Sucrose-Induced Stabilization of Domain-II and Overall Human Serum

Dec 5, 2018 - These signify that sucrose stabilizes the native state of the protein. Extent of thermal unfolding is also found to be minimized in the ...
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Article Cite This: ACS Omega 2018, 3, 16633−16642

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Sucrose-Induced Stabilization of Domain-II and Overall Human Serum Albumin against Chemical and Thermal Denaturation Sukanta Shil,† Nilimesh Das,* Bhaswati Sengupta,‡ and Pratik Sen*

ACS Omega 2018.3:16633-16642. Downloaded from pubs.acs.org by UNIV OF WINNIPEG on 12/14/18. For personal use only.

Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur, 208 016 Uttar Pradesh, India ABSTRACT: In this contribution, we have compared the stabilizing effect of sucrose on overall as well as on domain-II of human serum albumin (HSA) against unfolding by different denaturating agents. HSA was denatured thermally by raising the temperature and also chemically by guanidine hydrochloride (GnHCl) and urea. Circular dichroism spectroscopy was used to monitor the change in the overall structure of HSA, whereas tryptophan fluorescence has been used to investigate the local structural alteration within the domain-II of HSA. The degree of folding using different combinations of GnHCl−sucrose, urea−sucrose, and temperature-sucrose was investigated to calculate the relative stability of the native, intermediate, and denatured states of HSA with increasing sucrose concentration. In the presence of sucrose, the intermediate state, formed during GnHCl-induced denaturation, populated at higher GnHCl concentration (1.5 M for overall denaturation and 2.5 M for domain-II) compared to that in the absence of sucrose (1 M for overall denaturation and 2 M for domain-II). A similar effect has also been observed for urea. These signify that sucrose stabilizes the native state of the protein. Extent of thermal unfolding is also found to be minimized in the presence of sucrose. In a nut shell, sucrose stabilizes different parts of HSA differently and also the net stabilizing effect toward different denaturation profiles is different.

1. INTRODUCTION Protein is one of the most important biomolecules inside a living body and performs the necessary biological processes in a complex and confined environment.1−4 In such an environment, various forces act on the protein and the net effective force upon the protein determines whether it will be in its native form or in some extended or denatured form.5 Generally, proteins stay as a highly stable folded structure with certain extent of regular motifs.6 This stable state corresponds to the minima of the free-energy surface of the protein, which actually depends on the local environment around the protein.7−11 For example, in the polar medium, a structure with the hydrophilic amino acid residues exposed to outside environment corresponds to a lower energy state, whereas in the nonpolar solvent, the reverse is true.12 The most stable state of the protein in its normal biological environment is known as the native state. There are some specific external reagents that disrupt the protein structure. These are known as chemical denaturants such as urea, guanidine hydrochloride (GnHCl), proton, and so forth. On the other hand, there are also several molecules that can counteract the action of denaturation, for example, polyols, sugars, betaines, and macromolecular crowders.13−18 It has been reported that sugar molecules stabilize the proteins and the extent and mechanism of this stabilization are different for different sugars.13,15,19−21 Application of high temperature and pressure also leads to the perturbation of the native structure of a protein.22−25 © 2018 American Chemical Society

All of the protein functions depend strongly on its structural parameters and hence on its stability. Thus, the study of protein stability remains an interesting field among the researchers. The study of protein stability also holds high importance in understanding the fact of adaptability of some biomolecular mechanism in adverse conditions.26 In the current contribution, we have studied the effect of sucrose, one of the most important members of the sugar community, on the denaturation of human serum albumin (HSA). HSA is a transport protein present in human serum.27 It has a single chain of 585 amino acid residues and is divided into three distinct domains.28,29 It is involved in binding and transporting various kinds of molecules in the cellular system.30−34 In the physiological condition, HSA usually stays in a milieu of salt, sugar, and other biological small and macromolecules in blood having pH 7.4. To understand the effect of adverse condition on the structure and function of HSA, researchers have studied the chemical and thermal unfolding of HSA using several techniques among which the fluorescence of the single tryptophan residue (Trp-214 present in domain-II) is one of the most applied methods. The role of sucrose in the stabilization of proteins has been explored experimentally by several groups including number of reports on HSA.35−45 This is also to note that sucrose is the monomeric unit of the famous macromolecular crowder ficoll. Recently, our group is Received: July 31, 2018 Accepted: November 20, 2018 Published: December 5, 2018 16633

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Figure 1. (a) Some representative CD spectra at different GnHCl concentrations and in the presence of different concentrations of sucrose. (b) Degree of folding of HSA calculated from the CD spectra recorded at different GnHCl concentrations and in the presence of different concentrations of sucrose. The black dots on the contour represent the measured data points.

