, a protein’s structure in solution reveals the native form of the protein, and such gives a more realistic picture of the protein. Furthermore, using FTIR spectroscopy, for the first time, unfolding of secondary structural elements of PPO are presented depending to the thermal treatment in the temperature range of 25–80 °C. 2. Materials and methods 2.1. Chemicals Mushroom PPO (98% purity) (E.C 1.14.18.1), catechol (P99% purity), D2O (99.9 atom% D) were purchased from Sigma (St. Louis, MO, USA). The enzyme was used without further purification. All chemicals were obtained from commercial sources at the highest grade of purity available. 2.2. Enzyme preparation and activity assay Lyophilized PPO was dissolved in 50 mM phosphate buffer (pH 6.5). Catechol was used as substrate for the determination of the enzyme activity. An aliquot (2 ml) of 50 mM potassium
phosphate buffer (pH 6.5) and 0.3 ml of 0.2 M catechol solution in the phosphate buffer were incubated in a test tube at 25 ± 1 °C. Then the tube contents were transferred to a plastic cuvette (path length, 10 mm) and 0.3 ml of the enzyme solution (0.08 mg/ml) was added to the cuvette to initiate the enzyme reaction. PPO activity was determined using a UV–Vis spectrophotometer (BOECO Model S22, Germany) at 420 nm at room temperature (25 ± 1 °C). Absorbance was read every 5 s for 3 min. Enzyme activity was calculated from the slope of the initial linear section of the absorbance versus time curves. One unit of enzyme activity was defined as the amount of the enzyme which caused a change of 0.001 in absorbance unit per minute. Enzyme activities were measured 3 times and expressed as residual activity (Bayindirli et al., 2006; Sun & Song, 2003; Weemaes et al., 1997). 2.3. Thermal treatment at ambient pressure Thermal treatment of PPO was performed at varying temperatures in the range of 20–80 °C with a 10 °C increment for 0, 5, 10, 15, 20, 25 and 30 min. The enzyme solutions (5 ml) in a glass tube with an inner diameter of 16 mm and a depth of 50 mm were heated in a temperature controlled dry block heater (HBR-48, Daihan Scientific Co. Ltd., Seoul, Korea) at selected temperatures and times. They were immediately transferred to an ice water to stop thermal inactivation instantaneously and the enzyme activity measurement was performed after 10–60 min of storage. All the experiments and measurements were repeated in triplicate. 2.4. Fourier Transform Infrared (FTIR) spectroscopy In order to determine the conformational change of PPO after inactivation, the secondary structure of the enzyme was analyzed by using FTIR spectroscopy. Infrared spectra were collected using a Perkin-Elmer Spectrum 100 FTIR spectrometer (Perkin-Elmer Inc., Norwalk, CT, USA) equipped with a MIR TGS detector. The sample compartment was continuously purged with dry air to minimize atmospheric water vapor absorbance, which overlaps in the spectral region of interest, and carbon dioxide interference. The spectrum of air was recorded as background and subtracted automatically by using the appropriate software. In order to control the temperature, the cell was connected to a thermostated circulating water bath. A thermocouple was placed on the outside of the cell to monitor the temperature of the cell. For secondary structure analysis, measurements were performed in H2O buffer. Mushroom PPO (Sigma, St. Louis, MO, USA) was dissolved in 50 mM phosphate buffer (pH 6.5) to yield a final protein concentration of 70 mg/ml for FTIR measurements. A volume of sample (10 ll) was put between CaF2 windows with a 6 lm path length and the windows were inserted into the system. Each spectrum of enzyme solution samples and buffer were collected in the 1200–2800 cm 1 region at 25 °C. A total of 400 scans were taken for each interferrogram at 2 cm 1 resolution. For inactivation studies, measurements were performed in D2O buffer. Prior to infrared experiments, the enzyme was dissolved in 50 mM phosphate buffer (prepared with D2O). Enzyme solution was allowed to stand 24 h prior to measurement to allow H–D exchange. A volume of sample (10 ll) was put between CaF2 windows with a 50 lm path length and the windows were placed into the system as described above. Samples were heated over a linear temperature gradient from 25 to 80 °C with 2 °C intervals and then cooled back to the 25 °C. Infrared spectra were recorded continuously. Each spectrum of enzyme solutions and buffer were collected in the 1400–2200 cm 1 region. A total of 128 scans were taken for each interferrogram at 2 cm 1 resolution. Infrared spectra were recorded continuously. All of the experiments were repeated 3 times and identical results were obtained.
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2.5. Data analysis for FTIR spectra Collections of spectra and data manipulations were carried out using Spectrum 100 software (Perkin-Elmer). Infrared spectra of buffer solution were measured under identical conditions. The spectrum of buffer was subtracted from the spectra of enzyme solution at the same temperature to obtain protein bands. In the subtraction process the bulk water band located at 2125 cm 1 was flattened. Spectrum 100 software (Perkin-Elmer) was used for the subtraction procedure. For protein secondary structure determination, OPUSNT data collection software (Bruker Optics, Reinstetten, Germany) was used to generate Fourier self-deconvolution and second derivative of amide I. The second derivative spectra were obtained by applying a Savitzky–Golay algorithm with nine smoothing points and these derivatives were vector normalized at 1700–1600 cm 1 and then the peak intensity values were calculated. The peak minima of the second derivative signals were used, since they correspond to the peak positions of the original absorbance spectra. In the Fourier self-deconvolution, a half bandwidth of 14 cm 1 and resolution enhancement factor k = 2.4 were used for the absorbance spectra. The deconvolved spectra were fitted with Lorentzian band profiles. Transition temperatures (Tm) were determined in Microsoft Excel 2007 software (Microsoft Corp., Redmond, WA).
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PA). Mean separation was performed by Tukey test at p < 0.05 level. Analyses results were the mean with triplicate. 3. Results and discussion 3.1. PPO inactivation during thermal treatment The residual enzyme activity of mushroom PPO after heat treatment for different time intervals is shown in Fig. 1. When the stability of mushroom PPO after 30 min exposure at different temperatures was investigated, the enzyme remained fully active up to 40 °C (data not shown). There was a slight decrease during the period of 30 min at 40 °C and the activity of enzyme was recorded as 89.84 ± 2.70% after 30 min heat treatment at 40 °C. Between 50 °C and 70 °C, higher inactivation was achieved and 99% inactivation was detectable at 70 °C for 5 min (Fig. 1). Furthermore, no activity was observed over 80 °C. Residual enzyme
2.6. Protein secondary structure prediction 2.6.1. Secondary structure determination from crystal structure of PPO The crystal structure of tyrosinase from A. bisporus was obtained from Protein Data Bank (PDB; http://www.rcsb.org/pdb/) (PDB code, 2Y9W). Secondary structure is calculated using PDBsum bioinformatics tool through ProMotif program. 2.6.2. Secondary structure prediction by neural network (NN) analysis Protein secondary structure predictions in H2O were carried out using NN. NN were first trained using FTIR spectra of 18 water soluble proteins recorded in water whose secondary structures were known from X-ray Crystallography (Severcan et al., 2001). Before applying to the NN, the Amide I band (1700–1600 cm 1) was preprocessed which involves normalization and discrete cosine transformation (DCT) of the amide I band of the FTIR spectra. The size of the training data set was increased by interpolating the available FTIR spectra to improve the training of the NN. A separate NN was trained using Bayesian regularization whose number of inputs, i.e., the number of DCT coefficients, and the number of hidden neurons were optimized for each structure parameter. The trained NNs have standard error of prediction values of 4.19% for a-helix, 3.49% for b-sheet and 3.15% for turns. The secondary structure parameters of the new proteins were predicted by applying the inputs of the trained NNs the preprocessed FTIR data as reported in detailed in Severcan et al., 2004.
Fig. 1. Thermal stability of PPO.
2.6.3. Secondary structure prediction using curve-fitting analysis Curve-fitting analysis was applied to amide I region to obtain information about the changes in the secondary structure of the enzyme during thermal treatment in D2O. Curve-fitting analysis was performed by using Grams 32 (Galactic Industries, Salem, NH, USA) software. The center positions for each sub-band were determined by second derivative analysis and the shapes of the underlying bands were chosen as Gaussian. The iterations were performed until the correlation was better than 0.995 (Toyran, Zorlu, Dönmez, Ög˘e, & Severcan, 2004). 2.7. Statistical analysis The data were analyzed as a completely randomized design by analysis of variance using Minitab 16 (Minitab Inc., State College,
Fig. 2. The FTIR spectra of enzyme solution sample (A), buffer (B) and subtracted spectrum of PPO in H2O buffer at 25 °C (C).
