The objective of this study is to understand the effect of acidic pH and sugar-free on the ovalbumin stability using Fourier Transform Infrared Spectroscopy (FTIR) study. The FTIR spectra of phosphate buffer + Ovalbumin (OVA) + cosolvent (sugar-free) at 303K in various acidic pHs 2, 5 and 7 are reported and the second derivative and curve fitted spectra are carried out. The obtained result shows that pH and sugar free played vital role in content of secondary structure of ovalbumin. Destabilization of ovalbumin is observed due to the variation in the secondary structural content that is reflected in the result of presence of large amount of β-turns content compared to the core structure of β-sheet due to the addition of sugar-free. Presence of sugar-free destabilizes protein through the way of preferably bound with protein molecule.
Globular proteins are well-known for their distinctive functional properties in food, pharmaceutical and health-care products such as enzyme catalysis, ligand binding and transport, surface activity and self-association (McClements .D, 2002). The processing procedure involves in their protein products are often subjected to change in physical condition such as pH and heat etc. This procedure tends to affect the native state of protein hence proteins are got denatured. Many protein products also contain other functional ingredients such as low molecular weight water-soluble neutral cosolvent, for example, polyol, sorbitol, and sugar (Chanasattru.W, Decker. E.A, et al. 2008; McClements .D, 2002). Among these cosolvents, sugar solutions have large effects on the structural and functional properties of a protein. There are sample researchers and reports about the effect of sugar on the stability of protein (Velusamy.V, Palaniyappan.L, 2013; Palaniyappan.L, Velusamy.V, 2016; Mora Gutierrez et al., 2000).
Currently, Sugar-free is a commonly utilized artificial sweetener, and it is made using sucralose which is a derivative of sugar and taste also like sugar. It is widely used for cooking and backing as well as adding to beverages. However, the researcher reported that sucralose behaves very different compared to the sugar in terms of its interaction with biomolecules. It is interesting to note that sucralose destabilizes the protein secondary structure via electrostatic interaction like as urea (Lee Chan, Nimesh Shukla et al., 2015). In these aspects, the impact of artificial sweetener on protein stability is getting more attention in the food characteristics.
On analyzing structure and stability of proteins in aqueous and non-aqueous media, among in various techniques, Fourier transform infrared (FTIR) spectroscopy is acknowledged as a valuable tool (Carbonaro. M, Nucara.A, 2010; Youssef El Khoury Ruth Hielscher, et al., 2011; Dorr.S, et al. 2008). The most significant feature of FTIR spectra of biological samples can be obtained in a wide variety of environments. In connection with FTIR study of globular protein ovalbumin, it is of prime importance to understand why the secondary structure characterization is needed. Proteins are linear biological molecules for which the monomeric units are amino acids. The three-dimensional organization of proteins or conformation involves primary, secondary, tertiary and quaternary structures. The secondary structural composition is one of the most important pieces of information for a protein of unknown structure. The chemical environment in which a peptide or protein exists influences its secondary structure resulting its stability.
The present study is aimed at analyzing the stability of the protein in the presence of sugar-free with the change of pHs (2, 5 and 7) using FTIR technique at room temperature. Ovalbumin is chosen globular protein due to its wide application in food industry products (Velusamy.V, Palaniyappan.L, 2013). Use of sugar-free as the sugar substitute is obvious need not emphasized. Sugar-free is chosen as a cosolvent and phosphate buffers were prepared for pH changes.
Materials and methods
Powdered ovalbumin from chicken egg white purchased from Sigma Aldrich was used for sample preparation. Aqueous solutions of 0.2 M of both monobasic and dibasic sodium phosphates (NICE chemicals) were mixed in different proportions to prepare phosphate buffers of pH 2, 5 and 7. For the maintenance of the required pH with constant ionic strength, phosphate buffers were prepared as suggested by (Green A.A. 1933) and used for extreme pH values as done by Waris.B.N, et al. 2001. Also, it is absolutely necessary to maintain the ionic strength of the solution to be constant while varying the pH. Hence, the ionic strength as low as 0.005 mol dm-3 was chosen for the experiments so as to avoid the protein aggregation at these pH values (Antipova.A.S. Srmrnova.M.G, et al., 1999). The study of ovalbumin (5 mg/ml) in phosphate buffers of pH 2, 5 and 7 were reported by authors in their previous work (Velusamy.V, 2013). In the present study, sugar-free solutions of (~1M) prepared in phosphate buffers of pH 2, 5 and 7 were used as a solvent for ovalbumin (5 mg/ml). The pH of these solutions was measured by digital pH meter (HANNA Instruments, Model-HI 98107). After preparation, the stock solution was kept stored at 20oC overnight. These solutions were then degassed and each measurement was made after 20 minutes of thermal equilibration (30.00 ± 0.01oC).