Table 1. α-Helicity of HSA Calculated from CD Spectra Recorded in the Presence of Different GnHCl and Sucrose Concentrations

engaged in understanding the mechanism by which a crowder exerts its effect on a protein.46 We expect that the present study will be useful in elucidating the mechanism of crowderinduced changes of a protein. In our earlier reports, we have investigated the effect of sucrose on the unfolding of different domains I and III of HSA.36,41 Here, we will focus on the effect of sucrose on the denaturation of domain-II of HSA and compare it with the global denaturation. We have used Trp214 intrinsic fluorescence for studying of domain-II and circular dichroism (CD) spectroscopy to investigate the overall structural alteration.

sucrose concentration (M)

[GnHCl] (M)

2. RESULTS 2.1. CD Spectroscopy. The acquired CD data were analyzed using CDNN software (http://gerald-boehm.de) to get the information about the secondary structural parameters of HSA. In the absence of denaturant at room temperature, the α-helicity of HSA is found to be 65%, which matches well with the previous results.46 With increasing GnHCl concentration, the α-helicity gradually increases up to 73.5% at 1 M GnHCl and with the further increase of GnHCl concentration, the αhelicity value gradually decreases to 8.4% at 5.5 M GnHCl in the absence of sucrose. In the presence of sucrose, the nature of the denaturation curve remains similar. However, at higher sucrose concentration (≥0.6 M), the highest α-helicity value occurs at 1.5 M GnHCl. As for example, in the presence of 1 M sucrose, the denaturation starts with 70% α-helicity and increases to 83% in the presence of 1.5 M GnHCl and then the α-helicity value continuously decreases to 9% for 5.5 M of GnHCl (see Figure 1a and Table 1). Without sucrose, the αhelicity value increases slightly from 65 to 66% in the presence of 1 M urea and with further increase in the urea concentration, the α-helicity value decreases to 12% for 8 M of urea. However, in the presence of sucrose at a concentration greater than 0.4 M, the α-helicity value reaches a maximum value at 2 M. For example, in the presence of 1 M of sucrose, the denaturation profile starts with an α-helicity value of 68.5% and going through a maximum of 72.5% in the presence of 2 M urea, it reaches to 25% in the presence of 8 M urea (see Figure 2a and Table 2). HSA shows an α-helicity value of 66% at 5 °C without the presence of any denaturant or sucrose. Up to 45 °C, the value does not alter significantly. However, with the further increase in temperature, the α-helicity value gradually decreases to 25% at 80 °C. With the addition of sucrose, the slope of the denaturation profile gradually decreases. As for example in the presence of 1 M of sucrose, the denaturation profile spans in the range between 69.5% α-helicity at 5 °C and

