Impact of cosolvent (glucose) on the stabilization of ovalbumin

Food Hydrocolloids 30 (2013) 217e223

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Impact of cosolvent (glucose) on the stabilization of ovalbumin L. Palaniappan a, V. Velusamy b, * a b

Department of Physics (DDE), Annamalai University, TN 608002, India Department of Physics, Manonmaniam Sundaranar University, Abishekapatti, Tirunelveli, TN 627012, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 October 2011 Accepted 26 May 2012

Proteins are stabilized by glucose against denaturation due to extremes of pH. This was studied by means of density, ultrasonic velocity, viscosity and surface tension measurements in the ovalbumin (5 mg/ml) dissolved in phosphate buffer (pH 2, 5, 7, 9 and 12). Few thermo-acoustical parameters such as adiabatic compressibility, intermolecular free length, acoustic impedance, the partial apparent specific volume and the partial apparent specific adiabatic compressibility were calculated for the said systems. Obtained results suggest that the stabilization of ovalbumin occurs in the presence of glucose through strengthening of hydrophobic interactions supported by other non-covalent interactions and the steric exclusion effect of the cosolvent molecules. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Ovalbumin Glucose Sound velocity Viscosity Surface tension Hydrophobic interactions

1. Introduction Globular proteins are found to have various unique functional attributes such as surface activity, foaming capacity, emulsion stabilizing, thickening and gelling. These attributes make them to use as ingredients in industrial products of foods, cosmetics, health care products and pharmaceuticals. Many of these protein products also contain other functional ingredients, such as low molecular weight water-soluble neutral cosolvents, e.g., polyol, sorbitals and sugars (Chanasattru, Decker, & McClements, 2008; McClements, 2002). In general, cosolvents are able to change the conformation and functional performance of globular proteins in aqueous solutions (Chanasattru et al., 2008; McClements, 2002; Saunders et al., 2000). Sugar solutions in particular have large effects on the structural and functional properties of proteins including their solubility, denaturation etc. There are sample researches and reports (Anjum, Rishi, & Ahmad, 2000; Back, Oakenfull, & Smith, 1979; Demetriades & McClements, 1998; Mora Gutierrez & Farrell, 2000; Rishi, Anjum, Ahmad, & Pfeil, 1998) about the effect of sugars on the stability of proteins. Waris, Hasan, and Srivastava (2001) have reported that the proteins are stabilized by sugar against the denaturation due to extreme of pH. The extent of denaturation and renaturation are suddenly reflected in the packing (folding/unfolding) nature of protein

* Corresponding author. Tel.: þ91 98944 39295. E-mail address: [email protected] (V. Velusamy). 0268-005X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodhyd.2012.05.020

concerned. Such processes in globular proteins are always subjected to conformational and functional changes and accordingly their interactions with their neighboring components got affected. The variations in packing nature and molecular interactions can be best studied using ultrasonic techniques. This involves the measurement of kinematic properties such as sound velocity, density, etc and computation of related parameters. The review of literature shows that various attempts were made with varieties of kinematic properties such as surface tension, viscosity, density, sound velocity and with thermo-acoustical properties namely adiabatic compressibility, free length etc. to characterize the protein-cosovent system. In this regard, Chanasattru et al. (2008) have provided valuable information about the change in the molecular properties of globular proteins dispersed in cosolvent solutions. Waris et al. (2001) have obtained an important result that adiabatic compressibility of globular proteins are positive while the compressibility of the constituent amino acids are negative indicating the great contribution of the internal cavity in the structure of proteins. The volumetric and compressibility behavior of solutes in solution were used by various investigators (Chalikian & Filfil, 2003; Kharakoz, 1991; Taulier, Beletskaya, & Chalikian, 2005) for the characterization of protein conformational states at different pH environments. Awasthi and Shukla (2003) have used acoustic impedance and intermolecular free length to explore the inertial and elastic properties of the proteins. The viscometric studies by Waris, Bano, and Abdul Raziq (2003) and the surface tension studies by Lin and Timasheff (1996) have provided very useful information related to the stabilization of protein in aqueous solution by a variety of cosolvents.