The FTIR spectra of protein at various pHs 2,5 and 7 were recorded in the region 4000-450 cm-1 by a The Perkin Elmer Spectrum1 FT-IR instrument at Archbishop Casimir Instrumentation Center (ACIC), St. Joseph’s College(Autonomous), Tiruchirappalli, Tamilnadu, India.
The Perkin Elmer Spectrum1 FT-IR instrument consists of global and mercury vapor lamp as sources, an interferometer chamber comprising of KBr and mylar beam splitters followed by a sample chamber and detector. The spectrometer works under purged conditions. Solid samples are dispersed in KBr or polyethylene pellets depending on the region of interest. This instrument has a typical resolution of 1.0 cm-1. Signal averaging, signal enhancement, baseline correction, and other spectral manipulations are possible.
For recording FTIR spectra, aqueous samples (between 10 μl to 50 μl) are placed in a thermostated cell fitted with CaF2 windows with a 6 μm tin spacer. The interference pattern obtained from a two-beam interferometer as the path difference between the two beams is altered, when Fourier transformed, gives rise to the spectrum. The transformation of the interferogram into the spectrum is carried out mathematically with a dedicated online computer.
Acquisition of high signal-to-noise ratio spectra of peptides and proteins requires the elimination of water vapour from the sample compartment of the spectrometer since the narrow water vapour bands overlap with amide I band. So a pre-recorded water vapour absorption spectrum can be subtracted from the protein absorption spectrum to reduce the vapour bands still further.
Fourier transform infrared analysis of protein secondary structure
FTIR spectroscopy is a measurement of wavelength and intensity of the absorption of IR radiation by a sample. During this process of absorption, chemical bonds in the substance experience various forms of vibrations modes such as stretching, twisting and rotating. The energy of each molecular vibration corresponds to that of the infrared region of the electromagnetic spectrum. Many of the vibrations can be localized to specific bonds or groupings, such as the C=O and O-H groups. In this way, typical group frequencies include C=O, -COOH, COO-, O-H and S-H are being characterized. The IR spectrum of protein is characterized by a set of absorption regions recognized as the amide modes; they are namely amide A, B, and I-VII. Of all these, though amide II is unique, amide I band is the most prominent vibrational bands of the protein backbone (Krimm S., Bandekar J., 1986; Susi .H, Byler.D.M, 1986; Surewicz.W.K, Mantsch H, 1988, Banker.J, 1992).
The amide I band (1700-1600 cm-1) can be identified as the main sensitive spectral region to the protein, which is due to the C=O stretch vibrations of the peptide linkages (approximately 80%). The amide I band frequency components are found to be correlated closely to each secondary structural elements of the proteins. The amide II band, in contrast, originates mainly from in-plane NH bending (40-60% of the potential energy) and from the CN stretching vibration (18-40%) (Dong A., Meyer J. D. et.al 2000) showing much less protein conformational sensitivity than its amide I counterpart. Other amide vibrational bands are very complex depending on the details of the force field, the nature of side chains and hydrogen bonding, which therefore are of little practical use in the protein conformational studies. Banker (Banker. J, 1992) in his review, has described the nine amide vibration modes and some standard conformations in detail and they are listed in Table 1. The detailed characteristic IR bands of the proteins and are further confirmed by (Carbonaro et al. 2010) in their studies. Amide I frequencies assigned to protein secondary structure are given in Table 2 (Carbonaro et al. 2010; Dong.A, Huang P, 1990). These data are highly significant for the interpretations of the observed FTIR peaks in the present study.