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5

0

0.2

0.4

0.6

0.8

1.0

65.3 68.7 73.5 69.3 54.5 43.7 30.7 24.9 20.3 9.9 9.0 8.4

68.1 76.2 79.0 79.7 60.9 51.5 36.6 30.9 25.8 5.1 8.9 11.2

69.1 73.8 83.3 82.4 67.4 60.3 43.0 28.9 11.2 11.6 12.1 10.9

71.2 74.5 78.8 83.0 70.3 50.5 39.1 33.9 15.0 14.1 9.9 9.6

69.6 76.1 80.0 83.0 54.6 41.4 27.5 19.7 10.9 8.2 9.4 7.6

70.1 72.0 79.0 82.8 60.9 42.1 27.7 20.7 11.2 10.0 9.3 9.1

40% α-helicity at 80 °C (see Figure 3a and Table 3). To analyze our data, we have quantified the degree of folding as α − αU fi = i αN − αU (1) where αi is the α-helicity at a particular denaturating condition, αN and αU are the α-helicities of the native state (which is 65% in this case) and of the unfolded state (which is 8% in this case), respectively. With our nomenclature, extent of folding is 1 for native HSA. An increase in the α-helicity value compared to the native HSA will be reflected by the extent of folding greater than 1 and a decrease in the α-helicity value will be reflected by the extent of folding value less than one. By this nomenclature, we can capture the whole story in a uniform way. For instance, in the case of GnHCl-induced unfolding of HSA, the extent of folding, as per our nomenclature, starts from 1 and then first increases to 1.149 (at 1 M GnHCl) and then decreases to 0 (at 5.5 M GnHCl), whereas in the presence of 1 M of sucrose, the profile starts from 1.087 and decreased to 0.057 at 5.5 M GnHCl passing through a maximum of 1.359 at 1.5 M GnHCl (see Figure 1b). Similarly, for urea-induced denaturation, these values of highest and lowest points are 1.032 at 1 M urea and 0.07 at 8 M urea in the absence of sucrose and in the presence of 1 M sucrose, these points are 1.149 and 0.298 at 2 and 8 M urea, respectively (see Figure 2b). In the case of the thermal denaturation of HSA, the extent of folding gradually decreases with increasing temperature (Figure 3b). Thermal denaturation is not as prolific as 16634

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Figure 2. (a) Some representative CD spectra at different urea concentrations and in the presence of different concentrations of sucrose. (b) Degree of folding of HSA calculated from the CD spectra recorded at different urea concentrations and in the presence of different concentrations of sucrose. The black dots on the contour represent the measured data points.

Table 2. α-Helicity of HSA Calculated from CD Spectra Recorded in the Presence of Different Urea and Sucrose Concentrations

Table 3. α-Helicity of HSA Calculated from CD Spectra Recorded at Different Temperatures in the Presence Varying Concentrations of Sucrose

sucrose concentration (M)

urea concentration (M)

0 1 2 3 4 5 6 7 8

sucrose concentration (M)

0

0.2

0.4

0.6

0.8

1.0

65.0 66.2 61.0 56.7 46.9 36.2 25.9 18.1 12.1

65.8 69.2 67.9 61.0 47.8 39.8 33.5 20.3 14.1

66.5 72.1 68.3 63.5 54.5 36.9 28.6 23.0 16.4

68.6 73.2 73.6 71.1 63.2 55.7 39.9 28.3 19.6

69.3 73.3 75.6 71.8 68.0 54.5 44.5 34.5 26.1

66.5 70.7 65.9 59.2 62.0 54.0 40.9 37.4 25.2

temperature (°C)

the other two denaturation profile. In the absence of sucrose, the extent of folding starts from 1.017 at 5 °C and reaches to 0.298 at 80 °C, whereas in the presence of 1 M sucrose, the same starts from 1.096 at 5 °C and reaches to 0.561 at 80 °C. 2.2. Steady-State Spectroscopy. Upon excitation at 295 nm, the single tryptophan in HSA exhibits an emission maximum of 338 nm in the native state. In the presence of sucrose up to 1 M, which remains unchanged. Upon exposure to denaturating condition, the emission maximum shows a significant red shift. This shift is prominent even in the presence of sucrose, although the extent of shift varies with the sucrose concentration (Figures 4−6 and Tables 4−6). Here, to note that the tryptophan emission is only sensitive to its local environment and thus in the present case using the change in

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80

0

0.2

0.4

0.6

0.8

1.0

66.0 65.8 66.1 66.1 65.4 64.5 63.4 62.9 60.8 58.8 54.5 51.5 45.3 37.7 33.7 25.1

67.1 67.1 67.1 65.8 66.3 65.0 64.2 64.6 63.3 61.9 59.2 50.6 43.9 38.5 33.5 29.1

67.7 67.4 68.1 66.8 66.8 65.6 64.7 64.6 62.5 60.3 58.2 55.3 44.5 38.2 32.7 31.4

68.1 67.7 67.1 66.8 66.5 66.2 66.2 64.8 63.9 63.1 61.5 57.2 49.8 44.7 42.0 37.7

68.5 68.2 67.7 67.2 66.7 66.2 65.6 64.9 64.0 62.7 61.2 56.0 50.2 45.6 41.7 39.0

70.4 69.5 68.8 69.2 67.2 68.1 66.9 65.4 65.4 62.6 60.3 56.3 50.9 46.7 43.6 41.0

the tryptophan emission maximum, we are getting the information of only domain-II of HSA. With increasing GnHCl concentration, the emission maxima at first get blue shifted up to a certain concentration of GnHCl to reach 335 nm (blue shifted) and then get red shifted up to 347 nm. The blue shift in the tryptophan fluorescence is referred as the reduction of the polarity of the tryptophan local

Figure 3. (a) Some representative CD spectra at different temperatures and in the presence of different concentrations of sucrose. (b) Degree of folding of HSA calculated from the CD spectra recorded at different temperatures and in the presence of different concentrations of sucrose. The black dots on the contour represent the measured data points. 16635

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Figure 4. (a) Some representative normalized emission spectra of Trp-214 in HSA in the presence of different GnHCl and sucrose concentrations. (b) Degree of folding of HSA calculated from the wavelength maxima of tryptophan recorded at different GnHCl concentrations and in the presence of different concentrations of sucrose. The black dots on the contour graph represent the measured data points.