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activity after 30 min heat treatment at 50 °C and 60 °C were detected as 23.60 ± 1.26% and 0.42 ± 0.03%, respectively. From the graph, it can be seen that the rate of PPO inactivation depended on temperature and increased with increasing temperature and time (p < 0.05). Furthermore, there was a significant interaction (p < 0.05) among time and temperature. The trend of inactivation was in agreement with that reported McCord and Kilara (1983) who suggested that the mushroom PPO enzyme remained fully active up to 45 °C after 10 min exposure to different temperatures, and between 45 °C and 70 °C, a gradual decline in activity was observed and above 70 °C, no activity was detectable. Furthermore, our results were in agreement with previous studies (Gouzi et al., 2012; Ionita et al., 2014). 3.2. FTIR studies 3.2.1. Infrared spectroscopy of PPO in H2O The FTIR spectra of enzyme solution samples, buffer, subtracted spectrum and PPO in H2O buffer at 25 °C are shown in Fig. 2. The peak at 1653 cm 1 is assigned as the amide I band which arises
mainly from amide C@O stretching (80%) frequencies of the protein backbone. The peak at 1550 cm 1 is assigned as the amide II band, which is due to N–H bending (60%) and C–N stretching (40%) vibrations of the peptide backbone (Stuart, 2004; Haris & Severcan, 1999). Analysis of the amide I band (1700–1600 cm 1) by using Fourier self-deconvolution techniques gave qualitative information on the secondary structure of a protein (Haris & Severcan, 1999). The deconvoluted spectra revealed that the major amide I band at 1653 cm 1 is due to a-helix structure. The peak located at 1676 cm 1 is assigned to b-turn structure. The band at 1636 cm 1 is assigned to b-sheet structure. The peak at located around 1617 cm 1 is due to aggregated b-sheet structure. The amide II band, located at 1548 cm 1, is used to monitor hydrogen–deuterium exchange (Severcan & Haris, 2003; Murayama & Tomida, 2004). Quantitative secondary structure analysis of PPO in H2O buffer was carried out by using the NN analysis method, which revealed that PPO contains 42.33 ± 3.06% a-helix, 21.33 ± 4.73% b-sheet, 19.67 ± 1.15% turns and 16.67 ± 1.53% random coil structures.
Fig. 3. (A) Representative absorbance spectra of PPO in D2O buffer over a temperature range of 25–70 °C (solid lines) and spectrum of the PPO measured at 25 °C after cooling from 70 °C (dotted line), (B) representative Fourier self-deconvolution spectra of PPO in D2O buffer over a temperature range of 25–70 °C (solid lines) and spectrum of the PPO measured at 25 °C after cooling from 70 °C (dotted line).
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Furthermore, tyrosinase contains 38.1% a-helix, 4.1% 3–10 helix, 6.9% strand, and 50.9% other structures according to the analysis of crystal structure of the enzyme from the protein data bank. These analyses demonstrated that PPO enzyme is a-helix dominating enzyme. This finding agreed with that reported by Tse et al. (1997), who indicated the presence of a strong band at 1651 cm 1. Although the PDB results, as reported by Ionita et al. (2014) for helix structures (who reported 29.0% a-helix and 1.4% 3–10 helix), are not consistent with the results reported in our study, it is clear that the dominant structure of PPO is the a-helix. In the former study, the aim was not primarily to determine the secondary structure, but to characterize the heat-induced changes in enzyme dynamics and structure, including some information about salt bridge formation in the presence and absence of Ca, using the power of fluorescence spectroscopy coupled with molecular dynamics simulations. Spectroscopic techniques give information at different time scales and therefore, are used comparatively to each other. The current study used the well-known advantages of infrared spectroscopy in secondary structure determination of the proteins in their native environment (Turker, Ilbay, Severcan, & Severcan, 2014; Cakmak et al., 2011). Infrared spectroscopy is a powerful analytic technique based on the absorption of infrared photons that excite vibrations of molecular bonds. It is a rapid, low cost, operator independent, nondestructive (needs no probe) technique that requires small sample quantities. Furthermore, it monitors biomolecules in their native environment with high sensitivity. In FTIR spectroscopy, the data obtained are stored in digitally encoded formats, which facilitates spectral interpretation with the aid of post-acquisition data manipulation algorithms. This property of the technique provides the accurate detection of small changes even in weak absorption bands (Severcan & Haris, 2012; Ozek, Bal, Sara, Onur, & Severcan, 2014). 3.2.2. Infrared spectroscopy of PPO in D2O Representative absorbance and Fourier self-deconvolution spectra of PPO in D2O buffer at 25 °C are shown in Fig. 3A and B, respectively. It can be seen from the absorbance spectrum of PPO that the amide II band at 1550 cm 1, seen for PPO in H2O buffer, disappeared and shifted to the 1455 cm 1, suggesting hydrogen– deuterium exchange occurred. Fourier self-deconvolution analysis revealed that the band located at 1652 cm 1 is due to a-helix structure. The peak located at 1673 cm 1 is assigned to b-turn structure. The band at 1635 cm 1 is assigned to b-sheet structure. The peak at located around 1616 cm 1 is due to aggregated b-sheet structure. There was a shift towards lower frequencies due to the hydrogen– deuterium exchange in the sub bands. 3.2.3. Conformational change of PPO during thermal treatment Representative absorbance spectra of PPO in the 1750–1400 cm 1 region recorded at different temperatures are shown in Fig. 3A. It was observed that the intensity of the amide I band decreased as the temperature increased between 25 °C and 70 °C. This may indicate a conformational change in the enzyme. However, no change was observed for the spectral patterns between 70 °C and 80 °C (data not shown). The native spectrum was not observed when the enzyme was cooled back to room temperature, which indicates that heat induced structural changes in the enzyme are irreversible. Recovery of activity of the enzyme decreased with increasing temperature. Moreover, no recovery of enzyme activity was seen after thermal inactivation. To explore the temperature induced changes in the secondary structures of PPO in detail, Fourier self-deconvolution techniques applied to the FTIR spectra. Fig. 3B presents the representative deconvolved spectra of PPO solution in the 1700–1600 cm 1 region measured between temperature range of 25–70 °C and at 25 °C
Fig. 4. A plot of the temperature induced changes in the intensity of the amide I and its components for PPO (() 1652 cm 1, (j) 1616 cm 1, (N) 1635 cm 1 and (d) amide I).
after cooling. According to the deconvolved spectra of PPO, there was little change in the temperature range of 25–40 °C. Marked spectral changes were noted after this temperature and additional new bands due to aggregated b-sheet structures appeared at 1683 and 1616 cm 1. These bands are detected when proteins are denatured (Severcan & Haris, 2003). The intensity of these bands increased with increasing temperature. When the temperature was lowered back to 25 °C, from 70 °C, these bands were still observed, indicating an irreversible change in the structure of PPO. Moreover, the intensity of a-helix and b-sheet structures decreased and shifted to lower values while the temperature increased to 70 °C. These observations indicate the loss of main secondary structure elements of PPO during the thermal treatment. According to the X-ray analysis, mushroom tyrosinase is a tetrameric protein composed of two subunits (H and L). H subunit is the domain of the enzyme and contains 13 a-helices, eight, mostly, short b-strands and many loops. The active site of the enzyme is made up a bundle of four helices in the center of the domain. It is also reported that the L subunit consists of 12 antiparallel
Table 1A Transition temperatures (Tm) for different amide I components for PPO. Structure
Amide I band (cm
a-Helix
1652 1635 1616 1653
b-Sheet Aggregated b-sheet Amide I
1
)
Tm value (°C) 54.72 53.5 57.07 53
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the enzyme. Denaturation of the enzyme also started above 38 °C, since it was accompanied by an increase of the aggregation band located at 1616 cm 1. The differences in the thermal unfolding and protein secondary structure between the study by Ionita et al. (2014) and our current study might be mainly due to differences in the studied form of the protein, where crystalline and solution forms of the protein were studied, respectively. Our observation on thermal unfolding was consistent with the activity measurements in that activity loss was observed above 40 °C. As shown in our results, the structural conformation of the enzyme was easily detected even at lower temperatures by using FTIR spectroscopy, which is known to be very sensitive to conformational changes in the enzyme as previously mentioned (Haris & Severcan, 1999; Goormaghtigh et al., 2009; Yang et al., 2015). Simultaneously, the intensity of the aggregation bands at 1616 and 1683 cm 1 increased with increasing temperature. The band located at 1616 cm 1 first appeared at 42 °C. The intensity of the band increased linearly up to 64 °C and no significant change was observed after this temperature, with a midpoint temperature of 57 °C. These results clearly indicate denaturation of the enzyme since this band appears only when proteins are denaturated (Severcan & Haris, 2003). Similarly, Tse et al. (1997) found that the secondary structure began to change above 40 °C and a new band at 1616 cm 1 appeared at 45 °C for mushroom tyrosinase. Using the intensity profiles, transition temperature (Tm) for the different amide I components was determined and is represented in Table 1A. The Tm values for a-helix, b-sheet, aggregated b-sheet structure and amide I were determined as around 55 °C. This value was very close to that reported by McCord and Kilara (1983) and Weemaes et al. (1997), whom found Tm value at 54 °C and 51.8 °C, respectively. Fig. 5. Curve-fitting analysis of amide I band of PPO.
