The Effects of Temperature and pH on Secondary Structure and ...

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Nov 19, 2011 - Antioxidant activity Crocodylus siamensis Hemoglobin Secondary structure ..... Nakhon Ratchasima, Thailand, The Royal Golden Jubilee (RGJ) ...
Protein J (2012) 31:43–50 DOI 10.1007/s10930-011-9372-7

The Effects of Temperature and pH on Secondary Structure and Antioxidant Activity of Crocodylus siamensis Hemoglobin Jinda Jandaruang • Jaruwan Siritapetawee • Kanjana Thumanu Chomphunuch Songsiriritthigul • Chartchai Krittanai • Sakda Daduang • Apisak Dhiravisit • Sompong Thammasirirak



Published online: 19 November 2011 Ó Springer Science+Business Media, LLC 2011

Abstract Crocodylus siamensis hemoglobin (cHb) was purified by gel filtration chromatography and visualized by SDS-PAGE. Effects of temperature and pH on secondary structure and conformation changes of cHb were studied using circular dichroism spectropolarimeter and fourier transform infrared spectrophotometer. The secondary structure of intact cHb was mainly a-helices. cHb was not heat stable when heated at 65 °C and cooled down to original temperature, indicating the irreversible unfolding process. The stability of cHb at different pH ranging from 2.5 to 10.5 was determined. The maximum value of the a-helix content was found at pH 3.5 and tended to decrease at strong acid and strong base. The antioxidant activities of heat treated cHb and cHb in solution with pH range 2.5 to 10.5 were tested by J. Jandaruang  S. Daduang  S. Thammasirirak (&) Faculty of Science, Department of Biochemistry, Khon Kaen University, Khon Kaen 40002, Thailand e-mail: [email protected] J. Jandaruang  S. Daduang  S. Thammasirirak Protein and Proteomics Research Group, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand J. Siritapetawee School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand K. Thumanu  C. Songsiriritthigul Synchrotron Light Research Institute (Public Organization), Nakhon Ratchasima 30000, Thailand C. Krittanai Institute of Molecular Biology and Genetics, Mahidol University, Nakhon Pathom 73170, Thailand A. Dhiravisit Faculty of Humanities and Social Sciences, Khon Kaen University, Khon Kaen 40002, Thailand

DPPH radical scavenging assay. cHb at pH 4.5, having highest b-turn structure, showed highest radical scavenging activity. In contrast to pH, heat had no effect on antioxidant activity of cHb. Keywords Antioxidant activity  Crocodylus siamensis  Hemoglobin  Secondary structure Abbreviations Hb Hemoglobin cHb Crocodylus siamensis hemoglobin ATR Attenuated total reflectance FT-IR Fourier transform infrared CD Circular dichroism PBS Phosphate buffer saline CBB Coomassie brilliant blue TFA Trifluoroacetic acid ACN Acetonitrile MRW Mean residue weight MRE Mean residue ellipticity DPPH 1,1-Diphenyl-2-picrylhydrazyl

1 Introduction Hemoglobin (Hb) is the main component of the red blood cells and an oxygen transport protein. This molecule is a heterotetramer metalloprotein consisting of two identical a-globin and two identical b-globin polypeptides. Each globin associates with a heme group. Human a-and b-globin contain 141 and 146 amino acids residues, respectively [2]. All reptile Hb except crocodile Hb has two types of achains, which are aA-chain and aD-chain types [11]. Crocodile Hb has only aA-chain and b-chain types [11]. In the Alligator mississippiensis, the amount of a-and b-amino