Figure 5. (a) Some representative normalized emission spectra of Trp-214 in HSA in the presence of different urea and sucrose concentrations. (b) Degree of folding of HSA calculated from the wavelength maxima of tryptophan recorded at different urea concentrations and in the presence of different concentrations of sucrose. The black dots on the contour represent the measured data points.

Figure 6. (a) Some representative normalized emission spectra of Trp-214 in HSA at different temperatures and in the presence of different sucrose concentrations. (b) Degree of folding of HSA calculated from the wavelength maxima of tryptophan recorded at different temperatures and in the presence of different concentrations of sucrose. The black dots on the contour graph represent the measured data points. As the change is not clear from the contour plot, we have inserted an inset where the range of color scale is modified to clearly demonstrate the changes in the emission signal.

maximum of the native state (which is 338 nm in this case) and of the unfolded state (which is 347 nm in this case), respectively. In the presence of urea, the emission maxima show a behavior similar to that in the case of GnHCl, where a decrease in the tryptophan emission maximum was observed followed by an increase. In this case, the maximum blue shift appears at 4 M urea at lower sucrose concentration (till 0.4 M) and at 5 M urea at higher sucrose concentration. At 8 M urea, the emission maximum shows a 7.5 nm red shift from the native state and reaches to 345.5 nm. Figure 5 shows the extent of folding of HSA as a function of urea and sucrose. By comparing Figures 4 and 5, one can clearly conclude that the

environment in the presence of a certain concentration of GnHCl, which depends on the sucrose content. Below 0.4 M sucrose, this blue shift appears at 2.0 M GnHCl, whereas at higher sucrose concentration, it gets shifted to 2.5 M GnHCl. Beyond this concentration, the emission maximum increases steadily and reaches 346 nm at 5.5 M GnHCl irrespective of sucrose concentration. The extent of folding has been calculated for each sample using the following equation. fi =

λimax − λ umax λNmax − λ umax

(2)

where λimax is the emission maximum at a particular denaturating condition and λmax and λmax are the emission N u 16636

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Table 4. Tryptophan Emission Maxima of HSA Recorded in the Presence of Different GnHCl and Sucrose Concentrations sucrose concentration (M) 0

[GnHCl] (M)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5

338.0 337.0 336.5 336.0 335.0 339.5 342.5 346.5 347.0 347.0 346.0 347.0

0.2

0.4

0.6

0.8

1.0

339.0 338.0 337.0 336.5 336.0 340.0 341.0 343.0 345.5 346.0 347.0 347.0

339.0 339.0 338.0 337.0 334.5 338.0 342.0 343.0 346.0 346.5 347.0 347.0

339.0 339.0 338.0 337.0 336.0 334.0 337.0 342.5 343.5 345.0 346.0 346.0

339.0 338.5 338.0 337.0 336.0 334.0 337.5 341.0 343.0 344.5 346.0 346.0

339.0 338.0 337.0 336.0 335.5 334.5 337.5 341.0 343.0 344.0 345.0 346.0

Table 5. Tryptophan Emission Maxima of HSA Recorded in the Presence of Different Urea and Sucrose Concentrations sucrose concentration (M) 0 0 1 2 3 4 5 6 7 8

urea concentration (M)

338.0 337.5 337.5 337.0 334.5 337.0 341.5 343.5 345.5

0.2

0.4

0.6

0.8

1.0

338.0 337.5 338.0 337.0 336.0 337.0 341.5 343.5 343.6

338.0 338.0 338.0 338.0 336.5 336.0 338.0 341.0 345.0

338.0 338.0 338.5 338.5 336.5 334.5 336.5 336.5 345.0

338.0 338.0 338.0 338.0 337.5 334.5 336.0 340.5 345.0

337.5 337.5 337.5 337.0 334.5 334.5 334.5 339.0 345.0

Table 6. Tryptophan Emission Maxima of HSA Recorded at Different Temperatures in the Presence Varying Concentrations of Sucrose sucrose concentration (M) 0

temperature (°C)