b-strands assembled in a cylindrical barrel of six 2-stranded sheets and is not involved in the activation mechanism of the enzyme (Ismaya et al., 2011). As stated in X-ray analysis, reduction in a-helix structures affects the active site of the enzyme and caused structural deterioration. The intensities of the amide I band and its sub structures are plotted as a function of temperature in order to estimate the thermal unfolding of PPO more precisely (Fig. 4). As stated previously, the intensity of the 1652 and 1635 cm 1 bands decreased as the temperature increased and a multistep transition was represented for a-helix and b sheet. The first transition was observed at 45 °C and the higher transition occurred at 66 °C. A similar trend was also obtained for the thermal unfolding of un-deconvolved amide I band. However, Ionita et al. (2014) reported that no change was observed in the secondary structure of the enzyme up to 60 °C and the a-helix content decreased at temperatures over 60 °C after running molecular dynamics simulations at different temperatures. But according to our study, a-helix content started to decrease above 38 °C and the midpoint transition temperature located at 45 °C, indicating change in the secondary structure of
3.2.4. Curve-fitting analysis of PPO In order to determine secondary structural changes during thermal treatment in D2O, curve-fitting analysis was applied to the amide I region (1700–1600 cm 1). The contribution of each component band to the amide I band is shown in Fig. 5 for two different temperatures (25 and 70 °C). The bands at 1652 cm 1 due to the a-helix and that at 1635 cm 1 due to the b-sheet structure became weaker as temperature increases from 25 to 70 °C, whereas other bands due to the aggregated b-sheet (1683 and 1616 cm 1), turns (1676 cm 1) and random coil (1640 cm 1) structures became much stronger at 70 °C, indicating denaturation of the enzyme. Secondary structural change (%) of PPO during thermal treatment estimated by curve-fitting analysis is shown in Table 1B. According to the curve-fitting analysis, PPO contained 38.87% a-helix, 27.91% b-sheet, 14.93% b-turn, 15.01% random coil and 3.27% aggregated b-sheet structures in the untreated sample. These results are in accordance with the NN results, which indicated that PPO was an a-helix dominating enzyme. Previous circular dichroism (CD) studies reported values of 35.3% (Liu et al., 2013), 35.7% (Yi et al., 2012) and 41.3% (Liu et al., 2009) for a-helix content of PPO. a-Helix content decreased from 38.87% to 19.40% (p < 0.05) and b-sheet content decreased from 27.91% to 15.88% (p < 0.05), whereas b-turn, random coil and aggregated
Table 1B Secondary structure change (%) of PPO estimated by curve-fitting during thermal treatment. Temperature (°C)
a-Helix
b-Sheet
b-Turn
Random coil
Aggregated b-sheets
25 30 40 50 60 70 25 (after cooling from 70 °C)
38.87 ± 0.02 35.68 ± 0.06 26.91 ± 0.01 25.42 ± 0.10 24.01 ± 0.18 19.41 ± 0.16 19.40 ± 0.21
27.91 ± 0.02 25.14 ± 0.13 22.83 ± 0.01 19.50 ± 0.33 18.24 ± 0.14 16.25 ± 0.14 15.88 ± 0.02
14.93 ± 0.01 14.74 ± 0.02 18.07 ± 0.01 16.81 ± 0.07 17.67 ± 0.13 21.68 ± 0.18 21.93 ± 0.14
15.01 ± 0.01 19.94 ± 0.03 21.92 ± 0.01 22.22 ± 0.09 21.20 ± 0.16 22.48 ± 0.65 22.47 ± 0.60
3.27 ± 0.06 4.50 ± 0.01 10.28 ± 0.04 16.05 ± 0.07 18.88 ± 0.60 20.19 ± 0.17 20.31 ± 0.27
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b-sheet structures increased up to 21.93%, 22.47% and 20.31%, respectively (p < 0.05). This clearly showed that a-helix and bsheet structures decreased as temperature increase with the increase of aggregated b-sheets turns and disorder structure (1640 cm 1), which indicates denaturation of the enzyme at higher temperatures. As we have reported above, the enzyme started to denature at around 40 °C with a midpoint temperature of 55 °C. 4. Conclusion In the present study, changes in structure and activity of PPO during thermal treatment in the temperature range of 25–80 °C were investigated. According to the inactivation studies, the enzyme remained fully active up to 40 °C. Higher inactivation was achieved between 50 °C and 70 °C and the enzyme was completely inactivated at 70 °C for 5 min. Furthermore, no activity was observed over 80 °C. To explore the secondary structure and conformation change of PPO, Fourier self-deconvolution, NN and curve-fitting analysis were applied to the amide I region of the FTIR spectrum. FTIR results revealed that the PPO enzyme is an a-helix dominating enzyme and a-helix and b-sheet content decreased, aggregated b-sheet, turns and random coil increased at 55 °C, indicating protein denaturation. Moreover, the FTIR spectrum at 70 °C and that at 25 °C after cooling were very similar to each other and this was different than the original spectrum of 25 °C. In conclusion, denaturation of the enzyme was irreversible. This study gives information about the inactivation mechanism of polyphenoloxidase during thermal treatment in a model system. Further studies will be carried out to clarify the inactivation mechanism of the enzyme in food systems. Acknowledgments This work was supported by the State Planing Organization (DPT), Grant No: BAP-08-11-DPT2002K120510-GT-4 and Scientific and Technical Research Council of the Turkish Republic _ (TUBITAK), Project No: TOVAG-112O433. References Anthon, G. E., & Barrett, D. M. (2002). Kinetic parameters for the thermal inactivation of quality-related enzymes in carrots and potatoes. Journal of Agricultural and Food Chemistry, 50, 4119–4125. Bayindirli, A., Alpas, H., Bozoglu, F., & Hizal, M. (2006). Efficiency of high pressure treatment on inactivation of pathogenic microorganisms and enzymes in apple, orange, apricot and sour cherry juices. Food Control, 17, 52–58. Cakmak, G., Zorlu, F., Severcan, M., & Severcan, F. (2011). Screening of protective effect of amifostine on radiation-induced structural and functional variations in rat liver microsomal membranes by FT-IR spectroscopy. Analytical Chemistry, 83, 2438–2444. Fortea, M. I., López-Miranda, S., Serrano-Martínez, A., Carreño, J., & Núñez-Delicado, E. (2009). Kinetic characterization and thermal inactivation study of polyphenol oxidase and peroxidase from table grape (Crimson Seedless). Food Chemistry, 113, 1008–1014. Garip, S., Yapici, E., Simsek Ozek, N., Severcan, M., & Severcan, F. (2010). Evaluation and discrimination of simvastatin-induced structural alterations in proteins of different rat tissues by FTIR spectroscopy and neural network analysis. Analyst, 135, 3233–3241. Goormaghtigh, E., Gasper, R., Bénard, A., Goldsztein, A., & Raussens, V. (2009). Protein secondary structure content in solution, films and tissues: Redundancy and complementarity of the information content in circular dichroism,
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transmission and ATR FTIR spectra. Biochimica et Biophysica Acta, 1794, 1332–1343. Gouzi, H., Depagne, C., & Coradin, T. (2012). Kinetics and thermodynamics of the thermal inactivation of polyphenol oxidase in an aqueous extract from Agaricus bisporus. Journal of Agricultural and Food Chemistry, 60, 500–506. Haris, P. I., & Severcan, F. (1999). FTIR spectroscopic characterization of protein structure in aqueous and non-aqueous media. Journal of Molecular Catalysis B: Enzymatic, 7, 207–221. Ionita, E., Aprodu, I., Stanciuc, N., Rapeanu, G., & Bahrim, G. (2014). Advances in structure–function relationships of tyrosinase from Agaricus bisporus – Investigation on heat-induced conformational Changes. Food Chemistry, 156, 129–136. Ismaya, W. T., Rozeboom, H. J., Weijn, A., Mes, J. J., Fusetti, F., Wichers, H. J., et al. (2011). Crystal structure of Agaricus bisporus mushroom tyrosinase: Identity of the tetramer subunits and interaction with tropolone. Biochemistry, 50, 5477–5486. Liu, W., Liu, J., Liu, C., Zhong, Y., Liu, W., & Wan, J. (2009). Activation and conformational changes of mushroom polyphenoloxidase by high pressure microfluidization treatment. Innovative Food Science and Emerging Technologies, 10, 142–147. Liu, W., Zou, L., Liu, J., Zhang, Z., Liu, C., & Liang, R. (2013). The effect of citric acid on the activity, thermodynamics and conformation of mushroom polyphenoloxidase. Food Chemistry, 140, 289–295. Ludikhuyze, L., Van Loey, A., Smout, I. C., & Hendrickx, M. (2003). Effects of combined pressure and temperature on enzymes related to quality of