ata analysis- the second derivative method
During the early stages of the studying proteins by FTIR, data quality and interpretation were severely limited by factors such as low sensitivity of the instrument, interfering absorption from an aqueous solvent, and a lack of understanding of the correlation between specific backbone folding types and individual component bands. Further, the recording of IR spectrum in an aqueous solution of a sample is a difficult process because water absorbs strongly in the most significant spectral region at approximately 1640 cm-1 unless deuterium oxide was used as a solvent (Byler D.M, Susi .H, 1986). Even in D2O solution, usually only qualitative information was obtained because the components of absorption bands associated with specific substructures, such as α-helix and β-sheet, cannot be resolved. Later it was solved and the amide I band assignments for proteins in H2O solution were also made (Dong.A, Huang P, 1990, Venyaminov S.Y, 1990). Currently, the computerized FTIR instrumentation has improved the signal-to-noise (S/N) ratio and allowed extensive data manipulation.
Mathematical data analysis methods can be used to improve the resolution of the protein spectrum beyond that of instrument resolution (Smith B.C, 1996; Kauppinen J.K 1986). Several methods have been developed to estimate quantitatively the relative contributions of different types of secondary structures in proteins from their IR amide I spectra in solution, including FSD-curve fitting (Byler D.M, Susi H, 1986; Dong. A, Malecki, 2002), second derivative analysis (Susi H., Byler D. M., 1986; Dong.A, Huang P, 1990; Byler D.M, Susi H, 1986), partial least-squares analysis( Lee D. C., Haris P. I., et al. 1990), and database analysis (Sarver R. W., Krueger W. C., 1991). The FSD-curve fitting and second derivative analyses are the two most commonly used methods. In the present study, second derivative analyses are employed.
Results and Discussion
Figs. 1 shows the primary spectra of ovalbumin system with cosolvent respectively measured in pH 2, 5, and 7 at 303 K. The best information from infrared protein spectra are obtained in the amide I band, which appears between 1700 and 1600 cm-1. These bands arise mainly from C=O group stretching vibration. However, primary spectra of ovalbumin system directly provide no detailed information about protein structure. Hence to gain more detailed information regarding the secondary structure composition of protein, the second derivative spectra and curve fitting procedures are carried out for systems of ovalbumin with cosolvents. The second derivatives and curve fitting of all spectra were performed by using Origin 6.0 software. Figs 2 to 4 show the second-derivative curve-fitted spectra of ovalbumin with cosolvent system at pH 2, 5, and 7 respectively. The areas of peaks were calculated using Lorentz fit multi-peak analysis available in origin 6.0 software. The percentage of each secondary structure components such as α-helix, β-sheet, β-turn, and random coils was determined as follows (Nagarize S, 2004). % of the secondary structure = area of secondary structure/area of amid I band. Thus the relative area of the selected peak was performed.
On observing table 6, it can be found that a large amount of β-sheets are presented compared to α-helix in OVA irrespective of pH. This indicates that the major secondary structure of the OVA is β-sheets which had also been reported by (Jain et al. 2007).
The measurements of OVA in phosphate buffer at pH 2, 5 and 7 have been taken from literature (Velusamy. V, 2013). Hence the discussions about OVA in buffer with sugar-free system at pH 2, 5 and 7 are alone being made in the present study.
The perusal of table 6 shows that the decreasing trend of β-sheet and increasing trend of α-helix due to the addition of sugar-free. As the sucralose is a major ingredient in sugar-free, the properties of sucralose is mainly attributed to the structural stability of OVA. Researchers proved that the presence of sucralose in solution is found to be destabilizing the native structure of two model protein systems: The globular protein bovine serum albumin and an enzyme staphylococcal nuclease (Lee Chen, Nimesh Shukla et al. 2015). So these previous findings are good support for the present case.
