Interaction of Myoglobin with Cationic and Nonionic Surfactant in

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Interaction of Myoglobin with Cationic and Nonionic Surfactant in Phosphate Buffer Media Satyajit Mondal,† Maria Luz Raposo,‡ Gerardo Prieto,‡ and Soumen Ghosh*,† †

Centre for Surface Science, Department of Chemistry, Jadavpur University, Kolkata, 700 032, India Biophysics and Interfaces Group, Department of Applied Physics, Universidade de Santiago de Compostela, 15782 Santiago de Compostela A Coruña, Spain

‡

ABSTRACT: Interaction of myoglobin with cationic surfactants hexadecyltrimethylammonium bromide (HTAB), gemini surfactant 16-2-16 (dimethylene-1,2-bis(hexadecyldimethylammonium bromide)) and nonionic surfactant Mega 10 (N-decanoyl-N-methylglucamine) have been studied in phosphate buffer at pH 7.4 using surface tension, UV−visible, fluorescence, and circular dichroism spectroscopies and differential scanning calorimetry. With increasing concentration of HTAB, metal ion of the heme group changes its spin states; but in case of 16-2-16 and Mega 10, spin change does not occur. Fluorescence spectra clearly denote the unfolding process in HTAB media. With increasing HTAB concentration, α-helicity of myoglobin decreases with the appearance of β-sheet and random coil more rapidly than other two surfactants. Melting temperature of myoglobin is reduced drastically upon interaction with HTAB than their corresponding gemini and nonionic surfactants.

1. INTRODUCTION Protein is an important macromolecule in living systems and it is crucial in all biological processes. Protein can bind with a wide variety of compounds such as fatty acids, phospholipids, surfactants, and drugs.1,2 The interaction of protein with surfactant has been studied for a long time because it has important applications in biosciences, food, drug delivery, and biotechnological processes.3−6 Surfactants are attractive molecules to influence the three-dimensional structure of proteins. Surfactants and proteins both have hydrophilic and hydrophobic portions. The interactions between surfactants and proteins are very complex processes.7 Thus, well-characterized proteins are chosen to study the interaction with surfactants such as myoglobin which is suitable for many spectroscopic techniques.8,9 Myoglobin is a monomeric water-soluble heme protein found in muscle tissue. Myoglobin is a surface active protein. It contains two tryptophan, two tyrosine, seven phenylalanine residues, and one prosthetic group. It works as an intracellular storage site for oxygen. During periods of oxygen deprivation it releases bound oxygen which is used for metabolic purposes. Myoglobin is water-soluble globular protein which contains 153 amino acids residues. It has eight alpha helices and a hydrophobic core. Each myoglobin molecule contains one prosthetic group which is inserted into a hydrophobic cleft in protein.9 Each heme residue contains one central coordinately bound iron atom which is normally in the (2+) oxidation state. The oxygen carried by myoglobin is bound directly to the ferrous iron atom. HTAB is a single chain cationic surfactant, whereas 16-2-16 is a cationic gemini surfactant. Gemini is a dimeric surfactant © XXXX American Chemical Society

consisting of two identical amphiphilic moieties which are linked by a spacer group at the head groups position.1,9,10 The spacer group increases the hydrophobicity of the gemini surfactant, so the critical micelle concentration (CMC) of this surfactant is much lower than that of its monomeric analogue.11 Mega 10 is a sugar-based nonionic surfactant. It has a hydrophilic sugar moiety and a hydrophobic alkanoyl chain.12 Mega 10 is biodegradable and it is widely used in cosmetics, food, and cleaning products. In this paper we have studied the interaction between myoglobin and the above three surfactants individually using surface tension, UV−visible spectroscopy, fluorescence spectroscopy, circular dichroism (CD), and differential scanning calorimetry (DSC).

2. MATERIALS AND METHODS 2.1. Materials. Horse myoglobin is purchased from Sigma and used without further purification. Hexadecyl trimethylammonium bromide (HTAB) is obtained from Alfa Aesar (UK); gemini 16-2-16 (dimethylene-1,2-bis(hexadecyldimethylamonium bromide)) is a gifted sample, and Mega 10 is purchased from Sigma. All the surfactants are 99% pure. Sodium dihydrogen phosphate and sodium phosphate are obtained from S.D. Fine-Chem Ltd. and Loba Chemie, respectively. All experiments have been performed at constant temperature in a water bath maintained at 300 ± 0.05 K. Received: October 9, 2015 Accepted: January 20, 2016

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DOI: 10.1021/acs.jced.5b00858 J. Chem. Eng. Data XXXX, XXX, XXX−XXX