fruits and vegetables: From kinetic information to process engineering aspects. Critical Reviews in Food Science and Nutrition, 43(5), 527–586. McCord, J. D., & Kilara, A. (1983). Control of enzymatic browning in processed mushrooms (Agaricus bisporus). Journal of Food Science, 48, 1479–1483. Murayama, K., & Tomida, M. (2004). Heat-induced secondary structure and conformation change of bovine serum albumin investigated by Fourier transform infrared spectroscopy. Biochemistry, 43, 11526–11532. Ozek, N. S., Bal, B., Sara, Y., Onur, R., & Severcan, F. (2014). Structural and functional characterization of simvastatin-induced myotoxicity in different skeletal muscles. Biochimica et Biophysica Acta, 1840, 406–415. Severcan, M., Severcan, F., & Haris, P. I. (2001). Estimation of protein secondary structure from FTIR spectra using neural networks. Journal of Molecular Structure, 565–566, 383–387. Severcan, F., & Haris, P. I. (2003). Fourier transform infrared spectroscopy suggests unfolding of loop structures precedes complete unfolding of pig citrate synthase. Biopolymers, 69, 440–447. Severcan, M., Haris, P. I., & Severcan, F. (2004). Using artificially generated spectral data to improve protein secondary structure prediction from Fourier transform infrared spectra of proteins. Analytical Biochemistry, 332, 238–244. Severcan, F., & Haris, P. I. (2012). Vibrational spectroscopy in diagnosis and screening. IOS Press. Stuart, B. (2004). Infrared spectroscopy: Fundamentals and applications. John Wiley and Sons. Sun, N. K., & Song, K. B. (2003). Effect of nonthermal treatment on the molecular properties of mushroom polyphenoloxidase. Journal of Food Science, 68(5), 1639–1643. Toyran, N., Zorlu, F., Dönmez, G., Ög˘e, K., & Severcan, F. (2004). Chronic hypoperfusion alters the content and structure of proteins and lipids of rat brain homogenates: A Fourier transform infrared spectroscopy study. European Biophysics Journal, 33, 549–554. Tse, M., Kermasha, S., & Ismail, A. (1997). Biocatalysis by tyrosinase in organic solvent media: A model system using catechin and vanillin as substrates. Journal of Molecular Catalysis B: Enzymatic, 2, 199–213. Turker, S., Ilbay, G., Severcan, M., & Severcan, F. (2014). The investigation of compositional, structural and dynamical changes of PTZ-induced seizures on a rat brain by FTIR spectroscopy. Analytical Chemistry, 86(3), 1395–1403. Weemaes, C., Rubens, P., De Cordt, S., Ludikhuyze, L., Van Den Broeck, I., Hendrickx, M., et al. (1997). Temperature sensitivity and pressure resistance of mushroom polyphenoloxidase. Journal of Food Science, 62(2), 261–266. Yang, H., Yang, S., Kong, J., Dong, A., & Yu, S. (2015). Obtaining information about protein secondary structures in aqueous solution using Fourier transform IR spectroscopy. Nature Protocols, 10, 382–396. Yemeniciog˘lu, A., Özkan, M., & Cemerog˘lu, B. (1997). Heat inactivation kinetics of apple polyphenoloxidase and activation of its latent form. Journal of Food Science, 62(3). Yi, J., Jiang, B., Zhang, Z., Liao, X., Zhang, Y., & Hu, X. (2012). Effect of ultrahigh hydrostatic pressure on the activity and structure of mushroom (Agaricus bisporus) polyphenoloxidase. Journal of Agricultural and Food Chemistry, 60, 593–599.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Effect of thermal treatment on secondary structure and conformational change of mushroom polyphenol oxidase (PPO) as food quality related enzyme: A FTIR study Hande Baltacıog˘lu a,b, Alev Bayındırlı a, Mete Severcan c, Feride Severcan d,⇑ a
Department of Food Engineering, Middle East Technical University, 06800 Ankara, Turkey Department of Food Engineering, Ondokuz Mayis University, 55139 Samsun, Turkey Department of Electrical and Electronics Engineering, Middle East Technical University, 06800 Ankara, Turkey d Department of Biological Sciences, Middle East Technical University, 06800 Ankara, Turkey b c
a r t i c l e
i n f o
Article history: Received 24 November 2014 Received in revised form 25 March 2015 Accepted 21 April 2015 Available online 23 April 2015 Keywords: PPO Heat inactivation FTIR Spectroscopy Protein secondary structure
a b s t r a c t In order to understand the conformational changes of polyphenol oxidase (PPO), which is a food quality related enzyme, after thermal treatment, secondary structure changes of the enzyme were analyzed by using Fourier Transform Infrared (FTIR) spectroscopy and compared with the change in enzyme activity in the temperature range of 25–80 °C. Fourier self-deconvolution, neural network (NN) and curve-fitting analysis were applied to the amide I band of FTIR spectra for detail analysis of secondary structure elements. FTIR analysis indicated that PPO is an a-helix dominating enzyme. Detail analysis revealed that, as temperature increased, a-helix and b-sheet decreased, but aggregated b-sheet, turns and random coil increased. The marked changes were noted at 40 °C with the occurrence of new bands due to aggregated b-sheet structures, all of which indicate protein denaturation. These aggregation bands were still observed when the temperature was reduced back to 25 °C, from 70 °C, demonstrating an irreversible change in the structure. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Polyphenol oxidase (PPO, E.C 1.14.18.1) is a copper-containing enzyme that causes enzymatic browning in fresh fruit, vegetable products and also mushrooms. PPO from all of these species has six histidine residues that ligats the two copper ions of the active site (Yi et al., 2012). The mushroom PPO enzyme is commonly found as a tetrameric protein with a molecular mass of 120 kDa, composed of two subunits of 43 kDa (H subunit) and two subunits of 14 kDa (L subunit). The enzyme is a-helix dominating enzyme with the helices found in the H subunit. The H subunits contain a binuclear copper-binding site with each copper ion coordinated by three histidine residues. Binuclear copper-binding site locates in the center of the domain, between a bundle of four helices (a3, a4, a10 and a11). The L subunit consists of 12 antiparallel b-strands assembled in a cylindrical barrel of six 2-stranded sheets (Ismaya et al., 2011). Enzymatic browning is the result of the oxidation of o-diphenols into unstable quinones by PPO in the presence of molecular oxygen. o-Quinones are highly reactive ⇑ Corresponding author. E-mail address: [email protected] (F. Severcan). http://dx.doi.org/10.1016/j.foodchem.2015.04.097 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
compounds and react with other phenols and non-phenolic compounds to give brown pigments. This enzymatic browning affects color, flavor and nutritional quality of foods. Therefore, this enzyme is inactivated during food processing in order to prevent browning (Ludikhuyze, Van Loey, Smout, & Hendrickx, 2003). Thermal treatment is the most common and widely employed technique for the inactivation of food quality related enzymes in the food industry. The inactivation of enzymes during thermal treatment has been extensively studied over the past years and this process has been generally represented with simple kinetic approaches in the literature. Inactivation kinetics of PPO in apple (Yemeniciog˘lu, Özkan, & Cemerog˘lu, 1997), carrots and potatoes (Anthon & Barrett, 2002), table grape (Fortea, López-Miranda, Serrano-Martínez, Carreño, & Núñez-Delicado, 2009) and mushroom (Agaricus bisporus) (Gouzi, Depagne, & Coradin, 2012; Ionita, Aprodu, Stanciuc, Rapeanu, & Bahrim, 2014) were previously reported. However, less attention has been given to investigate the conformation changes of food enzymes during thermal treatment. There are different analytical tools to study protein conformation. Among these techniques FTIR spectroscopy has several advantages for determination of secondary structure of proteins