It is interesting to note that in buffer + glucose + OVA system, glucose interacts more strongly with water molecules than OVA by forming a hydrogen bond with a water molecule. This trend supports to stabilize the β- sheets and hence stabilize the protein. In the present case, sugar-free weakly interacts with water due to its less solubility compared to sugar. Further, it is studied that the chlorination of sucralose in sugar-free appears to enhance the hydrophobicity of the molecule (Shukla N., Pomarica E., et al. 2018). This hydrophobic trend of sucralose may lead to restricting the hydrogen bonding formation nearby protein-water interface. In other hand, researchers showed that sucralose would interact strongly with the permanent dipole moments of polar residues as well as the charged amino acids and possibly protein backbone. These cumulative effects may increase the affinity of sugar-free with protein molecule resulting in the destabilization of OVA.
Researchers also established (Lee Chen, Nimesh Shukla et al. 2015) that the sucralose is likely interacting with polar and charged portions of the protein structure in a manner consistent with known chemical denaturants such as urea. This strong electrostatic interaction may be responsible for the destabilization of the native structure of OVA through the decreasing trend in β- sheet content. In another side, it is anticipated that α-helix content is an increasing trend opposite to β- sheet.
The study was made using isothermal titration calorimeter (Shukla N., Pomarica E., et al. 2018) suggest that the interaction between sucralose and the protein must be the weak dipole-dipole type of interaction. Further, it is confirmed that sucralose destabilizes protein structures through its weak interaction with proteins due to in highly stressed environments. This may be the reason for more reduction of β- sheet at pH 2 (which analogous to highly stressed environment) due to adding sugar-free leads to destabilization of OVA.
As a concern of β-turns, the increasing trend is perceived at all pHs due to the addition of sugar-free. Turns affect the protein folding through the presence of specific structural features like the β- bulge reported in the globular protein ubiquitin (Marccelino. AMC, Gierasch. LM). The β-bulge in this turns to sample of stable β-hairpin conformations and prevents the formation of non- native hydrophobic interaction in ubiquitin. This implies that the local structure of β-turns affects the stability of the intact protein and that the contribution of tertiary interactions is important in β-sheet formation as well.
Sharpe (Sharpe et al. 2007) proposed that turn has two types of conformations: one that allows productive folding and the other that leads to a more aggregation-prone conformation. In this aspect, the increasing trend of β-turn with irrespective of pHs may affect the inherent nature of protein folding due to sugar-free addition leads to the destabilization of OVA.
It is observed from table 6, a higher value of β-turns at pH 2 and 5 compared to pH 7. This indicates more destabilization of OVA. The reason for this above trend may be narrated as follows: The surface of a globular protein is highly heterogeneous, consisting of functional groups of differing polarity, shape, and size. Each of these groups interacts differently with cosolvent and solvent molecules, depending on their molecular characteristic (Timasheft, S.N., 2002). Protein stability is affected by coexisting molecule or solute in the solution. It was reported that preferentially-excluded solute from the protein surface stabilizes proteins while preferentially-bound solute destabilizes proteins (Miyawaki.O, 2007). Preferential exclusion implies that there is no direct interaction between disaccharides and proteins. The addition of disaccharides to bulk water isolates water molecules away from the protein, decreasing its hydrated radius and increasing its compactness and consequently stability. It was reported that the chlorination of sucralose appears to slightly enhance the hydrophobicity of molecule (Jain N.K, Roy I., 2009) which may reduce the preferential exclusion of sucralose form the OVA-water interface. Simultaneously the sucralose preferentially bound with OVA through electrostatic interactions. The combination of these two effects may lead to destabilizing the protein structures.
As concern random coil, interesting results are observed at pH 2 and 5. Random coils refer the totally disordered and rapidly fluctuating conformations of proteins in solution. In the random coil form, the residues in the peptides are exposed to the solvent and hydrogen bond with solvent molecules. However, as stated earlier the sucralose leads to restricting the hydrogen bonding formation. Hence it is anticipated that no coil formation due to an addition of cosolvent. At the same time, the above trend is not observed at pH 7 as much of pH 2 and 5.
From literature survey (Velusamy. V, 2013), it observed that OVA highly resembles with native state of protein at pH 7. Generally, the minimum amounts of random coil conformations are expected in globular proteins at pH 7 due to the solvent- protein interaction.