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80

338.0 338.0 338.0 338.0 338.0 338.0 338.5 338.0 338.0 335.8 332.5 330.0 329.5 329.0 329.0 329.0

0.2

0.4

0.6

0.8

1.0

338.0 338.0 338.5 338.5 338.0 338.0 338.0 338.5 338.0 337.5 336.5 333.5 330.5 330.5 330.0 329.5

338.0 337.5 338.0 337.5 337.5 337.0 336.5 337.0 337.0 337.0 336.0 332.5 331.5 331.0 330.0 330.0

338.0 338.5 338.5 338.5 339.0 339.0 338.0 338.5 338.5 336.5 336.5 332.0 330.5 330.0 330.0 330.5

337.0 338.0 337.5 337.5 337.5 337.5 338.0 338.0 338.0 338.0 337.5 334.5 332.0 331.0 331.0 330.5

338.0 338.0 338.0 338.0 338.0 338.0 337.5 337.5 337.5 337.0 337.5 334.5 332.0 331.0 331.0 331.0

353 K, which shows the stabilization effect of sucrose. Figure 6 shows the extent of folding of domain-II HSA as a function of temperature and sucrose. In comparison to urea and sucrose, the effect of temperature is less prominent.

effect of sucrose is much profound in the case of urea than that in the case of GnHCl. With increasing temperature, however, an opposite trend has been observed. Unlike chemical denaturation, thermal denaturation does not proceed with a red shift in the emission spectrum. In this case, the emission maximum remains almost constant up to 323 K and with further increase in temperature, it gets blue shifted to reach the value of 329 nm at 353 K in the absence of sucrose. In the presence of high concentration of sucrose (>0.4 M), the emission maxima only reach 331 nm at

3. DISCUSSION As evident from the Results section, in this article, we have investigated the stabilizing effect of sucrose on the denaturation profile of HSA by monitoring the CD signal and tryptophan fluorescence. The change of α-helical content 16637

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presence of an intermediate state at 1 M GnHCl. The presence of such intermediate state in the overall unfolding of HSA by urea is previously reported by Ahmad et al.50 More interestingly, in this study, we have investigated the effect of sucrose on this denaturation profile. First, with the increase in the sucrose concentration, the extent of decrease of α-helicity reduces, which suggests that sucrose is acting as a stabilizing agent. Second, the presence of sucrose modifies the denaturation path by shifting the urea concentration at which the intermediate is formed. The profile of tryptophan emission maxima with increasing urea concentration is somewhat similar to the denaturation profile as caused by GnHCl. In this case also, the denaturation goes through an intermediate state. However, the concentration of urea at which the intermediate state is formed is different than that of GnHCl. For urea-induced denaturation, the intermediate is formed at relatively higher urea concentration (4 M). This fact also demonstrates that for a particular denaturant, different domains of a protein and the overall protein response in a different fashion. Whereas the overall unfolding path of HSA by urea goes through an intermediate state that is most populated at 1 M of urea, the unfolding path of domain-II involves an intermediate most populated at 4 M of urea. However, for both the cases, sucrose modifies the denaturation profile in such a way that the intermediate state is formed at higher urea concentrations. The change of α-helicity is least in the case of thermal denaturation of HSA. Whereas for chemical denaturation, the decrease in the α-helicity is ∼90%, for thermal denaturation, it is only 60%. Moreover, the monotonous decrease of α-helicity in the case of thermal denaturation proves that this denaturation profile does not involve any intermediate state. With increasing sucrose concentration, the slope of the denaturation profile decreases proving the counteraction of sucrose toward thermal denaturation. With increasing temperature, 9 nm of blue shift of the tryptophan fluorescence from 338 to 329 nm is observed, suggesting that the location of Trp214 residue goes to a more hydrophobic environment at higher temperatures. Although this is quite astonishing, there have been some reports on this phenomenon.51−54 The general expectation is that with increasing temperature, the protein will denature and the core (including Trp-214 residue) will be more accessible to water, leading to a red shift in the tryptophan fluorescence. However, it has been concluded that with increasing temperature, the unfolding of HSA occurs in such a way that the Trp-214 residue finds itself at the bottom of a deep narrow opening of domain-II of HSA and the local environment around the Trp-214 is in a more hydrophobic environment.51,53 In the absence of sucrose, 9 nm of blue shift in the tryptophan fluorescence has been observed, whereas at 1 M of sucrose, we have observed only a 7 nm blue shift. Thus, here also, the effect of sucrose is to oppose the effect of temperature. There are several theories regarding the stabilization mechanism of sucrose. Amongst them, the most common ones are (a) mechanical-entrapment hypothesis, where a particular state of protein is entrapped in highly viscous sugar matrix,55 (b) water replacement hypothesis, where hydrogen bonds are formed between sugar and protein, which stabilizes the protein,19,56 (c) water entrapment hypothesis, where water molecules are trapped between protein surface and sugar layer stabilizing the protein molecule,57 and (d) broken glass hypothesis, where sugar and protein interact directly assisted