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in solution (Severcan, Haris, & Severcan, 2004; Goormaghtigh, Gasper, Bénard, Goldsztein, & Raussens, 2009; Yang, Yang, Kong, Dong, & Yu, 2015) and in biological systems (Cakmak, Zorlu, Severcan, & Severcan, 2011; Garip, Yapici, Simsek Ozek, Severcan, & Severcan, 2010). It provides high-quality spectra with very small amounts of protein whose size is not important. It is a rapid technique with relatively low costs. Characteristic bands in an IR spectrum of a protein are very sensitive to conformational changes in the protein (Haris & Severcan, 1999). The amide I region is generally used for the analysis of secondary structures of proteins from FTIR spectra. Neural networks (NNs) are reported to be effective ways to predict the secondary structure of proteins from FTIR spectra (Severcan, Severcan, & Haris, 2001; Severcan et al., 2004). There are limited numbers of studies about the conformational changes of PPO. Among these, FTIR spectroscopic analysis of mushroom PPO was investigated in the temperature range of 25–45 °C. It was reported that tyrosinase was predominately a-helix in nature and above 40 °C the secondary structure of the enzyme started to change (Tse, Kermasha, & Ismail, 1997). Weemaes et al. (1997) determined the thermal stability of mushroom PPO by using FTIR spectroscopy. A temperature titration curve constructed in order to determine whether thermal inactivation was due to a small change in the active site or to a global conformation change of the enzyme was used in this study. However, detailed secondary structural analysis during thermal treatment was not reported in these studies. Ionita et al. (2014) studied the heat induced changes of tyrosinase from A. bisporus by using the combination of fluorescence spectroscopic measurements, inactivation kinetics, and in silico prediction. Changes in the enzyme during thermal treatment at different temperature values were studied. The thermal induced behavior of tyrosinase at single molecule level was investigated by means of numerical approach in this study and atomic details were provided after performing molecular dynamics simulations at different temperatures on the enzyme crystal structure. It was reported that significant changes in secondary structure of tyrosinase above 60 °C might lead to enzyme inactivation according to the molecular dynamics simulations. In the present study, in addition to the temperature induced changes in the activity of PPO, in order to understand the conformational changes of PPO after thermal inactivation, secondary structural content of the enzyme in solution was determined precisely for the first time, using FTIR spectroscopy. To achieve this, neural network (NN) and curve-fitting analysis based on amide I band (1700–1600 cm 1) were used. It is well known that, a protein’s structure in solution reveals the native form of the protein, and such gives a more realistic picture of the protein. Furthermore, using FTIR spectroscopy, for the first time, unfolding of secondary structural elements of PPO are presented depending to the thermal treatment in the temperature range of 25–80 °C. 2. Materials and methods 2.1. Chemicals Mushroom PPO (98% purity) (E.C 1.14.18.1), catechol (P99% purity), D2O (99.9 atom% D) were purchased from Sigma (St. Louis, MO, USA). The enzyme was used without further purification. All chemicals were obtained from commercial sources at the highest grade of purity available. 2.2. Enzyme preparation and activity assay Lyophilized PPO was dissolved in 50 mM phosphate buffer (pH 6.5). Catechol was used as substrate for the determination of the enzyme activity. An aliquot (2 ml) of 50 mM potassium
phosphate buffer (pH 6.5) and 0.3 ml of 0.2 M catechol solution in the phosphate buffer were incubated in a test tube at 25 ± 1 °C. Then the tube contents were transferred to a plastic cuvette (path length, 10 mm) and 0.3 ml of the enzyme solution (0.08 mg/ml) was added to the cuvette to initiate the enzyme reaction. PPO activity was determined using a UV–Vis spectrophotometer (BOECO Model S22, Germany) at 420 nm at room temperature (25 ± 1 °C). Absorbance was read every 5 s for 3 min. Enzyme activity was calculated from the slope of the initial linear section of the absorbance versus time curves. One unit of enzyme activity was defined as the amount of the enzyme which caused a change of 0.001 in absorbance unit per minute. Enzyme activities were measured 3 times and expressed as residual activity (Bayindirli et al., 2006; Sun & Song, 2003; Weemaes et al., 1997). 2.3. Thermal treatment at ambient pressure Thermal treatment of PPO was performed at varying temperatures in the range of 20–80 °C with a 10 °C increment for 0, 5, 10, 15, 20, 25 and 30 min. The enzyme solutions (5 ml) in a glass tube with an inner diameter of 16 mm and a depth of 50 mm were heated in a temperature controlled dry block heater (HBR-48, Daihan Scientific Co. Ltd., Seoul, Korea) at selected temperatures and times. They were immediately transferred to an ice water to stop thermal inactivation instantaneously and the enzyme activity measurement was performed after 10–60 min of storage. All the experiments and measurements were repeated in triplicate. 2.4. Fourier Transform Infrared (FTIR) spectroscopy In order to determine the conformational change of PPO after inactivation, the secondary structure of the enzyme was analyzed by using FTIR spectroscopy. Infrared spectra were collected using a Perkin-Elmer Spectrum 100 FTIR spectrometer (Perkin-Elmer Inc., Norwalk, CT, USA) equipped with a MIR TGS detector. The sample compartment was continuously purged with dry air to minimize atmospheric water vapor absorbance, which overlaps in the spectral region of interest, and carbon dioxide interference. The spectrum of air was recorded as background and subtracted automatically by using the appropriate software. In order to control the temperature, the cell was connected to a thermostated circulating water bath. A thermocouple was placed on the outside of the cell to monitor the temperature of the cell. For secondary structure analysis, measurements were performed in H2O buffer. Mushroom PPO (Sigma, St. Louis, MO, USA) was dissolved in 50 mM phosphate buffer (pH 6.5) to yield a final protein concentration of 70 mg/ml for FTIR measurements. A volume of sample (10 ll) was put between CaF2 windows with a 6 lm path length and the windows were inserted into the system. Each spectrum of enzyme solution samples and buffer were collected in the 1200–2800 cm 1 region at 25 °C. A total of 400 scans were taken for each interferrogram at 2 cm 1 resolution. For inactivation studies, measurements were performed in D2O buffer. Prior to infrared experiments, the enzyme was dissolved in 50 mM phosphate buffer (prepared with D2O). Enzyme solution was allowed to stand 24 h prior to measurement to allow H–D exchange. A volume of sample (10 ll) was put between CaF2 windows with a 50 lm path length and the windows were placed into the system as described above. Samples were heated over a linear temperature gradient from 25 to 80 °C with 2 °C intervals and then cooled back to the 25 °C. Infrared spectra were recorded continuously. Each spectrum of enzyme solutions and buffer were collected in the 1400–2200 cm 1 region. A total of 128 scans were taken for each interferrogram at 2 cm 1 resolution. Infrared spectra were recorded continuously. All of the experiments were repeated 3 times and identical results were obtained.
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2.5. Data analysis for FTIR spectra Collections of spectra and data manipulations were carried out using Spectrum 100 software (Perkin-Elmer). Infrared spectra of buffer solution were measured under identical conditions. The spectrum of buffer was subtracted from the spectra of enzyme solution at the same temperature to obtain protein bands. In the subtraction process the bulk water band located at 2125 cm 1 was flattened. Spectrum 100 software (Perkin-Elmer) was used for the subtraction procedure. For protein secondary structure determination, OPUSNT data collection software (Bruker Optics, Reinstetten, Germany) was used to generate Fourier self-deconvolution and second derivative of amide I. The second derivative spectra were obtained by applying a Savitzky–Golay algorithm with nine smoothing points and these derivatives were vector normalized at 1700–1600 cm 1 and then the peak intensity values were calculated. The peak minima of the second derivative signals were used, since they correspond to the peak positions of the original absorbance spectra. In the Fourier self-deconvolution, a half bandwidth of 14 cm 1 and resolution enhancement factor k = 2.4 were used for the absorbance spectra. The deconvolved spectra were fitted with Lorentzian band profiles. Transition temperatures (Tm) were determined in Microsoft Excel 2007 software (Microsoft Corp., Redmond, WA).