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acid residues are the same as human Hb [12]. The primary sequence of crocodile Hb has been reported in three species including A. mississippiensis (UniPortKB ID:P01999, UniPortKB ID:P02130), Crocodylus niloticus (UniPortKB ID:P01998, UniPortKB ID:P02129), and Caiman crocodiles (UniPortKB ID:P02000, UniPortKB ID:P02131) [20]. The percentage of identical amino acids of human Hb (UniPortKB ID:P69905, UniPortKB ID:P68871) to A. mississippiensis, C. niloticus and C. crocodiles are 67, 68 and 67% for a-subunits, 48, 54 and 49% for b-subunits, respectively. In contrast to mammals, reptiles have a rather inert basal metabolism and they rely on anaerobic metabolism during spurts of exercise [10]. Moreover, in the group of crocodiles, they are able to stay under water in excess of 1 h [17]. Hb of these reptiles has been found to hold oxygen molecules longer than human Hb. In addition, it is a source of endogenous bioactive peptides [14]. Some bioactive peptides isolated from the serum [27], plasma [28] and white blood cell [26] of Crocodylus siamensis inhibit the growth of microorganisms. Furthermore, human Hb, snake Hb, horse Hb and alligator Hb are very active against E. Coli [25]. In addition, porcine Hb [3, 4] and chicken Hb [6] show antioxidant properties. A secondary structure of bovine Hb (PCDDB ID: CD0000037000) has been reported [21] and the conformational changes at different pH values were studied by attenuated total reflectance FT-IR spectroscopy [7]. In addition, pH induced conformational change of Hb Langmuir–Blodgett film has been reported [23]. Circular dichroism (CD) spectroscopy was a useful technique for monitoring the thermal denaturation of many protein such as Streptomyces subtilisin inhibitor [18], P2 protein [30] and human Hb [1]. Artmann et al. [1] were evaluated structural change in Hb at the critical temperature. In the mentioned study, human hemoglobin A (HbA) and hemoglobin S (HbS) were denatured between temperatures of 35 °C and 39 °C and could be reversible at temperature below 39 °C. Furthermore, the effects of pH and temperature on antioxidant activity have been reported. For example, the effects of pH on antioxidant activity of tea saponins [22], r-Tocopherol and Trolox [13] and influence of heat treatment on the antioxidant activities of Shiitake mushroom [5]. The human and crocodile Hb have different abilities in oxygen binding, it would be interesting to determine the secondary structure as a function of temperature and pH of crocodile Hb. However, structural information on C. siamensis Hb very limited. Knowing the secondary structure as a function of temperature and pH would be the first basic information for further study including crystallography, NMR and applications on any bioactive fragments in C. siamensis Hb. In this study, the biochemical properties and secondary structure of C. siamensis are analyzed by CD and FT-IR

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spectroscopy. The effect of temperature and pH on structure and oxidative properties is investigated.

2 Materials and Methods 2.1 Chemicals and Reagents All analytical grade chemicals and solvents were from commercial sources. 2.2 Crocodile Blood The crocodile blood was supplied by Sriracha MODA Co., LTD, Chon Buri, Thailand. The crocodile blood was collected by the method of Pata [26] as follows. The crocodile blood was obtained from a slaughterhouse (Sriracha MODA Co., LTD). The blood samples were transferred immediately to sterile 15 mL centrifuge tubes containing 0.5 M EDTA. The tubes were stored not more than 24 h at 4 °C until used. 2.3 Isolation of Hemoglobin from C. siamensis cHb was isolated from red blood cells by a modification of the method of Deepthi [8]. The red blood cells were washed with phosphate buffer saline (PBS) pH 7.0 and centrifuged at 3,0009g for 10 min (25 °C). The cell pellet was gently re-suspended in PBS and centrifuged again. Cold dH2O was added to the red blood cell pellet for breaking the cells, and the solution was mixed and incubated at room temperature for 10 min. After centrifugation at 1,00009g for 20 min (4 °C), the supernatant (intact cHb) was collected and concentrated by speed vacuum concentrator (Savant Instruments, Inc. USA). 2.4 Purification of C. siamensis Hemoglobin The intact cHb was purified by gel filtration chromatography with a Sephadex G-50 resin column (1.5 cm 9 200 cm) which was equilibrated and eluted with 50 mM Tris–HCl buffer, pH 8.1 at a flow rate of 0.5 mL min-1 [8]. The fraction was detected at 280 nm and 410 nm. The eluted sample was concentrated on Amicon Centriprep centrifugal filtration units (MWCO 10,000 Da) for 20 min at 1,5009g. The protein concentration was determined by the Bradford method [19]. The purified cHb was stored at -20 °C for further experiments. 2.5 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was conducted according to the

