EFFECT OF HYDROSTATIC PRESSURE EFFECTS ON BOVINE …



Effect of Hydrostatic Pressure on Bovine Blood Protein and Lipid Constituents Revealed by Fourier Transform Infrared Spectroscopy

CAGATAY CEYLAN1, METE SEVERCAN2, FARUK BOZOGLU3,

FERIDE SEVERCAN4

1Biotechnology, 2Electrical and Electronics Engineering, 3Food Engineering,

4Biological Sciences

Middle East Technical University,

06531, Ankara, TURKEY

feride@metu.edu.tr

Abstract: -The objective of this study was to investigate the effects of high hydrostatic pressure (HHP) on protein component of blood cells. The pressure at 132 MPa was applied to blood cells at 25 0C for 5 minutes. Lipid bands in the C-H stretching region were analyzed by FTIR spectroscopy and the protein bands at amide I region were analyzed by neural network based on FTIR data. The results revealed that the pressure at 132 MPa does not induce any compositional, structural and functional changes in lipid constitutes at molecular level. HHP treated proteins display only minimal changes in their secondary structures of the proteins, possibly in the direction of stabilization of the protein. The results were further supported by atomic force microscopy.

Key-Words: Pressure, Blood, FTIR, Spectroscopy, Lipid, Protein, Artificial Neural Networks, Atomic Force Microscopy.

1 Introduction

High hydrostatic pressure (HHP) treatment has been used advantageously in many areas of biotechnology due to its isostatic pressure property applied [1]. One such area where industrial applications already exist is that of food processing [2]. High hydrostatic pressure has also been used to develop cancer vaccines to combat this deadly disease of the century [3]. High hydrostatic pressure application turns out to be one of the promising tools that scientists have been using to understand the protein-protein interactions and protein folding and dynamics since high pressure experiments on proteins provide opportunity to separate the effects of density and temperature [4]. High hydrostatic pressure has been proposed as an alternative technique to thermal processing to destroy food-borne pathogens since it can inactivate microorganisms without altering the flavor and nutrient content of foods [5]. Bacteria are observed to be injured or killed by hydrostatic pressure. Although high hydrostatic pressure was used advantageously in various scientific and industrial fields, its use in the blood-related fields has been limited [6, 7]. Therefore, the aim of the current study is to investigate the effect of elevated pressure on lipid and protein concentration and structure and lipid dynamics which to our best of knowledge has not been reported previously. To achieve this, two non invasive techniques, namely Fourier transform infrared (FTIR) spectroscopy and atomic force microscopy were used.

2 Materials and Methods

2.1 The High Pressure Unit

The high pressure experiments were carried out in a high pressure cell situated in the Ultra High Pressure Laboratory in the Food Engineering Department of Middle East Technical University. The system was designed to have a pressure chamber, a pressure generating unit and a control unit basically. The pressure generated by an electric motor system is transduced to the pressure chamber via hydrolic connectors containing grease and finally to a cylindric unit. Following the movement of the cylinder the pressure is generated in the pressure cell. The fluid used as the isostatic pressure transducing agent is water. The system has also a control unit to monitor and control the pressure, temperature of the cell and pressure level. Pressurization times reported in this study did not include the pressure increase and release times.

2.2 The Blood Material

The blood used in this research is post mortem bovine blood. The blood sample was taken into 100 ml glass bottle containing 1.2 mg/ml of anhydrous salt of Ethylene Diamine Tetra Acetic Acid (EDTA). The whole blood analyses and the analyses for the cells of blood were carried out with the blood samples containing EDTA. When necessary the whole blood was spun at a maximum of 2000 rpm to prevent cell disintegration.

2.3 HHP Teatment of Blood Cells

In pressurization, vials containing blood samples were placed inside the cylindirical vessel of the HHP unit and pressurized at 132 MPa for 5 minutes at 25 C.

2.4 Atomic Force Microscopy

Atomic Force Microscopy was used to see the effect of HHP on the outer structure of erythrocytes. For this purpose MMAFM-2/1700EXL model instrument within the contact mode was used. The whole blood samples were fixed on the glass slide surfaces and allowed to dry. Then, the glass slides were cut into appropriate sizes to fit into the device holder and the samples were analyzed.