tells us about the global structural change of HSA, whereas the change of tryptophan fluorescence tells us about the local structural change involving domain-II of HSA. The CD signal of protein is enriched with its secondary structural features such as α-helix, β-turn, β-sheet, and random coil, where α-helix is the most ordered secondary structure and random coil is the least ordered one. For our case, we have used α-helicity to see the structural changes of HSA in the presence of denaturants and sucrose. The increase in α-helicity generally means the increasing compactness of the protein and the decrease in αhelicity is referred to as the unfolding of the protein. For fluorescence measurement, we have used the fact that emission maximum of tryptophan depends greatly on the surrounding environment. Tryptophan as an amino acid shows emission maximum at 355 nm in phosphate buffer (pH 7.0).47 Inside different proteins, the emission maximum of tryptophan is different depending on its location within the protein. Depending upon the solvent exposure, its wavelength maximum may vary between 310 and 355 nm in various protein environments.48 When the tryptophan residue remains buried within the protein matrix, which is usually hydrophobic, the emission maximum is relatively blue shifted and when the tryptophan is located at the more exposed environment, it shows a red-shifted fluorescence. This is because the excitedstate dipole moment of tryptophan is higher than its ground state dipole moment. Therefore, a blue shift in the emission maximum implies the increase of hydrophobic environment around it. This high dependence of tryptophan fluorescence on its environment makes it a good measure of the folding− unfolding of the protein, within its local environment. In the absence of sucrose, the α-helical content shows a maxima at 1 M of GnHCl, suggesting that at 1 M of GnHCl, an intermediate state of HSA exists, which is even more compact than that in the native state. In a previous study by Anand et al., the existence of this intermediate state in the course of GnHCl-induced denaturation of HSA was also reported.49 With further increase in the GnHCl concentration, the helical content decreases and reaches to only 8% in the presence of 5.5 M of GnHCl, suggesting the gradual unfolding of HSA. In the present study, we have investigated the effect of sucrose on this denaturation profile. In the presence of higher sucrose content, the intermediate state is formed at 1.5 M GnHCl. The specific change of domain-II of HSA by GnHCl and sucrose is then studied by the tryptophan fluorescence as stated above. In the present case, native HSA shows emission maxima of 338 nm, suggesting that Trp-214 resides in an environment of intermediate polarity. In the absence of sucrose, with increasing GnHCl concentration, the emission maximum first blue shifted followed by a red shift. The initial decrease of emission maximum till 2 M GnHCl can be ascribed to the formation of an intermediate state at 2 M GnHCl. Understandably, in this state, the tryptophan residue reaches an environment of more hydrophobicity. In the unfolded state, the environment around tryptophan is hydrophilic, which is reflected in the red shift of the emission maximum. In the presence of sucrose, the signature of such intermediate state remains, but it is now observed at a higher GnHCl concentration (2.5 M). Urea-induced denaturation profile of overall HSA is somewhat similar to that of GnHCl-induced denaturation in the sense that in this case also, the α-helical content shows a maximum at 1 M of urea concentration followed by a steady decrease at higher urea concentrations. This suggests the 16638

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Figure 7. Plot of ΔG0 of overall denaturation of HSA as a function of sucrose concentration for (a) GnHCl-induced denaturation, (b) urea-induced denaturation, and (c) thermal denaturation. Here, ΔG01 is for N ⇌ I transition and ΔG02 is for I ⇌ U transition.