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method of Dimino [9] using 15% separating gel and 4% stacking gel. Samples were prepared by mixing with 29 solubilizing buffer (0.5 M Tris–HCl buffer, pH 6.8, 0.5% (w/v) bromophenol blue, 10% (v/v) glycerol, 2% (w/v) SDS, and 10% (v/v) b-mercaptoethanol) ratio of 1:1 (v/v) and then boiled for 5 min. The protein bands were stained with Coomassie brilliant blue R250 (CBB R-250). Low molecular weight calibration kit (Amersham Bioscience, Sweden) was used as the standard protein marker. The protein bands were analyzed by using the Quantity one TM software in the Gel Doc 2,000 system (BIO-RAD, USA). 2.6 High Performance Liquid Chromatography (HPLC) HPLC was used to analyze the purity of cHb. Purified cHb was injected onto a C4 reversed-phase HPLC column (4.6 mm 9 250 mm) and eluted with mobile phase A (0.1% Trifluoroacetic acid (TFA)) and mobile phase B (60% Acetonitrile (ACN) in 0.1% TFA). The column was initially washed with mobile phase B for 30 min. Then a linear gradient was run from 0 to 70% with was applied mobile phase B for 30 min. A second was linear gradient from 70 to 100% of mobile phase B for 35 min. Finally, the column was maintained at 100% mobile phase B for 20 min. The flow rate of the column was 1 mL min-1 and eluted materials were measured at 280 nm. 2.7 Effect of Temperature on the Secondary Structure of C. siamensis Hemoglobin Circular dichroism (CD) spectra were collected on a JASCO J-715 CD spectropolarimeter using solutions of protein concentration 2 mg mL-1 in a 0.2 mm cell. Each CD spectrum was the accumulation of three scans at 20 nm min-1 with a 2 nm bandwidth and a time constant of 2 s. Data was collected from 190 to 270 nm. Temperature regulation was carried out using the built-in temperature control device. The secondary structure of cHb was studied between 25 °C and 65 °C. The cHb solution was first adjusted to 25 °C and then the temperature was stepwise increased. Each temperature was allowed to equilibrate for 2 min. Afterwards a complete wavelength scan was carried out in the far-UV. At identical conditions to those used with cHb solutions, blank wavelength scans of pure buffer solutions were carried out. Blank spectra were subtracted from the cHb spectra at each temperature point. For far-UV CD spectra of proteins, the repeating unit is the peptide bond. The Mean Residue Weight (MRW) for the peptide bond is calculated from Eq. 1. MRW ¼ M=ðN  1Þ

ð1Þ

where M is the molecular mass of the polypeptide chain (Da), N is the number of amino acids in the chain and N - 1 is the

number of peptide bonds. The mean residue ellipticity (MRE) at wavelength k([h]mrw,k) is given by Eq. 2. ½hmrw;k ¼ MRWðhk Þ=10ðdcÞ

ð2Þ

where hk is the observed ellipticity (degrees) at wavelength k, d is the path length (cm) and c is the concentration (g mL-1). The units of MRE and molar ellipticity are deg cm2 dmol-1 [16]. To estimate the secondary structure composition of the protein, CD ellipticity in degrees was converted to molecular ellipticity using the path length of the cell and estimates of the number of amino acids in the cHb and protein concentration. The secondary contents structure such as a-helix and b-structure were then quantified by the K2D program which has an online server for protein secondary structure analyzes from CD data. 2.8 Effect of pH on the Secondary Structure of the C. siamensis Hemoglobin Conformational changes on secondary structure of cHb have been studied by using FT-IR spectroscopy. This technique was applied to investigate the effect of pH on cHb structure in the range 2.5–10.5 (25 mM glycine–HCl pH 2.5, 25 mM sodium citrate pH 3.5, 25 mM sodium acetate pH 4.5, 25 mM sodium acetate pH 5.5, 25 mM phosphate buffer pH 6.5, 25 mM Tris–HCl pH 7.5, 25 mM Tris–HCl 8.5, glycine–NaOH pH 9.5 and glycine–NaOH pH 10.5). Each sample of cHb solution (30 mg mL-1) was dissolved in buffer at different pH values. The ‘‘in situ’’ infrared cell is dedicated for the measurement of water soluble protein. It is composed of a CaF2 window of 6.5 lm which is optimized for studying aqueous solutions. In addition, the cell is integrated with on FT-IR system that is specially designed for including a water bath that allows control of temperature. Spectra were recorded at 25 °C by collecting 64 scans every 5 min to give 10 sets of 64 scans. Then, these spectra for each condition were averaged to give low noise level. The measurements were performed with a spectral resolution of 4 cm-1 with 64 scans co-added (Bruker Optics Ltd, Ettlingen, Germany). The difference spectra were calculated by subtraction absorbance spectra of cHb dissolved at each pH minus cHb dissolved in distilled water. The spectral changes of functional group were emphasized in the difference spectra. The relative integral areas of each peak were then found using OPUS 6.5 software (Bruker optics, Germany). 2.9 Antioxidant Activity Assays Antioxidant activity of the cHb was measured with 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging

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assay by the modified method of Shimada [29]. Briefly, cHb samples (6 lg lL-1) were prepared in each buffer pH range 2.5 to 10.5, treated with temperatures between 25 °C and 65 °C and reactivated at 65 °C and 25 °C. One hundred microliters of sample solution was mixed with 1,500 lL of freshly prepared 0.1 mM DPPH methanolic solution. The resulting solution was then left to stand for 20 min at room temperature and was centrifuged at 10,0009g for 5 min and measured at 515 nm. The control was prepared as above without sample and methanol used for the baseline correction. All samples were analyzed in triplicate. A low absorbance at 515 nm indicates a high DPPH scavenging activity. Radical scavenging activity was expressed as the inhibition percentage and was calculated using the Eq. 3. % Radical scavenging activity ¼ ½ðControl OD  Sample ODÞ=Control OD  100

ð3Þ

3 Results 3.1 Purification and Electrophoresis of C. siamensis Hemoglobin After crocodile red blood cells were washed and lysed, the supernatant was subjected to gel filtration chromatography using Sephadex G-50. The column was equilibrated with 50 mM Tris–HCl buffer, pH 8.1. The elution profile of cHb is showed in Fig. 1a, single peaks at A280 nm and at A410 nm were observed to overlay almost perfectly at 15–20 min. No tailing or other peak in the profile indicates the absence of any contaminant proteins, sheared cHb or free heme. The purity of the cHb was determined by SDSPAGE and the result shows three major protein bands (Fig. 1b). The molecular sizes of each band were estimated to be about 15, 16 and 30 kDa. HPLC of eluent from Sephadex G-50 column (Fig. 1c) could further dissociate the cHb tetramer, P1 (Fig. 1d) as free heme (the eluted fractions were checked at A410 to follow the heme profile, data not shown), P2 as b-chain, and P3 as a-chain. From the result, cHb is pure enough for further analysis. 3.2 Effect of Temperature on the Secondary Structure of C. siamensis Hemoglobin

estimate the secondary structure composition, the spectra (normalized to mean residue ellipticity (MRE)) were analyzed by K2D program. The calculation estimates the cHb secondary structural components to be 94% a-helix. Figure 3a shows the changes of CD spectra in the FarUV region, which is sensitive to change in the secondary structure, during temperature 25 °C and 65 °C. The results show that the MRE of cHb decreased when the temperature increased. Changes of ellipticity of cHb at 222 nm at different temperatures are shown in Fig. 3b. The molar ellipticity at 222 nm between 25 °C and 65 °C decreased approximately 18%, indicating the denaturation of the cHb molecules as a result of increasing temperature. The slope of the denaturation curve above 55 °C indicated stronger denaturation than those at lower temperature. The a-helical content of the cHb molecules at temperatures between 25 °C and 65 °C is shown in Fig. 3b. The a-helical content of cHb at temperatures above 45 °C decreased rapidly. Increasing from 25 °C and 65 °C caused cHb to lose about 14% of the a-helix content. We also studied the structural reversibility of the cHb after heat denaturation/renaturaltion by heating the protein at 25 °C and 65 °C and then cooled down the sample to 25 °C. When temperature increased the relative ellipticity decreased. After reactivation to 25 °C, relative ellipticity did not return to its original state, indicating that cHb was not heat stable (Fig. 4). 3.3 Effect of pH on the Secondary Structure of the C. siamensis Hemoglobin Effect of pH on the secondary structure of the cHb was analyzed by FT-IR. Figure 5 shows the changes of secondary structure in each pH buffer comparing within distilled water. The secondary structure of cHb reveals decrease a-helical content at pH 2.5, pH 4.5 and pH 10.5. In contrast, a-helical content was found to increase at pH 3.5, pH 5.5 and pH 8.5, whereas it mostly decreased at pH 6.5 and pH 7.5. The b-sheet content increased at lower and higher pH values (pH 2.5, 8.5 and 10.5) as well as the b-turn structure content which was highest at pH 4.5 and decreased as pH value increase. 3.4 Antioxidant Activity Assay

