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FTIR Spectroscopy Studies on the Bioprotective Effectiveness of Trehalose on Human Hemoglobin Aqueous Solutions under 50 Hz Electromagnetic Field Exposure Salvatore Magazu`,*,† Emanuele Calabro`,† and Salvatore Campo‡ Department of Physics and Department of Biochemical, Physiological and Nutritional Sciences, UniVersity of Messina, Messina, Italy ReceiVed: May 10, 2010; ReVised Manuscript ReceiVed: July 24, 2010
The effects of extremely low frequency electromagnetic field on the protein structure of hemoglobin were investigated by means of Fourier transform infrared spectroscopy. Three samples of different hemoglobin aqueous solutions (also in the presence of sucrose and trehalose) were exposed to a 50 Hz electromagnetic field at 1 mT, and FTIR measurements were performed after 3 h of exposure. Quantitative spectral analysis revealed an evident decrease in amide A band intensity for hemoglobin in bidistilled water and sucrose aqueous solutions, but not for hemoglobin in trehalose aqueous solution. In addition a low relative increase of β-sheet in amide I region was detected for hemoglobin in both bidistilled water and sucrose aqueous solutions, whereas no appreciable changes were evidenced in the infrared spectra of hemoglobin in trehalose aqueous solutions. These results led us to conclude that a 50 Hz electromagnetic field can affect the N-H plane bending and C-N stretching vibrations of peptide linkages, suggesting compensatory mechanisms by means of environmental biochemical agents, such as evidenced by a protective effect of trehalose toward a low-frequency electromagnetic field. 1. Introduction Homologues disaccharides as sucrose and trehalose, are cryptobiotic activating substances with different bioprotectant effectiveness and are directly comparable, since they possess the same chemical formula C12H22O11 and the same number of hydroxyl groups. In particular, trehalose is a disaccharide widely distributed in nature, capable of protecting many organisms in extreme life conditions, whose properties of stabilizing organic systems have been well recognized and used to protect proteins from several stress agents.1-4 The aim of this study is the evaluation of the bioprotective effectiveness of these disaccharides on hemoglobin with respect to another stress agent: the exposure to an extremely low frequency electromagnetic field (ELF-EMF). Several epidemiological studies have reported a relationship between an increased risk of cancer and exposure to ELF-EMFs. In particular, three studies of the World Health Organization (WHO) on EMF evidenced possible health effects from exposure to static and ELF magnetic fields.5-7 In response to public concern over health effects of EMF exposure, in 1996, the International EMF Project was established by WHO and the Radiation and Environmental Health Unit, which coordinated studies on EMF relative to the Environmental Health Criteria (EHC). The main objectives of EHC are to review the scientific literature on the biological effects of exposure to ELF-EMF fields to assess any health risks from exposure to these fields and to use this health risk assessment to make recommendations to national authorities on health protection programs. In particular, the International Agency for Research on Cancer (IARC) formally evaluated the evidence for carcinogenesis from * Corresponding author address: Department of Physics, University of Messina, Contrada Papardo, 31-98166 Messina, Italy. Phone: +39.090.6765025. E-mail:
[email protected]. † Department of Physics. ‡ Department of Biochemical, Physiological and Nutritional Sciences.