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In this regard the present investigation is aimed at analyzing the pH (2e12) denaturation of globular protein and its stabilization by a cosolvent using ultrasonic techniques at room temperature (30  C). Ovalbumin is the chosen globular protein due to its wide use in food products and relatively higher stability (axial factor less than 3), consisting of 385 amino acid residues in a single-chain phosphoglycoprotein and an isoelectric point (pI) of 4.8 (Doi & Kitabatakes, 1997). Use of glucose in food industry is obvious and need not be emphasized. Glucose is specifically chosen as it is also asymmetric like ovalbumin and freely dissolves in water. pH denaturation is considered so as to study the influence of zwitterions (pI) on the denaturation / renaturation process without disturbing the internal disulfide bond (Mine, 1995). For the maintenance of the required pH with constant ionic strength, phosphate buffers were prepared as suggested by Arda Alden Green (1933) and used for extreme pH values as done by Waris 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 dm3 was chosen for the experiments so as to avoid the protein aggregation at these pH values (Antipova, Semonova & Belyakova 1999). This is here achieved by suitably changing the composition of buffer systems utilizing both monobasic and dibasic sodium phosphates, as per the directions of Philip Elving, Markowitz, and Rosenthal (1956). Measured parameters in the present work include ultrasound velocity, density, viscosity and surface tension whereas the calculated parameters are adiabatic compressibility, intermolecular free length, acoustic impedance, the partial apparent specific volume and the partial apparent specific adiabatic compressibility. Structural conformational changes are best revealed by static properties (density and surface tension) while the other two dynamic properties reveal the associated functional changes. Among the calculated parameters, compressibility is the ease with which the medium can be compressed or the extent of available free space between the components. A well structured packing leads to a minimum compressibility value so that it cannot be further compressed. As the conformational changes are so fast, the compressibility exists as an adiabatic compressibility Such process can more easily be understand from the least average distance between two components, called as intermolecular free length. These two parameters viz., adiabatic compressibility and free length, reflects the structural modifications of the medium. However, to have more idea about the functional variations, a dynamic transport is needed and this is achieved by acoustic impedance, a measure of sound propagation and also by viscosity studies. The changes brought about in the medium by the sound propagation are purely transient and hence its effects are not absolute but apparent. Yet, the apparent changes such as apparent adiabatic compressibility, apparent volume made by the sound propagation can offer interesting results. Further as the size and contribution of the protein molecules are large compared to solvent / cosolvent molecules, the partial values of proteins are highly significant for the interpretation of observed changes. Thus partial apparent adiabatic compressibility and partial apparent volume were aimed in protein systems. These macroscopic observables are sensitive to structure and dynamics (Chalikian, Sarvazyan, & Breslauer, 1994; Kharakoz & Sarvazyan, 1993) of protein systems. Moreover to generalize and to standardize these two parameters, as per the definition (Robert, Don & James 1997), unit concentration is needed and hence cosolvent concentration is chosen as 1 M and hence the terms are aptly called as partial apparent adiabatic specific compressibility and partial apparent specific volume. Further this choice of 1 M glucose is a safe concentration that ensures that it will not rupture any of the amino acid residues in the chain (Campbell & Farrell, 2006).