The secondary structure of the cHb was investigated by CD spectroscopy. The CD spectrum was measured by monitoring the changes of the signal from 190 to 270 nm. The far—UV CD spectrum of the cHb is shown in Fig. 2. The CD spectrum showed the shape of the mainly a-helical secondary structure of the negative ellipticity bands near 222 nm and 208 nm and a positive band at 194 nm. To

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The antioxidant activity of the cHb was determined by DPPH radical scavenging. The radical scavenging activities (%) of the cHb at pH from 2.5 to 10.5 are shown in Fig. 6. cHb at pH 4.5 had highest radical scavenging activities of 47.3%. The activity decreased at pH 2.5, 5.5, 6.5 and 10.5.

The Effects of Temperature and pH on Secondary Structure

Fig. 1 Elution profile of cHb by gel filtration chromatography with a Sephadex G-50 resin column which was equilibrated and eluted with 50 mM Tris–HCl buffer, pH 8.1 at a flow rate of 0.5 mL min-1 (Filled black circles are absorbances at 280 nm and Filled black squares are absorbances at 410 nm) (a). 15% SDS-PAGE of fraction collected from gel filtration chromatography. Lane M (kDa) is the standard protein marker; Lane 1 is crude cHb (10 lg proteins); Lane 2

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is purified cHb (10 lg proteins) (b). Chromatogram of purified cHb by C4 RP-HPLC column. The column was eluted with mobile phase A (0.1% TFA) and mobile phase B (60% ACN in 0.1% TFA) at the flow rate 1 mL min-1 and eluted materials were measured at 280 nm (c). 15% SDS-PAGE analysis of cHb and its subunits obtained from C4 RP-HPLC column (d)

Fig. 2 Circular dichroism spectrum of 2 mg mL-1 of cHb in double distilled water. The far—UV wavelength was scanned at a temperature of 25 °C

The radical scavenging activities (%) of the cHb at temperature between 25 °C and 65 °C and reactivated from 65 °C and 25 °C are shown in Fig. 7. cHb had highest

radical scavenging activity when heated at 35 °C. However, radical scavenging activities at temperatures between 25 °C and 55 °C were not significantly different, but

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Fig. 4 Structural change of cHb with temperature, tested for its reversibility. The cHb sample was first equilibrated at 25 °C and the ellipticity was measured as wavelength scans. Then the sample was heated up to 65 °C and wavelength scans were taken at time points. After that the sample was cooled down to 25 °C. The relative ellipticity was calculated as: Erel = 100 9 [E(t) - E0]/E0 where E(t) is the ellipticity at 222 nm at time t and E0 is the ellipticity at t = 0 (25 °C) [1]

Fig. 3 The far—UV wavelength scans at temperature range of 25 °C and 65 °C of cHb (a). The change of ellipticity of cHb at 222 nm and estimated a-helical content of cHb at temperatures between 25 °C and 65 °C obtained with the CD spectra deconvolution software K2D (b)

slightly decreased at 65 °C. After cooling down the sample to 25 °C, the radical scavenging activity (%) of cHb was slightly decreased.

4 Discussion In this study, the Hb from crocodile (C. siamensis) red blood cell was purified by gel filtration chromatography. The elution profile showed two overlay peaks (Fig. 1a) which indicated high purity of intact cHb with bound heme molecules. No free heme was found. SDS–PAGE (Fig. 1b) of the eluent showed two major protein bands at 15 and 16 kDa, which were similar to a-chains and b-chains of A. mississippiensis Hb [12]. The majority of cHb migrated through the gel as a monomer (a-and b-globin chains) and some migrated as a dimer about 30 kDa, indicating that cHb might not be fully denatured from the tetramer form. Our result is in agreement with previous reports that show the protein band at 32 kDa of bovine Hb is an ab- dimer [9]. The HPLC profile assured the purity of cHb. Heme seemed to detach from both a-chains and b-chains in ACN,