2.5 Pretreatment of Blood Cells Prior to the FTIR Study

The whole blood was centrifuged to separate the two main parts of the blood, namely the cells of the blood and the plasma part. For this purpose, the blood was spun at 2000 rpm for 10 minutes at room temperature. The blood fractionated into three main components: the erythrocytes, the white layer comprising the white blood cells and platelets and the plasma. The plasma part was gently taken into a different tube for further analysis without disturbing the white layer and then vortexed briefly to mix the cells. The cells of the blood part were separated and analyzed.

2.6 Spectrum Accumulation and Data Processing

The spectral analysis was carried out by using a Perkin-Elmer spectrometer equipped with MIR TGS detector (Spectrum One Instrument, Perkin Elmer). 4 microliter of blood cell samples were placed onto clean ZnSe window without spacers. This amount of blood samples were optimized to obtain nice peaks with minimum scattering coming from water peaks. Each recording was carried out at 4 cm-1 resolution with 50 scan number. Prior to each set of experiments the background of the ZnSe window was taken along with the background absorption caused by water and carbon dioxide and the background spectrum was subtracted from the spectra of the samples automatically. Spectrum One (Perkin-Elmer) software was used for all of the data manipulations except for curve-fitting. ZnSe was obtained from Merck (Merck, Darmstadt, Germany). After smoothing with a width of 13 in the Perkin-Elmer Spectrum software the smoothed spectra were taken to Win Bomem Easy software (Galactic Industries Corporation). The smoothed spectra were baselined first and then the curve-fitting was carried out in the amide I region of the spectrum with Gaussian-Lorentzian components using Grams/32. The baseline was always linear. The spectrum of each pressure experiment was subtracted from the spectrum of water and the wavenumber values of all functional groups were recorded. The band positions were measured according to the center of weight.

2.7 Artificial Neural Network Analysis of Amide I Band

The amide-I band of the control and pressure treated samples were analyzed through the software developed by Severcan et al [8]. The software provides the predictions of the secondary structure content of the component proteins in the sample analyzed.

2. Statistical Analysis

The differences between the control and pressure treated groups were calculated by means of Mann-Whitney Test with the Minitab Statistical Software Release 13.0 program. The statistical results are expressed as means ( standard deviation (SD). Significance was accepted at p < 0.05.

3 Results and Discussion

3.1 FTIR studies

FTIR technique provides useful information about macromolecular content, structure and function of biological samples [9-11]. FTIR spectrum of a biological system is quite complex consisting of several bands which arises from different functional groups belonging to lipids, proteins, carbohydrates and nucleic acids. Advantage of FTIR spectroscopy is that we can monitor easily, rapidly and sensitively the lipid, protein, carbohydrate and nucleic acid bands simultaneously.

In the current study C-H stretching region which is located in between 3030-2800 cm-1 and amide I region which is located at 1700-1600 cm-1 were investigated. The assignment of the bands of interest was given in Table 1 [12, 13].

Table 1. The assignment of bands of interest in FT-IR spectrum of blood cells.

|Wave number |Definition of the spectral assignment |

|(cm-1) | |

|2959 |CH3 asymmetric stretch: mainly lipids, with the |

| |little contribution from proteins, carbohydrates |

| |and nucleic acids |

|2936 |CH2 asymmetric stretch: mainly lipids, and little|

| |contribution from, proteins, carbohydrates and |

| |nucleic acids |

|2872 |CH3 symmetric stretch: mainly proteins, with the |

| |little contribution from lipids, carbohydrates |

| |and nucleic acids |

|2852 |CH2 symmetric stretch: lipids with little |

| |contribution from proteins , carbohydrates and |

| |nucleic acids |

|1654 |Protein Amide I (mainly C=O stretching) |

3.1.1 The C-H stretching Region

Figure 1 shows FT-IR spectra of control (red) and 132 MPa HHP treated (blue) blood cells. The spectra were interactively baselined first from the two arbitrarily selected points. Then a normalization step was carried out with a manuel baseline option at one of the baseline point used in the previous step so that the spectra are baselined at the same two points for precise comparison of the HHP treated and control samples. As seen from the figure 1, the spectra of the control and HHP treated samples show almost no changes in this region. The figure and the results of statistical anaysis indicate that the pressure used in this study does not induce any variations in the lipid bands in terms of signal intensity, band-width and the peak-frequency which monitors the concentration of the functional groups, lipid dynamics and lipid order respectively [11,14].