Thus in this paper, we have studied the stabilization of ovalbumin dispersed in glucose solution against pH using ultrasonic techniques at 30  C. Analysis is made in the mixtures of ovalbumin in phosphate buffer and the same mixture dispersed in glucose solution, in the pH range 2e12 and at room temperature (30  C). 2. Experimental procedures At least six repeated reliable observations were made for the measurement of each property and the reported values correspond to the average of these six independent measurements. The standard deviation of all the trials for each property was found to be satisfactory. In the entire study, the temperature was controlled to  0.01  C.by water thermostatic bath provided by Ragaa Industries, Chennai, India. 2.1. Materials and sample preparation Powdered Ovalbumin from chicken egg white purchased from Sigma Aldrich (Product No. A5253, Grade II) was used for sample preparation. 0.2 M aqueous solutions of both monobasic and dibasic sodium phosphates (NICE chemicals) were mixed in different proportions to prepare phosphate buffers of pH 2,5,7,9 and 12. They were used as solvents for ovalbumin (5 mg/ml). For the second set, 1 M solutions of d-glucose (NICE chemicals) prepared in phosphate buffers of pH 2, 5, 7, 9 and 12 were used as 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 20  C overnight. These solutions were then degassed and each measurement was made after 20 min of thermal equilibration (30.00  0.01  C). 2.2. Measurement of density The density (r) of solvents and solutions were measured at room temperature (30  C) using 5 ml specific gravity bottle. The accuracy in the measurement was about 0.0001 kg m3. The specific gravity bottle was immersed in a thermostated paraffin bath to maintain the temperature of the system. 2.3. Measurement of ultrasonic velocity The ultrasonic velocity (u) in protein solutions was measured by a single frequency (2 MHz) ultrasonic interferometer (Mittal’s model F-81). The accuracy of sound velocity was 0.1 m s1. The interferometer consists of two main parts (i) the measuring cell and (ii) the high frequency generator. The measuring cell is a specially designed double walled cylindrical cell. Water is circulated through the annular space between the two walls in order to maintain the temperature of the sample in the cell as constant (30  C) during the experiment. A quartz crystal is fitted at the bottom, a reflector at the top of the cell and inner wall of the cell is corrugated to prevent wall reflections. A fine micrometer screw has been attached with the reflector which can raise or lower the reflector plate in the cell through a known distance. The high frequency generator consists of a radio frequency oscillator to excite the quartz plate fixed at the bottom of the measuring cell. The quartz plate generates ultrasonic waves when frequency of the oscillations of the high frequency generator is equal to natural frequency of the crystal. A micro-ammeter is provided in the plate circuit of the generator to observe the changes in current. The quartz crystal in the measuring cell is connected to the radio frequency generator through a shielded cable. The cell is filled with the experimental liquid. On exciting the crystal, standing waves are

L. Palaniappan, V. Velusamy / Food Hydrocolloids 30 (2013) 217e223

formed between the crystal and reflector. The micrometer screw is moved upward until the anode current in the micro-ammeter of R.F. generator shows minimum deflection. Initial reading of micrometer is noted. The screw is then rotated slowly in the same direction and successive minima are allowed to pass till the nth minima. Reading of the micrometer is again noted. Thus, the total distance (d) through which the reflector shifted for ‘n’ minima is noted. The wavelength (l) of the ultrasonic waves in the liquid is calculated by using the equation, l ¼ 2d/n. Knowing the value of (l) and the frequency of the quartz crystal (f), velocity of sound (u) is determined from the relation, u ¼ lf. 2.4. Measurement of viscosity The viscosity measurement of solvents and solutions were done by relative method using Ostwald’s viscometer of 10 ml capacity. The viscometer is calibrated with double distilled water and is immersed in the water bath to keep 30  C. The time of flow (s0 ) of water from first mark to second mark is noted. Then water is removed and is filled with experimental solution. The time of flow (s) of solution is measured after the viscometer has attained the temperature of the bath. By substituting the flow time of reference liquid (water) and solution, the viscosity of the solution can be determined using the relation h ¼ (rs/r’s’) h’ where, h, r and s are the viscosity, density and time of flow of solution respectively, h0 , r0 and s0 are the corresponding quantities for water. The measured viscosities are accurate to  0.001 mNs m2. 2.5. Measurement of surface tension The surface tension (g) was measured at 30  C by drop weight method. Platinumeirridium Du Nouy ring is used for identical drop formations with a drop formation time of 60 s. Two sets of drops viz., 20 drops and 30 drops were attempted and the average measure of all the reliable observations is taken as final. 2.6. Calculation of thermo-acoustical parameters Measurement of the protein solution density (r) and that of the sound velocity (u) allows the determination of the solution adiabatic compressibility (b), using Laplace’s equation:



b ¼ ru2

1

(1)

Free length (Lf) and acoustic impedance (Z) can be calculated using the following standard expressions (Palaniappan & Velusamy, 2004; Velusamy, Nithyanandham, & Palaniappan, 2007): 1=2

Lf ¼ KT b

(2)

Z ¼ ur

(3)

where KT is the temperature-dependent constant having a value 199.53  108 in S.I system. The partial apparent specific volume (4v) and the partial apparent specific adiabatic compressibility (4k) of ovalbumin of the protein are calculated using the well-known relationship (El Kadi et al., 2006)

4v ¼ 1=ro þ ðro  rÞ= Cp $ro




4k ¼ bo þ ð24V  2½u  1=ro Þ

(4) (5)

where r and ro are the densities of the protein solution and of the reference solvent respectively, Cp is the protein concentration, bo is

219

the adiabatic compressibility of the solvent and [u] is the relative specific sound velocity increment given as.