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2.2. Preparation of Buffer Solution. Phosphate buffer solution at pH 7.4 is prepared by mixing sodium dihydrogen phosphate and sodium phosphate solutions using standard protocol. Ionic strength of the buffer solution is 0.236 m. All the protein and surfactant solutions are prepared in this buffer medium. 2.3. Methods. 2.3.1. Tensiometry. Surface tension measurements are taken in a calibrated du Noüy Tensiometer (Krüss, Germany) by ring detachment technique. A 5 mL sample of myoglobin solution in phosphate buffer, pH = 7.4 is taken in a container and concentrated surfactant solution in the same buffer is added stepwise using Hamilton microsyringe as required. The extent of dilution of myoglobin solution during the experiment is 2−3%. Measurements are taken allowing 15 min intervals for equilibration. Accuracy of surface tension value is within ±0.1 mN m−1. 2.3.2. UV−Visible Absorption Studies. Absorbance measurements have been performed in a UV 1601 Shimadzu (Japan) spectrophotometer using 10 mm path length quartz cuvette. The spectra have been measured in 250−700 nm wavelength range. A 2.5 mL sample of 0.005 g% myoglobin solution of fixed pH is taken in the cuvette, and then a concentrated surfactant solution in the same buffer is added stepwise, using Hamilton microsyringe, varying from a lower to a higher value than that of critical micelle concentration or CMC. The extent of dilution of myoglobin solution during the experiment is 2− 3%. The absorbance intensity is measured at 409 nm wavelength. 2.3.3. Fluorescence Emission Studies. The spectra and intensity of fluorescence emission of myoglobin have been measured in a PerkinElmer LS 55 fluorescence spectrometer using a 10 mm path length quartz cuvette. Fluorescence spectra are recorded from 300 to 450 nm with excitation and emission slit widths fixed at 9 and 4 nm, respectively. The excitation wavelength is 280 nm. A concentrated surfactant solution in the same buffer is stepwise added using a Hamilton microsyringe and the emission spectra are recorded after excitation. The extent of dilution of myoglobin solution during the experiment is 2−3%. The scan time is fixed at 250 nm per minute. The fluorescence intensity is measured at 339 nm wavelength. 2.3.4. Circular Dichroism (CD). Far-UV circular dichroism experiments are performed using JASCO J-815 CD spectrometer attached to a water bath to control the temperature of the electronic circuit. A 2.5 mL sample of 0.0025% myoglobin solution is taken in the cuvette of 10 nm path length for measuring the CD spectra in the range between 200 to 250 nm. The scan speed is 50 nm per minute. Then concentrated surfactant solution in the same buffer is added stepwise using a Hamilton microsyringe. The extent of dilution of myoglobin solution during the experiment is 2−3%. 2.3.5. Differential Scanning Calorimetry (DSC). DSC experiments were done with a VP-DSC (MicroCal Inc., Northampton, MA) calorimeter using 0.542 cm3 twin cells for the reference and sample solutions. Initially, the samples and the references were degassed under vacuum condition while being stirred. Thermograms were plotted between the temperatures of 15 and 100 °C at a scan rate of 60 °C per hour. Each experiment was repeated thrice to create a good reproducibility. The baseline reference, obtained with both cells filled with water, was subtracted from the thermograms of the samples.

3. RESULTS AND DISCUSSION 3.1. Surface Tension. Initially, surface tension of the solution decreases owing to adsorption of the amphiphile at the air/solution interface, and after complete saturation of the interface, a micelle is formed, and the surface tension value is more or less constant. The CMC and the surface tension at the CMC for pure surfactants were determined from the break point in the surface tension versus logarithm of surfactant concentration profile. Myoglobin has pronounced surface activity, and its presence reduces surface tension. The profile for the surface tension versus log[surfactant] plot for myoglobin−surfactant solution exhibited three discontinuities in the case of HTAB shown in Figure 1a. A sharp break of the interaction profile is obtain at the low [HTAB] regime which corresponds to the critical aggregation concentration or CAC, representing the monomeric HTA+ adsorption on the oppositely charged protein backbone because of electrostatic interaction. After CAC, a more or less constant surface tension region is observed until the protein backbone is saturated with respect to surfactant adsorption and the corresponding concentration is called the protein saturation concentration or Cs. Beyond this, the activity of monomeric surfactant starts to increase again resulting in a decrease of surface tension with further addition of HTAB to the mixture, and eventually, an inflection marks the appearance of “free” micelles. This concentration is known as extended CMC or CMCe which is higher than the CMC of pure HTAB solution. Such types of curvatures are observed for the systems of pepsin−HTAB,13 NaCMC−HTAB,14 myoglobin−gemini surfactants,9 lysozyme−SDS,15 and many other systems.16−25 Figure 1b shows the surface tension curves of pure 16-2-16 as well as that in the presence of myoglobin, where the characteristic trend of the mixture is different to that noted previously for HTAB. Some surface tension change of the solution in the premicellar region is observed, but the presence of inflection points cannot be assessed with surface tensiometry. In the case of the myoglobin-Mega 10 system, the initial surface tension value is lower than that of Mega 10 itself in the buffer medium, indicating the surface activity of myoglobin. At high concentration of Mega 10, the surface tension value of the solution remained more or less constant, indicating that Mega 10 cannot interact with the myoglobin at this concentration range [25] shown in Figure 1c. Thus, initial break or CAC signifies the starting of interaction of Mega 10 with protein. The CMC values of the surfactants are listed in Table 1. 3.2. UV−Visible Absorption Spectra. The UV−vis spectra provide the important information about the heme environment of myoglobin. Figure 2a shows the absorption spectra of myoglobin with an increase of [HTAB]. The bands of the spectra of myoglobin appear at 409, 505, 544, and 635 nm. These bands indicate the presence of a six-coordinated high-spin heme having a histidine residue and a water molecule attached at the fifth and the sixth coordinated positions of the iron atom, respectively.26 With the addition of HTAB, intensity of the mixture decreases and the band shifts from 409 to 400 nm. At high concentration of HTAB, the spectra give maxima at 400 and 607 nm. This signifies the formation of 5coordinated high spin species attached with a hydroxyl group as the fifth ligand,8,9 since the Fe−histidine bond is broken here. With increasing [HTAB], absorbance decreases rapidly owing to the binding of the monomeric HTA+ on the oppositely charged myoglobin backbone. After 0.3 mM concentration, B