2. The Amid I region

The amide bands that arise from the vibration of the peptide groups provide information on the secondary structure of polypeptides and proteins. The amide I arises principally from the C=O stretching vibrations of the peptide group. Amide I absorption occurs in the region 1600-1700 cm-1. Figure 2 shows the baseline corrected, normalized average FT-IR spectra of control (red), 132 MPa HHP treated (blue) blood cells in the 1700-1600 cm-1 region.

The amide I bands of proteins consists of many overlapping component bands that represent different structural elements such as (-helices (1650-1655 cm-1), (-sheet (1620-1640 cm-1), turns and non-ordered or irregular structures (1670-1695 cm-1) and random coil (1640 cm-1) [13].

[pic]

Fig. 1. The FTIR spectra of the control (red) and 132 MPa HHP treated (blue) cells of blood in the 3010-2830 cm-1 region at 25 0C for 5 minutes. The spectra were normalized with respect to the 2958 cm-1 band.

Fig. 2. Amide I Region of blood cells, control (red), 132 MPa (blue) HHP Treated at 25 0C for 5 minutes.

Several methods have been developed for quantitative analysis of infrared spectra of proteins which provide information on the fractional content of different secondary structural elements. Recently an accurate method using neural networks for determining the secondary structure of proteins was developed by Severcan et al [8]. Using this procedure, we predicted the relative amounts of several secondary structural elements of blood cells for control and pressure treated samples. The results are shown in the Table 2.

Table 2. The Effect of HHP on the Secondary Structural Elements of Cells of Blood Studied by the Artificial Neural Network Algorithm Method.

|Pressure |α-Helix |β-Sheet |Turns |Random Coil |

|Control |62.04 |17.42 |10.27 |10.27 |

|132 MPa |65.01 |15.32 |10.24 |9.43 |

The results revealed that in pressure treated samples α-Helix content increases, β-Sheet decreases, no change in turn structure is observed and random coil structure slightly decreases. It is known that if there is a loss in α-helix or β-sheet structure and an increase in random coil structure, protein denaturates [15]. In the current study no increase in the random coil structure was observed due to HHP treatment. This shows that the pressure used in the current study does not denaturate proteins. However a slight decrease in β-sheet structure and a slight increase in α-helix structure were observed. Previous studies showed that in some disease states α-helix structure decreased but β-sheet structure increased [16]. However in our case we observed opposite results: α-helix structure increased and β-sheet structure decreased. This may indicate that HHP treatment may stabilize protein structure.

3.2. Atomic Force Microscopy Results

Atomic Force Microscopy (AFM) experiments were carried out to see any morphological changes on the red blood cells in more detail. Figure 3 shows the results of AFM study which clearly indicates that pressure does not induce any changes in the blood cells.

[pic]

a . Control

[pic]

b.132 MPa

Fig. 3. The effect of pressure on original Red Blood Cell morphology studied with Atomic Force Microscopy at 25 0C for 5 minutes of HHP treatment; (a) Control; (b)132 MPa.

4 Conclusion

The FTIR results clearly indicated that pressure application used in this study does not induce significant variations in lipid content, structure and function. However some changes towards protein stabilization were observed in proteins. 132 MPa pressure was used in the present study since this pressure could be used to inactivate viruses and bacteria under favorable conditions. The results of this study are especially important, because if a considerably high reduction in the number of pathogens could be achieved using high hydrostatic pressure without giving too much harm to the blood or blood products it should be considered a success especially today in the age of HIV or hepatitis or other bacteriemic/viremic diseases severely injuring people’s health upon blood transfusion.