½u ¼ ðu  uo Þ=uo Cp

(6)

where u and uo are the ultrasound velocity in the solution and the solvent respectively. 3. Results and discussion The measured values and the respective standard deviation values of density, sound velocity, viscosity and the surface tension of the ovalbumin system with and without cosolvent for various pH at room temperature (30  C) are summarized in Table 1. The trend of calculated parameters viz., the adiabatic compressibility, intermolecular free length, acoustic impedance, the partial apparent specific volume and the partial apparent specific adiabatic compressibility against various pH values were shown in Figs. 1e5 respectively. 3.1. Measured parameters It can be observed from Table 1 that all the measured parameters are found to increase with the addition of cosolvent (glucose) in the ovalbumin þ phosphate buffer system irrespective of pH. However no such linear trend is obtained for pH variations irrespective of the presence of cosolvent. i.e. the pH variations are sharply reflected in the protein systems. The least value of the measured parameters at pH 5 or 7, in general, suggests that the protein’s native state allows more free space around it. Extremities of pH, viz., 2 and 12, recorded higher density values and they reflect the highly denatured states of ovalbumin. It is interesting to note that extreme pH aids tight packing and this reveals that denaturation leads to coiling of protein primary structure to a-helix and b sheets. As ovalbumin belongs to globular type, b-sheets form the core structure (Satyanarayana, 2003) and hydrogen bonds form interchain as well as intrachain of the coil. As the isoelectric point (pI) of ovalbumin is 4.8 (Doi & Kitabatakes, 1997), the values presented here for pH 5 (without cosolvent) resembles largely that of native state of ovalbumin. As regards density, the least value is found at pH 7. An increase in density is an indication of coiling of structure whereas the reduction may be taken as a rupture of structure. Neither of these two effects can be ascertained for the density at pH 7 as rupture of rigid peptide bonds cannot be claimed for a small change in pH. Hence the least density at pH 7 (without cosolvent) suggests that the primary helical structure remains intact but are supposed to extend

Table 1 Experimental (Expt) values and their standard deviation (SD) of measured parameters in the mixtures at various pH. pH

Density Expt kg m3

Without cosolvent 2 1023.2 5 1021.2 7 1017.7 9 1019.3 12 1027.9 With cosolvent 2 1082.9 5 1073.8 7 1075.4 9 1069.3 12 1081.7

Velocity

Viscosity

Surface tension

SD %

Expt m s1

SD %

Expt mNs m2

SD %

Expt N m1

SD %

0.30 0.48 0.30 0.42 0.40

1557.5 1540.1 1557.2 1546.0 1565.5

0.33 0.28 0.50 0.19 0.38

0.8354 0.9118 0.7723 0.7870 0.8128

0.21 0.16 0.23 0.40 0.31

0.2235 0.2439 0.1785 0.2413 0.1951

0.13 0.20 0.15 0.10 0.18

0.38 0.29 0.36 0.25 0.33

1605.5 1600.8 1593.3 1604.2 1605.3

0.41 0.30 0.27 0.75 0.52

1.2901 1.1859 1.1384 1.1456 1.1864

0.05 0.21 0.11 0.28 0.14

0.2393 0.2498 0.2462 0.2525 0.2137

0.16 0.17 0.21 0.11 0.13

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No cosolvent

With cosolvent

Z x 10 -6 kgm -2s-1

1.80

1.70

1.60

1.50 2

4

6

8

10

12

pH Fig. 3. Plots of acoustic impedance versus pH.