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PA). Mean separation was performed by Tukey test at p < 0.05 level. Analyses results were the mean with triplicate. 3. Results and discussion 3.1. PPO inactivation during thermal treatment The residual enzyme activity of mushroom PPO after heat treatment for different time intervals is shown in Fig. 1. When the stability of mushroom PPO after 30 min exposure at different temperatures was investigated, the enzyme remained fully active up to 40 °C (data not shown). There was a slight decrease during the period of 30 min at 40 °C and the activity of enzyme was recorded as 89.84 ± 2.70% after 30 min heat treatment at 40 °C. Between 50 °C and 70 °C, higher inactivation was achieved and 99% inactivation was detectable at 70 °C for 5 min (Fig. 1). Furthermore, no activity was observed over 80 °C. Residual enzyme
2.6. Protein secondary structure prediction 2.6.1. Secondary structure determination from crystal structure of PPO The crystal structure of tyrosinase from A. bisporus was obtained from Protein Data Bank (PDB; http://www.rcsb.org/pdb/) (PDB code, 2Y9W). Secondary structure is calculated using PDBsum bioinformatics tool through ProMotif program. 2.6.2. Secondary structure prediction by neural network (NN) analysis Protein secondary structure predictions in H2O were carried out using NN. NN were first trained using FTIR spectra of 18 water soluble proteins recorded in water whose secondary structures were known from X-ray Crystallography (Severcan et al., 2001). Before applying to the NN, the Amide I band (1700–1600 cm 1) was preprocessed which involves normalization and discrete cosine transformation (DCT) of the amide I band of the FTIR spectra. The size of the training data set was increased by interpolating the available FTIR spectra to improve the training of the NN. A separate NN was trained using Bayesian regularization whose number of inputs, i.e., the number of DCT coefficients, and the number of hidden neurons were optimized for each structure parameter. The trained NNs have standard error of prediction values of 4.19% for a-helix, 3.49% for b-sheet and 3.15% for turns. The secondary structure parameters of the new proteins were predicted by applying the inputs of the trained NNs the preprocessed FTIR data as reported in detailed in Severcan et al., 2004.
Fig. 1. Thermal stability of PPO.
2.6.3. Secondary structure prediction using curve-fitting analysis Curve-fitting analysis was applied to amide I region to obtain information about the changes in the secondary structure of the enzyme during thermal treatment in D2O. Curve-fitting analysis was performed by using Grams 32 (Galactic Industries, Salem, NH, USA) software. The center positions for each sub-band were determined by second derivative analysis and the shapes of the underlying bands were chosen as Gaussian. The iterations were performed until the correlation was better than 0.995 (Toyran, Zorlu, Dönmez, Ög˘e, & Severcan, 2004). 2.7. Statistical analysis The data were analyzed as a completely randomized design by analysis of variance using Minitab 16 (Minitab Inc., State College,
Fig. 2. The FTIR spectra of enzyme solution sample (A), buffer (B) and subtracted spectrum of PPO in H2O buffer at 25 °C (C).
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activity after 30 min heat treatment at 50 °C and 60 °C were detected as 23.60 ± 1.26% and 0.42 ± 0.03%, respectively. From the graph, it can be seen that the rate of PPO inactivation depended on temperature and increased with increasing temperature and time (p < 0.05). Furthermore, there was a significant interaction (p < 0.05) among time and temperature. The trend of inactivation was in agreement with that reported McCord and Kilara (1983) who suggested that the mushroom PPO enzyme remained fully active up to 45 °C after 10 min exposure to different temperatures, and between 45 °C and 70 °C, a gradual decline in activity was observed and above 70 °C, no activity was detectable. Furthermore, our results were in agreement with previous studies (Gouzi et al., 2012; Ionita et al., 2014). 3.2. FTIR studies 3.2.1. Infrared spectroscopy of PPO in H2O The FTIR spectra of enzyme solution samples, buffer, subtracted spectrum and PPO in H2O buffer at 25 °C are shown in Fig. 2. The peak at 1653 cm 1 is assigned as the amide I band which arises
mainly from amide C@O stretching (80%) frequencies of the protein backbone. The peak at 1550 cm 1 is assigned as the amide II band, which is due to N–H bending (60%) and C–N stretching (40%) vibrations of the peptide backbone (Stuart, 2004; Haris & Severcan, 1999). Analysis of the amide I band (1700–1600 cm 1) by using Fourier self-deconvolution techniques gave qualitative information on the secondary structure of a protein (Haris & Severcan, 1999). The deconvoluted spectra revealed that the major amide I band at 1653 cm 1 is due to a-helix structure. The peak located at 1676 cm 1 is assigned to b-turn structure. The band at 1636 cm 1 is assigned to b-sheet structure. The peak at located around 1617 cm 1 is due to aggregated b-sheet structure. The amide II band, located at 1548 cm 1, is used to monitor hydrogen–deuterium exchange (Severcan & Haris, 2003; Murayama & Tomida, 2004). Quantitative secondary structure analysis of PPO in H2O buffer was carried out by using the NN analysis method, which revealed that PPO contains 42.33 ± 3.06% a-helix, 21.33 ± 4.73% b-sheet, 19.67 ± 1.15% turns and 16.67 ± 1.53% random coil structures.
Fig. 3. (A) Representative absorbance spectra of PPO in D2O buffer over a temperature range of 25–70 °C (solid lines) and spectrum of the PPO measured at 25 °C after cooling from 70 °C (dotted line), (B) representative Fourier self-deconvolution spectra of PPO in D2O buffer over a temperature range of 25–70 °C (solid lines) and spectrum of the PPO measured at 25 °C after cooling from 70 °C (dotted line).
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Furthermore, tyrosinase contains 38.1% a-helix, 4.1% 3–10 helix, 6.9% strand, and 50.9% other structures according to the analysis of crystal structure of the enzyme from the protein data bank. These analyses demonstrated that PPO enzyme is a-helix dominating enzyme. This finding agreed with that reported by Tse et al. (1997), who indicated the presence of a strong band at 1651 cm 1. Although the PDB results, as reported by Ionita et al. (2014) for helix structures (who reported 29.0% a-helix and 1.4% 3–10 helix), are not consistent with the results reported in our study, it is clear that the dominant structure of PPO is the a-helix. In the former study, the aim was not primarily to determine the secondary structure, but to characterize the heat-induced changes in enzyme dynamics and structure, including some information about salt bridge formation in the presence and absence of Ca, using the power of fluorescence spectroscopy coupled with molecular dynamics simulations. Spectroscopic techniques give information at different time scales and therefore, are used comparatively to each other. The current study used the well-known advantages of infrared spectroscopy in secondary structure determination of the proteins in their native environment (Turker, Ilbay, Severcan, & Severcan, 2014; Cakmak et al., 2011). Infrared spectroscopy is a powerful analytic technique based on the absorption of infrared photons that excite vibrations of molecular bonds. It is a rapid, low cost, operator independent, nondestructive (needs no probe) technique that requires small sample quantities. Furthermore, it monitors biomolecules in their native environment with high sensitivity. In FTIR spectroscopy, the data obtained are stored in digitally encoded formats, which facilitates spectral interpretation with the aid of post-acquisition data manipulation algorithms. This property of the technique provides the accurate detection of small changes even in weak absorption bands (Severcan & Haris, 2012; Ozek, Bal, Sara, Onur, & Severcan, 2014). 3.2.2. Infrared spectroscopy of PPO in D2O Representative absorbance and Fourier self-deconvolution spectra of PPO in D2O buffer at 25 °C are shown in Fig. 3A and B, respectively. It can be seen from the absorbance spectrum of PPO that the amide II band at 1550 cm 1, seen for PPO in H2O buffer, disappeared and shifted to the 1455 cm 1, suggesting hydrogen– deuterium exchange occurred. Fourier self-deconvolution analysis revealed that the band located at 1652 cm 1 is due to a-helix structure. The peak located at 1673 cm 1 is assigned to b-turn structure. The band at 1635 cm 1 is assigned to b-sheet structure. The peak at located around 1616 cm 1 is due to aggregated b-sheet structure. There was a shift towards lower frequencies due to the hydrogen– deuterium exchange in the sub bands. 3.2.3. Conformational change of PPO during thermal treatment Representative absorbance spectra of PPO in the 1750–1400 cm 1 region recorded at different temperatures are shown in Fig. 3A. It was observed that the intensity of the amide I band decreased as the temperature increased between 25 °C and 70 °C. This may indicate a conformational change in the enzyme. However, no change was observed for the spectral patterns between 70 °C and 80 °C (data not shown). The native spectrum was not observed when the enzyme was cooled back to room temperature, which indicates that heat induced structural changes in the enzyme are irreversible. Recovery of activity of the enzyme decreased with increasing temperature. Moreover, no recovery of enzyme activity was seen after thermal inactivation. To explore the temperature induced changes in the secondary structures of PPO in detail, Fourier self-deconvolution techniques applied to the FTIR spectra. Fig. 3B presents the representative deconvolved spectra of PPO solution in the 1700–1600 cm 1 region measured between temperature range of 25–70 °C and at 25 °C
Fig. 4. A plot of the temperature induced changes in the intensity of the amide I and its components for PPO (() 1652 cm 1, (j) 1616 cm 1, (N) 1635 cm 1 and (d) amide I).