Figure 8. Plot of ΔG0 of the denaturation of domain-II of HSA as a function of sucrose concentration for (a) GnHCl-induced denaturation, (b) urea-induced denaturation, and (c) thermal denaturation. Here, ΔG01 is for N ⇌ I transition and ΔG02 is for I ⇌ U transition.

denaturations are fitted with a three-state model. The two and three state models can be represented as

by water entrapment.58 Apart from these hypothesis, there are several reports published on the stabilization of proteins by sucrose.35−45,59−63 Back in 1969, Hinton et al. reported the decreased activity of several enzymes in the presence of sucrose.59 Kulmyrzaev et al. established that sucrose stabilizes the molten globule state of the protein in the course of thermal unfolding, and as a result, the denaturation temperature increases by a certain extent.60 Ruan et al. proposed that the volume change (ΔV) associated with the addition of sucrose leads to the stabilization of the protein against pressureinduced unfolding.61 Kim et al. reported that in the presence of sucrose, the native state of protein becomes more compact and structured.62 In 1981, Timasheff et al. and in 1997 Kendrick et al. published two similar articles to prove that thermodynamic stabilization of proteins by sucrose results from the increased chemical potential in the presence of sucrose because of the sugar’s non-preference for the protein surface.38,63 Because of this effect, the structure having the smallest surface area is the most stable. In the present case, we see the effectiveness of sucrose to resist the effect of thermal and chemical denaturation of HSA. Protein denaturation is a process which is generally accompanied by the opening of the protein secondary structure. In this process, the size of the protein increases and solvent can access more surface area of the protein. The distribution of sucrose (or basically any sugar) in protein solution is such that the concentration of sucrose near the protein surface is lower than that of the bulk.37,38,62,63 Thus, addition of sucrose will affect the folding−unfolding equilibrium of a protein to the direction in which the surface area of the protein is minimum as per the La Chateliar principle. In this way, sucrose counteracts the action of unfolding of HSA by chemical and thermal denaturation. To better understand the unfolding pathway of HSA and its modification by sucrose, we have extracted the thermodynamic parameters related to the transition. The thermal denaturation profiles are fitted with a two-state model, whereas the chemical

NFU

NFIFU where N stands for the native HSA, that is, HSA without the presence of any chemical denaturant or sucrose, I stands for the intermediate state and U stands for the unfolded or denatured state. In order to extract the thermodynamic parameters, we have fitted the variation of α-helicity with denaturant and sucrose concentration as per the following method. Let, Y be the spectroscopic signal at some conditions, whereas YN, YI, and YU are the same type of spectroscopic signals for the native, intermediate, and unfolded states, respectively. For a two-state model, we can write64 Y=

YN + YU × e−x 1 + e −x

(3)

In addition, for a three-state model, the equation becomes64 Y=

YN + YI × e−x + YU × e−y 1 + e −x + e −y

(4)

x and y are defined as x=

(ΔG 0 − m[denaturant]) RT

y=

(ΔG 0 − (m1 + m2)[denaturant]) RT

(5)

(6)

In the above equations, ΔG is the free-energy change associated with the concerned transition, and m denotes the slope of the free-energy change. R and T are the universal gas constant and temperature in K, respectively. Variation of αhelicity will deliver information regarding the overall structural change of HSA under the influence of denaturant and stabilizer and fitting of the tryptophan intensity data will furnish 0

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studied and the relative effect of sucrose on these changes has been summarized and quantified. Real biological environment is very much complexed with the presence of various salts, osmolytes, crowders, amino acids, and so forth. All of them exert their effect on a protein in a complex manner. Some may cause denaturation of the protein, some may be inert and some may be protective in nature. Our study will somewhat help to understand the complexity of the biological system where more than one component exerts their effect simultaneously on some proteins. More studies are intended involving different types of sugars such as, monosachharide, disachharide, trisachharide, polysachharide, and so forth to understand the complete behavior.

information about structural change involving domain-II of HSA. The ΔG0 has been estimated for each of the chemical and thermal denaturation processes for various sucrose concentrations both for overall and domain-II denaturation. For GnHCl- and urea-induced denaturation, we got two ΔG0 values: ΔG01 for N ⇌ I transition and ΔG02 for I ⇌ U transition. For thermal denaturation profile, we got only one ΔG0 as this process does not involve any intermediate state. For the unfolding of overall HSA, the variations of ΔG0s in the presence of different sucrose concentrations are shown in Figure 7. In the case of overall denaturation of HSA by GnHCl, N state is found to be less stable than I state up to 0.4 M of sucrose. Beyond 0.4 M sucrose, we observed that the N state is relatively more stabilized than the I state. However, relative stability of the I and U states is not altered much with the increase in sucrose concentration. In the case of overall denaturation of HSA by urea, the I state gradually becomes more stable than the N and U states with increasing sucrose concentration. On the other hand, the thermal denaturation profile of overall HSA is not much altered in the presence of sucrose. For the unfolding of domain-II of HSA, the variations of ΔG0s in the presence of different sucrose concentrations are shown in Figure 8. As it can be seen clearly, for GnHClinduced denaturation of domain-II of HSA, up to 0.4 M, sucrose stabilizes the N and U states more as compared to the I state, whereas the trend is reversed beyond 0.4 M sucrose. For urea-induced denaturation of domain-II of HSA, on the other hand, the I state is more stabilized by sucrose, up to a concentration of 0.4 M, compared to the N and U states. Beyond this concentration of sucrose, the relative stabilization is similar for all three states. For thermal denaturation, with increasing sucrose concentration, the relative stability of U state gradually increases, though the effect is not as prominent as compared to the chemical denaturation.