Fig. 1. Plots of adiabatic compressibility versus pH.

further for compact folding by the alkaline nature of the medium, as supported by the following discussions. When the medium becomes acidic (pH < pI) or basic (pH > pI) the native helical structure of the protein is lost and the structure coiled to form a-helix and b sheets. However this denaturation takes place differently in these two cases. The primary structure is full of peptide bonds which are rigid and planar with partial double bond in character. The free amino group is at left end (N-terminal) and the free acid group is at the right end (C-terminal). In strong acidic pH, the amino acid is positively charged. As positive charges are heavier with lesser mobility, they cannot defold / extend further and so coiling of structure starts instantly though the coils will be larger in radius. But in the case of strong alkaline pH, the amino acid is negatively charged and can exhibit larger mobility. So, the primary helical peptide chain undergoes further extension and then started coiling. This leads to a relatively good degree of compactness with coils of low radius. Thus alkaline extremity is expected to provide a higher density than that by acidic extremity, as obtained in the present work. This explains the observed reduction of density at pH 7 and the enhancement at other pH values. For the same reasons, the sound velocity at pH 5 is least, acidic pH is higher whereas in alkaline pH, it fluctuates. The trend observed for sound velocity supports the above view. Apart from the effects of pH, the asymmetric nature ovalbumin in general, a-helix in particular, also supports the difference in mobility of N-terminal and C-terminal. Because of its asymmetric nature, a-helix is actually a dipole with slight positive charge at its N-terminal and slight negative charge at its C-terminal (Clark, 2009) that promotes the stability of a-helix. Unlike density, sound velocity is a dynamic property that needs cohesion between the components. In alkaline pH range, the further extension of

No cosolvent

helical structure at pH7 offers sufficient cohesion whereas the formation of inter/intra hydrogen bonds due to coiling at pH 9 reduces cohesion. So sound velocity decrease at pH 9. As extremes of pH can convert amides of the amino acid residues into corresponding acids (Clark, 2009) which are highly interactive and cohesive, pH 12 records a higher sound velocity. But for the explanation of viscosity parameter, the surface area of the layers and the interlayer distance of separation are the key factors. At pI, the protein is a single-chain with no layer formation and hence viscosity is more. In other pH values, interlayer distance exists due to coiling and viscosity tends to decrease. But in acidic pH coils are having a larger area due to larger radius. As a net effect, viscosity will be smaller than that at pI, but larger than that in alkaline pH. As denaturation increases with pH, more and more coils are formed. As the surface area increases, viscosity also increases in alkaline pH range. As the native state is the highly stable state, surface tension is higher at pH 5. Shape of the surface plays a vital role in deciding surface tension. Helical structure is more or less linear, having a very large radius and hence the native state records highest surface tension. The observed surface tension values provide an excellent support to the suggestions made earlier. Acidic pH is having coils of larger radius and hence they have high surface tension than at alkaline pH. Further pH extreme converts amides into acids, which can largely interact and forms compact sizes that lower the surface tension. In general pH effects are highly reflected in the observed values for the system without cosolvent. On adding cosolvent the medium becomes denser and hence all the parameters show an increase in their values. The values at pH 5 correspond to that of native state of ovalbumin. As stated earlier, due to increased mobility in alkaline pH situations, exclusion of material takes place and this form many

With cosolvent

No cosolvent 2.40

φ V x 10 3 m 3kg-1

Lf x 10 11m

4.20

With cosolvent

4.00

3.80

3.60

1.60 0.80 0.00 -0.80 -1.60

2

4

6

8

10

pH Fig. 2. Plots of intermolecular free length versus pH.

12

2

4

6

8

10

pH Fig. 4. Plots of partial apparent specific volume versus pH.

12

L. Palaniappan, V. Velusamy / Food Hydrocolloids 30 (2013) 217e223

No cosolvent

With cosolvent

φ k x 10 12 m 3kg-1Pa -1

3.20 2.40 1.60 0.80 0.00 -0.80 -1.60 2

4

6

8

10

12

pH Fig. 5. Plots of partial apparent specific adiabatic compressibility versus pH.