after cooling. According to the deconvolved spectra of PPO, there was little change in the temperature range of 25–40 °C. Marked spectral changes were noted after this temperature and additional new bands due to aggregated b-sheet structures appeared at 1683 and 1616 cm 1. These bands are detected when proteins are denatured (Severcan & Haris, 2003). The intensity of these bands increased with increasing temperature. When the temperature was lowered back to 25 °C, from 70 °C, these bands were still observed, indicating an irreversible change in the structure of PPO. Moreover, the intensity of a-helix and b-sheet structures decreased and shifted to lower values while the temperature increased to 70 °C. These observations indicate the loss of main secondary structure elements of PPO during the thermal treatment. According to the X-ray analysis, mushroom tyrosinase is a tetrameric protein composed of two subunits (H and L). H subunit is the domain of the enzyme and contains 13 a-helices, eight, mostly, short b-strands and many loops. The active site of the enzyme is made up a bundle of four helices in the center of the domain. It is also reported that the L subunit consists of 12 antiparallel
Table 1A Transition temperatures (Tm) for different amide I components for PPO. Structure
Amide I band (cm
a-Helix
1652 1635 1616 1653
b-Sheet Aggregated b-sheet Amide I
1
)
Tm value (°C) 54.72 53.5 57.07 53
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the enzyme. Denaturation of the enzyme also started above 38 °C, since it was accompanied by an increase of the aggregation band located at 1616 cm 1. The differences in the thermal unfolding and protein secondary structure between the study by Ionita et al. (2014) and our current study might be mainly due to differences in the studied form of the protein, where crystalline and solution forms of the protein were studied, respectively. Our observation on thermal unfolding was consistent with the activity measurements in that activity loss was observed above 40 °C. As shown in our results, the structural conformation of the enzyme was easily detected even at lower temperatures by using FTIR spectroscopy, which is known to be very sensitive to conformational changes in the enzyme as previously mentioned (Haris & Severcan, 1999; Goormaghtigh et al., 2009; Yang et al., 2015). Simultaneously, the intensity of the aggregation bands at 1616 and 1683 cm 1 increased with increasing temperature. The band located at 1616 cm 1 first appeared at 42 °C. The intensity of the band increased linearly up to 64 °C and no significant change was observed after this temperature, with a midpoint temperature of 57 °C. These results clearly indicate denaturation of the enzyme since this band appears only when proteins are denaturated (Severcan & Haris, 2003). Similarly, Tse et al. (1997) found that the secondary structure began to change above 40 °C and a new band at 1616 cm 1 appeared at 45 °C for mushroom tyrosinase. Using the intensity profiles, transition temperature (Tm) for the different amide I components was determined and is represented in Table 1A. The Tm values for a-helix, b-sheet, aggregated b-sheet structure and amide I were determined as around 55 °C. This value was very close to that reported by McCord and Kilara (1983) and Weemaes et al. (1997), whom found Tm value at 54 °C and 51.8 °C, respectively. Fig. 5. Curve-fitting analysis of amide I band of PPO.
b-strands assembled in a cylindrical barrel of six 2-stranded sheets and is not involved in the activation mechanism of the enzyme (Ismaya et al., 2011). As stated in X-ray analysis, reduction in a-helix structures affects the active site of the enzyme and caused structural deterioration. The intensities of the amide I band and its sub structures are plotted as a function of temperature in order to estimate the thermal unfolding of PPO more precisely (Fig. 4). As stated previously, the intensity of the 1652 and 1635 cm 1 bands decreased as the temperature increased and a multistep transition was represented for a-helix and b sheet. The first transition was observed at 45 °C and the higher transition occurred at 66 °C. A similar trend was also obtained for the thermal unfolding of un-deconvolved amide I band. However, Ionita et al. (2014) reported that no change was observed in the secondary structure of the enzyme up to 60 °C and the a-helix content decreased at temperatures over 60 °C after running molecular dynamics simulations at different temperatures. But according to our study, a-helix content started to decrease above 38 °C and the midpoint transition temperature located at 45 °C, indicating change in the secondary structure of
3.2.4. Curve-fitting analysis of PPO In order to determine secondary structural changes during thermal treatment in D2O, curve-fitting analysis was applied to the amide I region (1700–1600 cm 1). The contribution of each component band to the amide I band is shown in Fig. 5 for two different temperatures (25 and 70 °C). The bands at 1652 cm 1 due to the a-helix and that at 1635 cm 1 due to the b-sheet structure became weaker as temperature increases from 25 to 70 °C, whereas other bands due to the aggregated b-sheet (1683 and 1616 cm 1), turns (1676 cm 1) and random coil (1640 cm 1) structures became much stronger at 70 °C, indicating denaturation of the enzyme. Secondary structural change (%) of PPO during thermal treatment estimated by curve-fitting analysis is shown in Table 1B. According to the curve-fitting analysis, PPO contained 38.87% a-helix, 27.91% b-sheet, 14.93% b-turn, 15.01% random coil and 3.27% aggregated b-sheet structures in the untreated sample. These results are in accordance with the NN results, which indicated that PPO was an a-helix dominating enzyme. Previous circular dichroism (CD) studies reported values of 35.3% (Liu et al., 2013), 35.7% (Yi et al., 2012) and 41.3% (Liu et al., 2009) for a-helix content of PPO. a-Helix content decreased from 38.87% to 19.40% (p < 0.05) and b-sheet content decreased from 27.91% to 15.88% (p < 0.05), whereas b-turn, random coil and aggregated
Table 1B Secondary structure change (%) of PPO estimated by curve-fitting during thermal treatment. Temperature (°C)
a-Helix
b-Sheet
b-Turn
Random coil
Aggregated b-sheets
25 30 40 50 60 70 25 (after cooling from 70 °C)
38.87 ± 0.02 35.68 ± 0.06 26.91 ± 0.01 25.42 ± 0.10 24.01 ± 0.18 19.41 ± 0.16 19.40 ± 0.21
27.91 ± 0.02 25.14 ± 0.13 22.83 ± 0.01 19.50 ± 0.33 18.24 ± 0.14 16.25 ± 0.14 15.88 ± 0.02
14.93 ± 0.01 14.74 ± 0.02 18.07 ± 0.01 16.81 ± 0.07 17.67 ± 0.13 21.68 ± 0.18 21.93 ± 0.14
15.01 ± 0.01 19.94 ± 0.03 21.92 ± 0.01 22.22 ± 0.09 21.20 ± 0.16 22.48 ± 0.65 22.47 ± 0.60
3.27 ± 0.06 4.50 ± 0.01 10.28 ± 0.04 16.05 ± 0.07 18.88 ± 0.60 20.19 ± 0.17 20.31 ± 0.27