5. EXPERIMENTAL SECTION 5.1. Materials and Methods. HSA, GnHCl, and urea were purchased from Sigma-Aldrich and used as received. Analytical grade disodium hydrogen phosphate and sodium dihydrogen phosphate were purchased from Merck, India, and used to prepare phosphate buffer. All solutions were prepared in 50 mM sodium phosphate buffer (pH = 7.4). For steadystate measurements, the protein concentration is kept around 5 μM and for CD measurements, the concentration is kept around 2 μM. Sucrose concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1 M are used for the study. GnHCl concentrations are varied from 0 to 6 M with an interval of 0.5 M for each sucrose concentrations except for 1 M sucrose, where until 5.5 M GNHCl concentration can only be reached. Urea concentrations are varied from 0 to 9 M with an interval of 1 M. In this case also, at highest sucrose concentration, it was impossible to reach up to 9 M urea and hence, the data are recorded up to 8 M only. For thermal denaturation study, a temperature range of 5−80 °C is used and the data are recorded with every 5 °C increment. For the measurement of GnHCl- and urea-induced changes, all of the samples were incubated for 6 h at 5 °C and for thermal denaturation, the samples are prepared with respective sucrose concentration and are incubated for 6 h at 5 °C and then the sample is equilibrated at the desired temperature for 20 min. Emission spectra are recorded by exciting the sample at 295 nm to probe only the tryptophan residue. 5.2. Instrumentation. All of the absorption spectra were recorded in a commercial UV−visible spectrophotometer (UV-2450, Shimadzu, Japan). Steady-state emission spectra were recorded in a commercial fluorimeter (Fluoromax-4, Jobin-Yvon, USA). A commercial CD spectrometer (J-815, Jasco, Japan) was used to measure CD spectra.

4. CONCLUSIONS In this report, the combined effect of a protective reagent (sucrose) and denaturating agents (GnHCl, urea, and temperature) is studied on the unfolding of HSA. We observed a significant role of sucrose to counteract the denaturation effect. This counteraction is perceived in two ways: first, the chemical denaturants and temperature cannot induce much alteration to the protein structure in the presence of sucrose and second, the denaturation profile is modified in such a way that in the presence of sucrose, the intermediate state is formed at higher denaturant concentration. These observations have been explained on the basis of sucrose’s nonpreference for the HSA surface. However, the counteraction ability is found to be different for different denaturation profiles. Here, one point is to note that though temperature is less efficient compared to the chemical denaturants, sucrose brings least change to the thermal denaturation profile. Our result also proves that for a big multidomain protein such as HSA, the stabilizing effect of sucrose is different for the overall protein and domain-II. We have quantified our data by calculating ΔG0 as a function of sucrose concentration for each denaturation profile and predicted the relative stability of not only native and unfolded states but also intermediate state is predicted involved therein. One interesting point to note is that sucrose modifies the domain-II denaturation profile to a greater extent than overall denaturation profile. As a whole, the global and local changes of HSA induced by GnHCl, urea, and temperature have been

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (N.D.) *E-mail: [email protected] (P.S.) ORCID

Pratik Sen: 0000-0002-8202-1854 Present Addresses †

Surface Department, Oil and Natural Gas Corporation, Avani Bhaban, Chandkheda, Ahmadabad 380 005, India. ‡ Department of Chemistry, Pennsylvania State University, 104 Chemistry Building, University Park, PA 16802, USA. Notes

The authors declare no competing financial interest. 16640

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ACKNOWLEDGMENTS S.S., B.S., and N.D. acknowledge Council of Scientific and Industrial Research (CSIR, Government of India) for providing fellowship. This work is financially supported by Science and Engineering Research Board, Government of India (grant number EMR/2016/006555), and Indian Institute of Technology Kanpur.

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