number of cavities. The added cosolvent molecules may fill up these cavities and hence compactness increases. However the effect of pH and the asymmetric nature of a-helix decrease the compactness. Combination of these two opposite effects, summed over the entire protein surface, results in the macroscopic preferential binding (Lin & Timasheff, 1996) or exclusion that can be attributed to all the observed non-linear changes in alkaline pH. However the extreme of pH (2 or 12) converts the amides into acids, the added cosolvent forms a weak binding system and hence density increases. The presence of cosolvent leads to a larger increase in sound velocity at all pH values. The increase in ultrasonic velocity in a solution indicates the greater association among the components (Hornowski, Józefczak, qabowski, & Skumiel, 2008; Miecznik, Golebiewski, & Mielcarek, 2004), which may be due to the intermolecular hydrogen bonding between the solute and the cosolvent molecules. In the chosen systems the weak as well as the strong interactions are found to exist. Weak interactions occur between all atoms, either polar or nonpolor. These are essentially the electrostatic interactions or van der Waals interactions that arise because the distribution of electronic charges will, at any instant of time, be localized in some asymmetric geometry. Further the aqueous solutions of glucose have lower dielectric constant than pure water indicating that the electrostatic interactions are stronger in these solutions than in pure water as reported in the literature (Waris et al., 2001). The evidences for the existence of the strong intermolecular interactions can be realized by thinking of an ion-dipole interaction. In a protein, charged amino acids are available in ample numbers and they provide lot of dipoles or ions. The surrounding water molecules which are polar will offer the counterpart for the strong interactions. Moreover the ions dissolved in water enhance the situation for strong interactions. It is to be noted that the protein conformational equilibrium involves ionic interactions (Lins & Brasseur, 1995). Thus the cumulative effect of non-covalent interactions such as hydrophobic, hydrogen bonds, ionic and van der Waals interactions are taking vital role in the observed increase in sound velocity. The observed increase in viscosity in the protein solutions may be attributed to the fact that the addition of glucose to the protein stabilizes it through increased hydrophobic interactions. As the solubility of glucose in water is 18% w/w at 20  C which is very low (Eliel, 1985), its interactions with protein molecules would be comparatively high. This may be supposed to protect the protein from the denaturation effects of extreme pH. In the absence of sugar, the extremes of pH cause denaturation of protein and the random coils are formed. Due to random coiling of protein, the effective surface area of protein increases and hence viscosity also increases.

221

The trend of surface tension is more interesting. The surface tension of ovalbumin solutions, with or without glucose, is found to be more than three times higher than that of pure water. It is to be remembered that in the present experiment, ovalbumin is not dissolved in pure water but in aqueous buffer solutions of monobasic and dibasic sodium phosphates as stated earlier. Surface tension of pure water is found to be 0.072 N m1, that of buffer (pH 2) is 0.2521 N m1 and for the added ovalbumin is 0.2235 N m1 as reported in Table 1. The same is true at all pH values with or without cosolvent. The components of buffer drastically reduces the potential difference in the surface layer thereby largely increases the surface tension of buffer. However the addition of ovalbumin decreases the surface tension. This reduction of surface tension with the addition of protein is quiet expectable. Further, this simply reveals that ovalbumin molecules are highly depleted from the water surface thereby opposes the effect of hydrolysis. In the analysis of surface phenomena, Gibbs (1878) showed that substances that lower the surface tension of water accumulate at the surface those that raise it are depleted from the surface. As the surface tension of water got raised in the present analysis due to ovalbumin, it is evident that ovalbumin molecules are depleted from the surface and form equal number of cavities in the solvent system. Belton and Twidle (1940) have noticed that the maximum of surface tension was at the isoelectric point of the ovalbumin. In order for a protein to dissolve in solution, the protein must form a tiny cavity, or pocket, within the solution. The higher the solution’s surface tensions the more difficult for the protein to form a cavity. Breslow and Guo (1990) have shown that cosolvents can affect the energy required to produce a cavity in the solvent, which is reflected in their effect on surface tension and salvation energy of the solute. In the present study, the addition of cosolvent increase the surface tension, thereby reduces the cavity forming tendency of ovalbumin and hence increases their stability. It is to be remembered that the protein surfaces in native and denatured states are similar in nature (Lin & Timasheff, 1996) with respect to the surface tension effect. Thus in the absence of cosolvent, the cavities remain unfilled and they can disturb the ovalbumin by means of hydrogen or van der Walls or hydrophobic interactions. These are attributed to the observed surface tension at other pH values without cosolvent. But with the addition of cosolvent, as the cavities got filled up, ovalbumin is not disturbed and hence its stability got improved, that shows almost same surface tension at all pH values. 3.2. Calculated parameters The perusal of Fig. 1 indicates that b value for all the systems studied are positive suggesting the presence of highly compressible cavities in the protein molecules and that the effect of cavity has overcome the solvation effect. If the compressibility values are higher it implies that the medium is loosely packed which is due to the denaturation of the protein, where as the lower compressibility is an indication of maximum interaction among the molecules in the protein solution from which the stabilization of protein can be obtained. Analyzing Figs.1 & 2, it is found that Lf reflects a similar trend as that of b. Denaturation makes the medium to be more flexible and this tends to increase the intermolecular free length in the solution. But on addition of glucose, a decrease in intermolecular free length is observed due to the raising of compact structure of protein. Further the resemblance of native state or the extension of primary helical structure of ovalbumin at pH 7 is confirmed. When an acoustic wave travels in a medium, there is a variation of pressure from particle to particle. The ratio of the instantaneous pressure excess at any particle of the medium to the instantaneous velocity of that particle is known as acoustic impedance (Z) of the