exposure to static and ELF fields.8 The reviews concluded that ELF-EMFs are possibly carcinogenic to humans. Nevertheless, there have been few studies carried out on the effects of ELF-EMFs on hematological organic systems. Milham and Ossiander9 have suggested that the appearance of the peak incidence at around age 3 in childhood acute lymphocytic leukemia is linked to electrification and, therefore, to exposure. Otherwise, it is possible that exposure to electromagnetic fields of living matter can alter the structure of some proteins that perform an important role in metabolism processes of organic systems. In experiments evaluating differential white blood cell counts, exposures ranged from 2 µT to 1 mT. No consistent effects of acute exposure to low-frequency magnetic fields or to combined ELF electric and magnetic fields have been found in either human or animal studies. Therefore, the evidence for the effects of ELF-EMF on the immune and hematological system is considered inadequate. In the present paper, we focused our attention on EMF effects on hemoglobin because of the previous large literature set concerning the effects on human and in vitro red blood cells induced by microwaves and radiofrequencies, such as erythrocyte hemolysis, diminution of sedimentation speed, and globulin fraction changes.10-14 In particular, the effects of the exposure of hemoglobin to ELF-EMF, analyzing vibration bands of protein linkages in the infrared spectroscopy region from 4500 to 1200 cm-1, are taken into account. FTIR can be considered as a valuable tool for analyzing protein structure in H2O-based structure or in deuterated form.15-19 Unlike UV-vis spectroscopy, which probes transitions between electronic states, IR spectroscopy detects transitions between rotational and vibration energy levels, yielding much more information on molecular structure. Infrared absorption bands that are notoriously characteristic for proteins are
10.1021/jp104226p 2010 American Chemical Society Published on Web 08/30/2010
Bioprotective Effectiveness of Trehalose amide A, amide B, and amide I, amide II and amide III.20-22 Amides IV-VII, instead, are less prominent protein vibration bands. In this paper, we reported the effects of ELF-EMF on hemoglobin structure by means of FTIR, evaluating bioprotective effects of sucrose and trehalose. Previous research carried out up to now on protein thermal denaturations have confirmed a bioprotection mechanism by disaccharides.23,24 Several hypotheses have been proposed to explain why trehalose is particularly effective. Crowe et al.25 formulated the so-called “water replacement hypothesis”, for which a direct trehalosebiostructure interaction occurs. This hypothesis is supported by a simulation of Donnamaria et al.,26 who infer that the structure of trehalose is perfectly adaptable to the tetrahedral coordination of pure water, whose structural and dynamical properties are not significantly affected. Cryptobiosis, from the Greek kriptos, which means both “hidden” and “coated”, refers to a particular state of organisms, during which undetectable hidden levels of metabolic functions are maintained for mending prohibitive environmental conditions. As a matter of fact, experimental data show not only that the structural and dynamical properties of water are strongly perturbed by disaccharides but also that these cosolvents are preferentially excluded from the hydration shell of proteins. Inelastic neutron spectra reveal a more marked downshift of the OH stretching mode and a narrower bandwidth for the trehalose/H2O mixture, which are indicative of a higher disaccharide-water interaction and of a higher order.27 Other experimental data point out that the structural properties of water are locally perturbed more strongly by trehalose with respect to sucrose and maltose. To mention a result, the intramolecular OH stretching Raman spectra of disaccharide aqueous solutions shows that, at the same concentration and temperature value, the area of the tetrahedric contribution is smaller in the case of trehalose, thus indicating that a more marked destructuring effect occurs. This result, which is confirmed in all the investigated concentration ranges by the trend of the integrated intensity of the low-density contribution that is constantly smaller for trehalose with respect to its homologus sucrose and maltose, constitutes a conclusive, crystal-clear proof and furnishes an accounting of the fact that trehalose frustrates ice formation better than the other disaccharides.28 Experiments on trehalose/H2O, maltose/H2O, and sucrose/H2O mixtures, through the intensity profile of the bending modes identified by a density functional simulation, appears more structured, indicating that trehalose forms with water molecule entities that show a more “cryptocrystalline” behavior: the macroscopically glassy protective shell hides a local crystalline character.4,29,30 2. Materials and Methods Hemoglobin Samples. Ten healthy human subjects (ages 18-25 years, 4 males and 6 females) living in southern Italy were recruited for this study, and written authorizations were collected. Blood samples were obtained by venipuncture and anticoagulated by using Na2EDTA (ethylenediaminetetraacetic acid bisodium salt) 0.5 M (10 nmol/mL blood sample). After centrifugation at 1500 rpm for 5 min, each sample was immediately washed five times with a physiological solution (NaCl 0.9%), whereas the pellet of white blood cells was removed by using a water pump. The erythrocyte pellet obtained was treated with an equal volume of bidistilled water and lysed by vigorously shaking and freezing/thawing. The hemoglobin solution was centrifuged at 6000 rpm (model MR 23; Thermo