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Fig. 5 Variation of secondary structure of cHb in the buffer pH range 2.5–10.5 comparing within the distilled water

as shown in P1 (Fig. 1c, d). Spike peak of b-chain in P2 and flat peak a-chain in P3 indicated different conformation of these two chains as they migrated through the hydrophobic environment. The CD spectrum (Fig. 2) showed that the overall secondary structure of intact cHb was mainly a-helix, with more than 90% of the total secondary structure. The a-helix content of cHb was more than that in bovine Hb (69% a-helix, PCDDB ID: CD0000037000) [21]. This difference might explain the extent of the Hb function about whether the animal is on land or under water. The effect of

The Effects of Temperature and pH on Secondary Structure

Fig. 6 Effects of pH at 2.5–10.5 on radical scavenging activity of cHb using the DPPH assay and measured at wavelength 515 nm. cHb samples (6 lg lL-1) were prepared in each buffer

temperature on the secondary structure (Fig. 3a, b) of the cHb resulted in a decreasing in a-helix content and the process seemed to be irreversible (Fig. 4). Inside the crocodile Hb molecule, the structure of the protein was stabilized by the sulfhydryl bond and weak interactions of the hydrogen bond, electrostatic interaction and hydrophobic effect. Hydrogen bonding is the main force of a-helix secondary structure of Hb and can be broken by heat. Therefore, decreasing of a-helix content of cHb has caused from increasing temperature. The effect of pH on the secondary structure of the cHb showed that, at acidic pH (pH 2.5 and pH 4.5) and basic pH (pH 10.5) cHb had decrease a-helix content (Fig. 5). cHb at pH 3.5 and pH 5.5 had highest a-helix content. Such a result is similar to the maximum value of the a-helix content of bovine Hb at pH 4.8 [7]. The a-helix content of cHb was lowest at pH 6.5

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and pH 7.5 since cHb was precipitated at the isoelectric point of the a-and b-chains (pI at 7–8). Moreover, the b-sheet content was abundant at pH 2.5, pH 8.5 and pH 10.5, showing similar content to that of bovine Hb and giving a maximum value at pH 2.5. In addition, the b-turn structure was highest at pH 4.5. The effect of pH on the secondary structure indicated that the secondary structure of cHb is very sensitive to the environment. Chang [3], reported that porcine Hb shows antioxidant properties. It has high reducing power with ferrous ion chelating ability and 21% DPPH radical scavenging activity [3]. In this study, cHb also has antioxidant activity. When pH was 4.5, cHb had highest antioxidant activities (Fig. 6). The results revealed that cHb perhaps contains domain/functional groups acting as electron donors and reacting with free radicals to be more stable products and eventually terminating the radical chain reaction. cHb antioxidant property might attributed to the presence of some amino acids in their primary structure. This resulted may from protonated glutamate at pH 4.5 that could donate protons to free radicals. The antioxidant activity of cHb was similar to that of Trolox which was highest at pH 4 [13]. Moreover, at pH 4.5 cHb had the highest b-turn structure (Fig. 6). b-turn structures are implicated in protein folding [24] as an appropriate structure for antioxidant activity. In this study, b-turn structure is a contributing factor on antioxidant activity, similar to what has been reported in Jiang et al. [15] about the essential of the b-turn/b-sheet secondary structure on antioxidant activity. Regarding antioxidant activity as a function of temperature, cHb gave highest antioxidant activity at 35 °C (Fig. 7). Generally, there is high concentration of protonated residues when protein is at a

Fig. 7 Effect of heat treatment at 25 °C–65 °C on radical scavenging activity of cHb using the DPPH assay and measured at wavelength 515 nm. cHb samples (6 lg lL-1) were treated with temperatures between 25 °C and 65 °C and reactivated at 65 °C and 25 °C

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temperature of 37 °C–40 °C, which is suitable for all chemical reactions inside the cells at body temperature. Antioxidant activity is effective at this temperature range as well.

5 Conclusions The secondary structure of the cHb was largely a-helices. Its structure could be modified and denatured by heat treatment. Its secondary structure was sensitive to strong acidic and basic environments leading to change in secondary structure. However, at acidic conditions (pH 4.5), the cHb had the highest antioxidant properties which corresponded with increased b-turn structure. In vitro antioxidant activities of cHb were temperature and pH dependent. Acknowledgments This investigation was supported by the Synchrotron Light Research Institute (Public Organization), Nakhon Ratchasima, Thailand, The Royal Golden Jubilee (RGJ) Ph.D. Program of Thailand Research Fund, and Protein and Proteomics Research Group, Faculty of Science, Khon Kaen University, Thailand.