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medium (Awasthi. & Shukla, 2003). This factor is governed by the inertial and elastic properties of the medium. The increasing trend of Z (Fig. 3) on addition of glucose suggest that the strengthening of interaction among the components in the solutions. The interaction may be solute e solute or solute e solvent or solvent e solvent type. This trend of increasing Z of the solution reveals the reduction of protein denaturation. Further the figure shows that, when there is no cosolvent the Z of ovalbumin at pH 5 and 9 are less than that at pH 2 and 12. This suggests that large amount of denaturation of protein is occurred at pH extremes. But the addition of cosolvent removes the effects of pH as noticed from the other curve of the same figure. An examination of Figs. 4 and 5 shows that 4v and 4k of ovalbumin at pH 7 is less than that of at pH 2, 5, 9 and 12. This again confirms that the extension of primary helical structure is existing at pH 7. It will be significant to note that at pH5 4k is least that indicates the undisturbed native state of ovalbumin. The extreme of pH cause denaturation of protein and random coils are formed. Due to the random coiling of protein, the values of 4v and 4k are increased and are highly reflected at other values of pH. It has been observed that the addition of glucose to the protein solutions decreases the values of 4v and 4k of the solutions. This may be attributed due to the interactions between protein, water and additive (glucose) molecules. i.e., the additives are interacting more strongly with protein and favor the stabilization of protein molecules as explained by Monsan and Combes (1988) in their systems. Proteins are biopolymers forming chain-like structures of small units of amino acids and stabilized by a combination of hydrogenbonding, electrostatic and hydrophobic interactions (Lins & Brasseur, 1995). Many amino acids have side chains or active groups which are essentially hydrophobic. They have little attraction for water molecules in comparison to strong hydrogen bonding between water molecules and also many hydrogen bonds form between glucose and water molecules. This non-polar side chain groups in the native protein molecules would have a tendency to enter into the interior of protein due to the polar environment produced by sugar molecules. This phenomenon would be responsible for higher stability of the protein molecule in these solvents and would reduce the extent of denaturation of protein molecules induced by extremes of pH. In another view, this may be concluded 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 characteristics (Timasheff, 2002). Protein stability is affected by coexisting solute in the solution. Also it was reported that preferentially-excluded solute from the protein surface stabilizes proteins while preferentially-bound solute destabilizes proteins (Miyawaki, 2007). Further, it was found that the cosolvents of larger dimension compared to solvent should be preferentially excluded from the protein surface due to steric exclusion effect (Timasheff, 2002). In our case, firstly the ovalbumin is a globular protein and secondly the cosolvent (glucose) has larger dimension than solvent (water). Hence the cosolvent molecules would be preferentially-excluded from the ovalbumin surface so as to reduce the extent of denaturation of protein molecules by extremes of pH. Carmen Romero & Alberto Albis (2010) have also analyzed the influence of glucose for the stability of bovine a-lactalbumin in aqueous solution and concluded that linear correlations can be obtained for parameters other than surface tension for globular proteins. Of course, the present work deviates largely from their work in respect of aim, techniques, type of globular protein, etc the present conclusion that glucose can offer a good degree of protection to protein denaturation is confirmed by them.

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