J. Phys. Chem. B, Vol. 114, No. 37, 2010 12145 Electron Corporation; Waltham, MA) at 4 °C to precipitate the insoluble stroma. The quantity of each hemoglobin solution was acquired by spectrophotometric analysis (model T70 UV/vis; Fulltech Instruments) at 420 nm against a calibrator. For each sample, three hemoglobin solutions were prepared at 150 mg/ mL concentration in bidistilled water, 50 mg/mL sucrose, and 50 mg/mL trehalose, respectively, and immediately subjected to the following assays. Experimental Design. The exposure system consisted of a couple of Helmholtz coils, with pole pieces of round parallel polar faces, to produce a uniform magnetic field at the center of the coils distance. This device was used to generate timevarying electromagnetic fields at a frequency of 50 Hz by means of an AC voltage regulating up to 230 V, which enabled us to change the magnetic flux density up to 1 mT between the polar faces of the coils. Samples were placed at the center of a uniform field area between the coils. The magnetic field was continuously monitored by a magnetic field probe (GM07 Gmeter of HIRSTMagnetic Instruments Ltd., UK). Infrared Spectroscopy. FTIR absorption spectra were recorded at room temperature by a spectrometer, Vertex 80v, from Bruker Optics. The attenuated total reflection (ATR) method was chosen for spectrum collection. In fact, transmission FTIR requires the placement of proteins between two calcium fluoride windows with thin path length spacers, with a potential for solvent interference. The ATR technique is the desired method to overcome solvent masking, since the penetration depth of infrared light is inherently limited to a fraction of the wavelength, estimated to be λ/10, permitting rapid secondary structure analysis on small volumes of protein in H2O solution.31 Therefore, the effective path length for ATR-FTIR is sufficiently short to enable the analysis of protein in H2O solution. Hemoglobin solution (150 mg/mL) samples, in the absence or presence of sucrose and trehalose, were placed between a pair of CaF2 windows separated with a 25 µm Teflon spacer. For each spectrum, 128 interferograms were collected and coadded by Fourier transform employing a Happ-Genzel apodization function to generate a spectrum with a spectral resolution of 4 cm-1 in the range 7500-350 cm-1. IR spectra of the water solution were subtracted from the spectra of the hemoglobin at the corresponding temperature. Each measure was performed under vacuum to eliminate minor spectral contributions due to residual water vapor. However, a smoothing correction for atmospheric water background was performed. In addition, the IR spectra were baseline-corrected and areanormalized for exposed hemoglobin solutions and control samples. In particular, vector normalization was used, calculating the average value of the spectrum and subtracting from the spectrum decreasing the midspectrum. The sum of the squares of all values was calculated, and the spectrum was divided by the square root of this sum. The automatic baseline scattering correction function was used to subtract baselines from spectra, which allows getting spectra with band edges of up to the theoretical baseline. In addition, interactive baseline rubberband correction was used for the spectra shown in Figure 2a. This method also uses a rubberband that is stretched from one spectrum end to the other, and the band is pressed onto the spectrum from the bottom up with varying intensity. This method performs iteratively, depending on the number of iterations in the algorithm and the baseline as a frequency polygon consisting of n baseline points. The resulting spectrum will be the original spectrum minus the baseline points manually set and a subsequent concave rubberband correction. We used the default value of n ) 64 baseline