Reference:

[1] Knorr, D., Effects of High-Hydrostatic-Pressure Processes on Food Safety and Quality, Food Technology, 1993, pp.156-161.

[2] Tedford, L.A., Smith, D.,Schaschke, C.J., High pressure processing effects on the molecular structure of ovalbumin, lysozyme and β-lactoglobulin, FoodResearch International, 32, 1999, pp.101-106.

[3] Goldman, Y., Shinitzky, M., , Immunotheraphy of Cancer With a Pressurized, Surface Reduced Tumor-Cell Vaccine, Drug Development Research, 50, 2000, pp.272-284.

[4] Heremans, K., Smeller, L., Protein structure and dynamics at high pressure, Biochimica et Biophysica Acta, 1386, 1998, pp. 353-370.

[5] Hoover, D. G., Metrick, C., Papineau, A. M., Farkas, D. F.and Knorr, D., Biological effects of high hydrostatic pressure on food microorganisms, Food Technology, 43 (3), 1989, pp. 99-107.

[6] Pares, D., Saguer, E., Toldra, M., Carretero, C., Effect of High Pressure Processing at Different Temperatures on Protein Functionality of Porcine Blood Plasma, Journal of Food Science, 65, 2000, pp. 486-490.

[7] Bradley, D. W., Hess, R. A., and Tao, F., Sciaba-Lentz, L., Remaley, A. T., Laugharn, J. A., Manak, M., Pressure cycling technology: a novel approach to virus inactivation in plasma, Transfusion, 40, 2000, pp.193-200.

[8] Severcan M. , Haris P. I., Severcan F., Using artificially generated spectral data to improve protein secondary structure prediction from Fourier transform infrared spectra of proteins, Analytical Biochemistry, 332 (2004) 238-244.

[9] Toyran N., Zorlu F., Dönmez G., Öğe K., Severcan F., 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, 2004. pp. 549-554,

[10] Boyar H., Zorlu F., Mut M., and Severcan F., “The effects of chronic hypoperfusion on rat cranial bone mineral and organic matrix: A fourier transform infrared spectroscopy study” Analytical and Bioanalytical Chemistry, 379 (3), 2004, pp. 433-438.

[11] Severcan F., Sahin I., Kazancı. N., “Melatonin strongly interacts with zwitterionic model membranes-evidence from Fourier transform infrared spectroscopy and differential scanning calorimetry” Biochimica et Biophysica Acta (BBA) - Biomembranes, 1668 (2): 2005, pp.215-222.

[12] Çakmak, G., Togan, I., Uğuz, C., Severcan, F., FT-IR Spectroscopic Analysis of Rainbow Trout Liver Exposed to Nonylphenol, Applied Spectroscopy, 57, 2002, pp. 835-841.

[13] Haris, P., Severcan, F., FTIR spectroscopic characterization of protein structure in aqueous and non-aqueous media, Journal of Molecular Catalaysis B : Enzymatic 7, 1999, pp.207-221.

[14] Toyran N. and Severcan F., “Competitive effect of vitamin D2 and Ca2+ on phospholipid model membranes: An FTIR study” Chemistry and Physics of Lipids, 123, 2003, pp.165-176,.

[15]Freifelder, D., Physical Biochemistry Applications to Biochemistry and Molecular Biology, W. H. Freeman and Company, Newyork, 1982.

[16] Kneipp, J., Miller, L. M., Joncic, M., Kittle, M., Lasch, P., Beekes, M., Naumann, D., In situ identification of protein structural changes in prion- infected tissue, Biochimica et Biophysica Acta, 1639, 2003, pp.152-158.

-----------------------

HHP Treated

Control

2852,90

2872,34

2935,47

2958,63

Wavenumber (cm-1)

Absorbance (Arbitrary Units)

3000 2980 2960 2940 2920 2900 2880 2860 2840

HPP

Control

Wavenumber (cm-1)

Absorbance

(Arbitrry Units)

1700 1675 1650 1625 1600

Absorbance

(Arbitrary Units)

................
................

In order to avoid copyright disputes, this page is only a partial summary.

Google Online Preview   Download