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b-sheet structures increased up to 21.93%, 22.47% and 20.31%, respectively (p < 0.05). This clearly showed that a-helix and bsheet structures decreased as temperature increase with the increase of aggregated b-sheets turns and disorder structure (1640 cm 1), which indicates denaturation of the enzyme at higher temperatures. As we have reported above, the enzyme started to denature at around 40 °C with a midpoint temperature of 55 °C. 4. Conclusion In the present study, changes in structure and activity of PPO during thermal treatment in the temperature range of 25–80 °C were investigated. According to the inactivation studies, the enzyme remained fully active up to 40 °C. Higher inactivation was achieved between 50 °C and 70 °C and the enzyme was completely inactivated at 70 °C for 5 min. Furthermore, no activity was observed over 80 °C. To explore the secondary structure and conformation change of PPO, Fourier self-deconvolution, NN and curve-fitting analysis were applied to the amide I region of the FTIR spectrum. FTIR results revealed that the PPO enzyme is an a-helix dominating enzyme and a-helix and b-sheet content decreased, aggregated b-sheet, turns and random coil increased at 55 °C, indicating protein denaturation. Moreover, the FTIR spectrum at 70 °C and that at 25 °C after cooling were very similar to each other and this was different than the original spectrum of 25 °C. In conclusion, denaturation of the enzyme was irreversible. This study gives information about the inactivation mechanism of polyphenoloxidase during thermal treatment in a model system. Further studies will be carried out to clarify the inactivation mechanism of the enzyme in food systems. Acknowledgments This work was supported by the State Planing Organization (DPT), Grant No: BAP-08-11-DPT2002K120510-GT-4 and Scientific and Technical Research Council of the Turkish Republic _ (TUBITAK), Project No: TOVAG-112O433. References Anthon, G. E., & Barrett, D. M. (2002). Kinetic parameters for the thermal inactivation of quality-related enzymes in carrots and potatoes. Journal of Agricultural and Food Chemistry, 50, 4119–4125. Bayindirli, A., Alpas, H., Bozoglu, F., & Hizal, M. (2006). Efficiency of high pressure treatment on inactivation of pathogenic microorganisms and enzymes in apple, orange, apricot and sour cherry juices. Food Control, 17, 52–58. Cakmak, G., Zorlu, F., Severcan, M., & Severcan, F. (2011). Screening of protective effect of amifostine on radiation-induced structural and functional variations in rat liver microsomal membranes by FT-IR spectroscopy. Analytical Chemistry, 83, 2438–2444. Fortea, M. I., López-Miranda, S., Serrano-Martínez, A., Carreño, J., & Núñez-Delicado, E. (2009). Kinetic characterization and thermal inactivation study of polyphenol oxidase and peroxidase from table grape (Crimson Seedless). Food Chemistry, 113, 1008–1014. Garip, S., Yapici, E., Simsek Ozek, N., Severcan, M., & Severcan, F. (2010). Evaluation and discrimination of simvastatin-induced structural alterations in proteins of different rat tissues by FTIR spectroscopy and neural network analysis. Analyst, 135, 3233–3241. Goormaghtigh, E., Gasper, R., Bénard, A., Goldsztein, A., & Raussens, V. (2009). Protein secondary structure content in solution, films and tissues: Redundancy and complementarity of the information content in circular dichroism,
269
transmission and ATR FTIR spectra. Biochimica et Biophysica Acta, 1794, 1332–1343. Gouzi, H., Depagne, C., & Coradin, T. (2012). Kinetics and thermodynamics of the thermal inactivation of polyphenol oxidase in an aqueous extract from Agaricus bisporus. Journal of Agricultural and Food Chemistry, 60, 500–506. Haris, P. I., & Severcan, F. (1999). FTIR spectroscopic characterization of protein structure in aqueous and non-aqueous media. Journal of Molecular Catalysis B: Enzymatic, 7, 207–221. Ionita, E., Aprodu, I., Stanciuc, N., Rapeanu, G., & Bahrim, G. (2014). Advances in structure–function relationships of tyrosinase from Agaricus bisporus – Investigation on heat-induced conformational Changes. Food Chemistry, 156, 129–136. Ismaya, W. T., Rozeboom, H. J., Weijn, A., Mes, J. J., Fusetti, F., Wichers, H. J., et al. (2011). Crystal structure of Agaricus bisporus mushroom tyrosinase: Identity of the tetramer subunits and interaction with tropolone. Biochemistry, 50, 5477–5486. Liu, W., Liu, J., Liu, C., Zhong, Y., Liu, W., & Wan, J. (2009). Activation and conformational changes of mushroom polyphenoloxidase by high pressure microfluidization treatment. Innovative Food Science and Emerging Technologies, 10, 142–147. Liu, W., Zou, L., Liu, J., Zhang, Z., Liu, C., & Liang, R. (2013). The effect of citric acid on the activity, thermodynamics and conformation of mushroom polyphenoloxidase. Food Chemistry, 140, 289–295. Ludikhuyze, L., Van Loey, A., Smout, I. C., & Hendrickx, M. (2003). Effects of combined pressure and temperature on enzymes related to quality of fruits and vegetables: From kinetic information to process engineering aspects. Critical Reviews in Food Science and Nutrition, 43(5), 527–586. McCord, J. D., & Kilara, A. (1983). Control of enzymatic browning in processed mushrooms (Agaricus bisporus). Journal of Food Science, 48, 1479–1483. Murayama, K., & Tomida, M. (2004). Heat-induced secondary structure and conformation change of bovine serum albumin investigated by Fourier transform infrared spectroscopy. Biochemistry, 43, 11526–11532. Ozek, N. S., Bal, B., Sara, Y., Onur, R., & Severcan, F. (2014). Structural and functional characterization of simvastatin-induced myotoxicity in different skeletal muscles. Biochimica et Biophysica Acta, 1840, 406–415. Severcan, M., Severcan, F., & Haris, P. I. (2001). Estimation of protein secondary structure from FTIR spectra using neural networks. Journal of Molecular Structure, 565–566, 383–387. Severcan, F., & Haris, P. I. (2003). Fourier transform infrared spectroscopy suggests unfolding of loop structures precedes complete unfolding of pig citrate synthase. Biopolymers, 69, 440–447. Severcan, M., Haris, P. I., & Severcan, F. (2004). Using artificially generated spectral data to improve protein secondary structure prediction from Fourier transform infrared spectra of proteins. Analytical Biochemistry, 332, 238–244. Severcan, F., & Haris, P. I. (2012). Vibrational spectroscopy in diagnosis and screening. IOS Press. Stuart, B. (2004). Infrared spectroscopy: Fundamentals and applications. John Wiley and Sons. Sun, N. K., & Song, K. B. (2003). Effect of nonthermal treatment on the molecular properties of mushroom polyphenoloxidase. Journal of Food Science, 68(5), 1639–1643. Toyran, N., Zorlu, F., Dönmez, G., Ög˘e, K., & Severcan, F. (2004). Chronic hypoperfusion alters the content and structure of proteins and lipids of rat brain homogenates: A Fourier transform infrared spectroscopy study. European Biophysics Journal, 33, 549–554. Tse, M., Kermasha, S., & Ismail, A. (1997). Biocatalysis by tyrosinase in organic solvent media: A model system using catechin and vanillin as substrates. Journal of Molecular Catalysis B: Enzymatic, 2, 199–213. Turker, S., Ilbay, G., Severcan, M., & Severcan, F. (2014). The investigation of compositional, structural and dynamical changes of PTZ-induced seizures on a rat brain by FTIR spectroscopy. Analytical Chemistry, 86(3), 1395–1403. Weemaes, C., Rubens, P., De Cordt, S., Ludikhuyze, L., Van Den Broeck, I., Hendrickx, M., et al. (1997). Temperature sensitivity and pressure resistance of mushroom polyphenoloxidase. Journal of Food Science, 62(2), 261–266. Yang, H., Yang, S., Kong, J., Dong, A., & Yu, S. (2015). Obtaining information about protein secondary structures in aqueous solution using Fourier transform IR spectroscopy. Nature Protocols, 10, 382–396. Yemeniciog˘lu, A., Özkan, M., & Cemerog˘lu, B. (1997). Heat inactivation kinetics of apple polyphenoloxidase and activation of its latent form. Journal of Food Science, 62(3). Yi, J., Jiang, B., Zhang, Z., Liao, X., Zhang, Y., & Hu, X. (2012). Effect of ultrahigh hydrostatic pressure on the activity and structure of mushroom (Agaricus bisporus) polyphenoloxidase. Journal of Agricultural and Food Chemistry, 60, 593–599.