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points and 50 iterations. Either exposed or control samples were located in the same room at a temperature of 20 °C. ATR spectra were smoothed by the Loess algorithm and the deconvolved spectra fitted with Gaussian band profiles. Initial values for the peak heights and widths were estimated from the deconvolved spectra. To enhance the fine spectral structure, the second-derivative analysis of infrared spectra was performed. In the second-derivative spectrum, the intrinsic shape of an infrared absorbance is approximated by a Lorentzian function,16 and the peak frequency is practically identical to the original peak frequency, but the half-bandwidth is reduced. The height of a second-derivative peak is proportional to the original peak height with an opposite sign, and the half-bandwidth of the second-derivative peak is proportional to the original halfbandwidth.21 3. Results and Discussion Structural changes of vibration bands of human hemoglobin induced by exposure to ELF-EMF in the absence or presence of sucrose and trehalose were studied by FT-IR. Samples of 250 µL of hemoglobin in bidistilled water and in sucrose and trehalose aqueous solutions were exposed for 3 h to a uniform electromagnetic field of 1 mT at a frequency of 50 Hz at a room temperature of 20 °C. Analogue unexposed samples at the same room temperature were used as the control. Measurements relative to an exposure of 3 h can be considered quite reliable, whereas the relatively low surface tension of the investigated system, the hydration value of the investigated water solutions, remarkably could produce changes after a long time of exposure to air, providing less reliable results.32,33 On the contrary, the presence of disaccharides (i.e., sucrose and trehalose) in hemoglobin aqueous solutions considerably increases the surface tension of the system, remarkably decreasing water evaporation.28,30,34 The magnetic flux density selected for this study corresponded to the reference level proposed in the European Community for occupational exposure, based on the guidelines of the International Commission of Non-Ionizing Radiation Protection.35 ATR spectra were collected, as described in the preceding section, for several samples. Typical spectra from 4000 to 2600 cm-1, obtained after 3 h of exposure, are showed in Figure 1a, b, and c for hemoglobin in bidistilled water and sucrose and trehalose aqueous solutions, respectively, and show the amide A and B regions of hemoglobin, partially overlapped by the water vibration band around 3500 cm-1. It appears that after 3 h of exposure, the intensity of amide A band decreased strongly for hemoglobin in bidistilled water and sucrose aqueous solutions, whereas it was unchanged for hemoglobin samples in trehalose aqueous solution. The interactive baseline concave rubberband correction was applied to the acquired spectra using the parameters indicated in section 2 to exclude the water vibration band shape around 3500 cm-1. The relative infrared spectra from 4000 to 1400 cm-1 of hemoglobin in bidistilled water and in sucrose and trehalose aqueous solutions acquired after 3 h of exposure to 50 Hz frequency EMF at 1 mT are shown in Figure 2A. The spectra exhibited an intense amide I band centered at ∼1654 cm-1, corresponding mainly to an R-helix structure content due to CdO stretching vibration and a N-H bending mode, a low intensity amide II, coupling of the N-H bending and C-N stretching modes, and a strong amide A band centered at 3293 cm-1, whereas the amide B is not evident.
Figure 1. Representative infrared spectra from 4000 to 2600 cm-1 of hemoglobin in bidistilled water (A) and sucrose (B) and trehalose (C) solutions after 3 h of exposure to 50 Hz frequency EMF at 1 mT (red lines represent exposed samples spectra). The decrease in the amide A band at 3293 cm-1 (indicated by arrow) appears clearly for exposed samples of hemoglobin in water and sucrose aqueous solutions, but not for that in trehalose aqueous solution.
Such spectra were zoomed in from 3400 to 3200 cm-1 and are represented in Figure 2B, in which the spectra of hemoglobin in bidistilled water and in sucrose and trehalose solutions are represented in blue, purple, and green, respectively (dashed lines refer to exposed samples). The interactive concave rubberband correction allowed evidence of the double peaks of amide A band close to 3293 and 3307 cm-1. Furthermore, the decrease in the amide A vibration band for hemoglobin in bidistilled water and sucrose solutions after exposure was evidenced, confirming that no appreciable change occurred for hemoglobin in trehalose solution. The second-derivative spectra of hemoglobin solutions revealed the presence of three bands centered at 1654, 1635, and 1685 cm-1 in the amide I region. The band at 1654 cm-1 was due to R-helix structures, and around 1635 and 1685 cm-1 can be associated with β-sheet structures.36,37 This analysis relative to water aqueous solution revealed a loss of R-helical and short-
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Figure 2. (A) Infrared spectra from 4000 to 1400 cm-1 of hemoglobin in bidistilled water and in sucrose and trehalose aqueous solutions acquired after 3 h of exposure to 50 Hz frequency EMF at 1 mT, and baseline-corrected by a concave rubberband interactive method. (B) The same spectra zoomed in from 3400 to 3200 cm-1, in which the spectra of hemoglobin in bidistilled water and in sucrose and trehalose solutions are represented in blue, purple, and green, respectively (dashed lines refer to exposed samples). The concave rubberband correction allowed evidence of the double peaks of amide A band close to 3293 and 3307 cm-1, clearly showing their decrease for hemoglobin in bidistilled water and sucrose solutions after exposure.