References 1. Artmann GM, Burns L, Canaves JM, Temiz-Artmann A, SchmidSchonbein GW, Chien S, Maggakis-Kelemen C (2004) Eur Biophys J 33:490–496 2. Braunitzer G, Gehring-Muller R, Hilschmann N, Hilse K, Hobom G, Rudloff V, Liebold-Wittmann B (1961) Hoppe-Seyler’s Z Physiol. Chem 325:283–286 3. Chang CY, Wu KC, Chiang SH (2005) Food Chem 100: 1537–1543 4. Chang CY, Wu KC, Lin ZY (2004) Food Chem Antioxid Bioact Agents 23:79–85 5. Choi Y, Lee SM, Chun J, Lee HB, Lee J (2006) Food Chem 99:381–387 6. Dafre AL, Brandao TAS, Reischl E (2007) NRC Canada 85:404–412 7. Damian G, Canpean V (2005) Romanian J Biophys 15:67–72 8. Deepthi S, Johnson A, Sathish R, Pattabhi V (2000) Biochim Biophys Acta 1480:384–387

123

J. Jandaruang et al. 9. Dimino ML, Palmer AF (2007) J Chromatogr B Anal Technol Biomed Life Sci 856:353–357 10. Dyson J (1986) Hemoglobin: molecular, genetic and clinical aspects In: Animal hemoglobin. W.B. Saunders Company, Philadelphia 11. Gorr TA, Mable BK, Kleinschmidt T (1998) J Mol Evol 47:471–485 12. Hoffman BF, Key B, Ofer B, Kiryat T (2002) Reptilian-derived peptides for the treatment of microbial infections. In: United States Patent (USA) 6,340,667 13. Huang SW, Frankel EN, Schwarz K, German JB (1996) J Agric Food Chem 44:2496–2502 14. Ivanov VT, Karelin AA, Philippova MM, Nazimov IV, Pletnev VZ (1997) Biopolymers 43:171–188 15. Jiang J, Kurnikov I, Belikova NA, Xiao J, Zhao Q, Amoscato AA, Braslau R, Studer A, Fink MP, Greenberger JS, Wipf P, Kagan VE (2007) J Pharmacol Exp Ther 320:1050–1060 16. Kelly SM, Jess TJ, Price NC (2005) Biochim Biophys Acta 1751:119–139 17. Komiyama NH, Miyazaki G, Tame J, Nagai K (1995) Nature 373:244–246 18. Komiyama T, Miwa M, Yatabe T, Ikeda H (1984) J Biochem 95:1569–1575 19. Kruger NJ (1984) In: John MW (ed) The Bradford method for protein quantitation, 2nd edn. W.B. Saunders Company, Philadelphia 20. Leclercq F, Schnek A, Braunitzer G, Stangl A, Schrank B (1981) Hoppe-Seyler’s Z Physiol Chem 362:1151–1158 21. Lees JG, Miles AJ, Wien F, Wallace BA (2006) Bioinformatics 22:1955–1962 22. Li Y, Du Y, Zou C (2009) Eur Food Res Technol 228:1023–1028 23. Mahato M, Pal P, Kamilya T, Sarkar R, Talapatra GB (2010) J Phys Chem. B 114:495–502 24. Marcelino AMC, Gierasch LM (2008) Biopolymers 89:380–391 25. Parish CA, Jiang H, Tokiwa Y, Berova N, Nakanishi K, McCabe D, Zuckerman W, Xia MM, Gabay JE (2001) Bioorg Med Chem 9:377–382 26. Pata S, Yaraksa N, Daduang S, Temsiripong Y, Svasti J, Araki T, Thammasirirak S (2011) Dev Comp Immunol 35:545–553 27. Preecharram S, Daduang S, Bunyatratchata W, Araki T, Thammasirirak S (2008) Afr J Biotechnol 7:3121–3128 28. Preecharram S, Jearranaiprepame P, Daduang S, Temsiripong Y, Somdee T, Fukamizo T, Svasti J, Araki T, Thammasirirak S (2010) Anim Sci J 81:393–401 29. Shimada K, Fujikawa K, Yahara K, Nakamurat T (1992) J Agric Food Chem 40:945–948 30. Stuart BH (1996) Biochem Mol Biol Int 38:153–160