segment connecting R-helix segments in the amide I region and a low increase in β-sheet bands at 1635, comparing exposed and unexposed spectra, after 3 h of exposure, as shown in Figure 3a. These features can be attributed to the formation of aggregates.38 No increase in β-sheet bands was detectable in the samples in trehalose aqueous solution after ELF exposure, as appeared in the IR spectrum of the amide I region (Figure 3c). On the contrary, a slight increase in the R-helix component was evidenced there, suggesting a refolding action by trehalose. To explore the ELF-EMF-induced changes in the infrared spectrum of hemoglobin in detail, we concentrated on the amide A vibration in the IR spectra, one of the most prominent spectral features at ∼3300 cm-1, that is due mostly to the peptide linkage N-H stretching mode; the weaker one is the amide B band around 3100 cm-1. These bands are present in the infrared spectra of peptides, polypeptides, and proteins. Previous studies have showed that local environments and hydrogen bonding configurations can play a role in determining the line shape of amide A vibration.39-41 A reasonable explanation of the observed amide A band, which is considered too strong to be only an overtone or a combination of lower frequency bands and is widely accepted, is that it derives from an overtone of amide II (NH in-plane angle bend) or a combination of amide I and amide II that interacts with the strong NH s fundamental through a cubic anharmonic potential, acquiring enough intensity to be observable through a Fermi
Figure 3. Second-derivative spectra of amide I and amide II for hemoglobin in bidistilled water (A) and in sucrose (B) and trehalose aqueous solutions (C) after 3 h of exposure (red lines represent the spectra of exposed samples). The relative increase in β-sheet close to 1635 cm-1 in the amide I region as to R-helix content (both localized by arrows) is visible for water solutions, but it is absent for trehalose aqueous solution.
resonance and resulting in two bands (amide A and amide B) whose separation is proportional to the interaction strength.42,43 Amide A is a useful indicator of secondary structure and often appears as a doublet band arising from two different structures, the stronger of the two components being associated with the standard R-helical structure in the chain.44,45 A comparison of data acquired after exposure to ELF-EMF showed a substantial difference in the evolution of the amide A features for hemoglobin in water and sucrose aqueous solutions. We calculated the ratio between the amide A integrated area from 3380 to 3260 cm-1 with respect to the integrated area from 3800 to 2800 cm-1 of exposed samples and that relative to not exposed samples (this ratio must be 1 before exposure, obviously). The computation performed for a relevant number of spectra provided the average value of 0.77 ( 0.05 and 0.86 ( 0.04 for water and sucrose aqueous solutions, respectively, after 3 h of exposure, as represented in Figure 4. These levels were
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Figure 4. Amide A integrated area ratio between exposed and control samples for hemoglobin in bidistilled water and in sucrose and trehalose aqueous solutions after 3 h of exposure to 50 Hz frequency EMF at 1 mT. Its value remains close to 1 after exposure for hemoglobin in trehalose aqueous solutions.
significantly different in comparison to the controls (p < 0.001). On the other hand, the amide A integrated area ratio relative to samples of hemoglobin in trehalose aqueous solutions provided the average value of 0.99 ( 0.05, proving that no significant effects occurred after 3 h of exposure when compared to controls. These results led us to consider the possibility of a loss of NH linkage in the secondary structure of hemoglobin due to ELF-EMF exposures, suggesting the hypothesis that trehalose can preserve the protein from eventual structural changes due to ELF-EMF. 5. Conclusion The effects of 50 Hz EMF at 1 mT on hemoglobin aqueous solutions were studied by means of FTIR techniques, showing that ELF-EMF can affect infrared vibration bands of hemoglobin. Some changes, such as the relative increase in β-sheet with respect to R-helix content in the amide I region, appeared barely perceptible, whereas an evident decrease in the amide A intensity band was observed after 3 h of exposure for water and sucrose aqueous solutions. In contrast, ELF-EMF did not affect the infrared spectrum of hemoglobin in trehalose solution, suggesting the hypothesis of possible protective effects against ELF-EMF of disaccharides such as trehalose. However, further research is needed to confirm such bioprotection properties of trehalose. References and Notes (1) Carpenter, J. F.; Prestrelski, S. J.; Arakawa, T. Arch. Biochem. Biophys. 1993, 303, 456. (2) Strickley, R. G.; Anderson, B. D. Solid-state stability of human insulin. II. Effect of water on reactive intermediate partitioning in lyophiles from pH 2-5 solutions: stabilization against covalent dimer formation. J. Pharm. Sci. 1997, 86, 645–653. (3) Kaushik, J. K.; Bhat, R. J. Biol. Chem. 2003, 278, 26458. (4) Magazu`, S.; Migliardo, F.; Vadala`, M.; Mondelli, C. Correlation between bioprotective effectiveness and dynamic properties of trehalosewater, maltose-water and sucrose-water mixtures. Carbohydr. Res. 2005a, 340 (18), 2796–2801. (5) WHO: World Health Organization. Extremely low frequency (ELF) fields. Environmental Health Criteria; World Health Organization: Geneva, 1984, Vol. 35. (6) WHO: World Health Organization. Magnetic fields. Environmental Health Criteria; World Health Organization: Geneva, 1987; Vol. 69.
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