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Figure 2. (a) Absorption spectra of 0.005 g% myoglobin solution at increasing concentration of HTAB in phosphate buffer at pH = 7.4. Inset A shows the long wavelength region for better visualization and inset B shows absorbance vs [HTAB] plot of 0.005 g% myoglobin solution in phosphate buffer at pH = 7.4. (b) Absorbance vs wavelength plot spectra of 0.005 g% myoglobin solution with increasing concentration of 16-2-16 in phosphate buffer at pH = 7.4. Inset shows absorbance vs [16-2-16] plot of 0.005 g% myoglobin solution in phosphate buffer at pH = 7.4. (c) Dependence of absorption spectra of 0.005 g% myoglobin solution at increasing [Mega 10] in phosphate buffer at pH = 7.4, Inset shows absorbance vs [Mega 10] plot of 0.005 g% myoglobin solution in phosphate buffer at pH = 7.4.

Figure 1. Tensiometric profiles of (a) pure HTAB and 0.03 g% myoglobin−HTAB system, (b) pure 16-2-16 and 0.03 g% myoglobin− 16-2-16 system, (c) pure Mega 10 and 0.03 g% myoglobin−Mega 10 combination in phosphate buffer at pH = 7.4.

Table 1. Critical Micelle Concentration (CMC) Values of Surfactants in Different Media CMC (mM) surfactant

pure water

phosphate buffer

0.03 g% myoglobin

HTAB 16-2-16 Mega 10

0.80 0.032 6.18

0.48 0.028 6.18

1.76a 0.028 6.18

a

CMCe of HTAB in the presence of 0.03 g% myoglobin. Uncertainty of CMC is ±0.1 mM.

absorbance becomes constant over the concentration of surfactant. The break point denotes the unfolding of protein. In the case of the myoglobin−16-2-16 system, absorption maxima at 409 nm decreases with increasing concentration of C

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gemini surfactants, but no shifting occurs as shown in Figure 2b. With increasing concentration of 16-2-16, all the bands of myoglobin except the band at 409 nm disappear. This indicates that 16-2-16 cannot change the spin state of the metal ion of the heme group, which remains in the six-coordinated high-spin state. 8 With the increase in surfactant concentration, absorbance decreases linearly without any break point. This indicates that the binding of surfactant occurs with protein molecule as intensity decreases, but we cannot detect the concentration where the actual unfolding of protein starts due to lack of any clear break point. Hence it can be concluded that 16-2-16 has a weak interaction with myoglobin. In the case of the myoglobin−Mega 10 combination, with increasing [Mega 10] absorbance decreases but all bands remain at the same position shown in Figure 2c. This suggests that myoglobin has the six-coordinated high-spin heme with a histidine residue, and a water molecule bound at the fifth and sixth coordinated positions of the iron atom remains at the same positions after the gradual addition of Mega 10. A plot similar to the absorbance vs [16-2-16] is observed where absorbance decreases linearly with [Mega 10] without any break point. This indicates that protein−surfactant cooperative interaction occurs, but as there is no break point, the actual concentration at which the unfolding of protein occurs cannot be detected. Thus, we conclude that myoglobin has a weak interaction with nonionic surfactant Mega 10. 3.3. Fluorescence Spectra. Myoglobin has two tryptophan, two tyrosine, and seven phenylalanine residues.27 Tryptophan residues dominate the fluorescence of myoglobin, because tyrosine can be quenched in the presence of tryptophan. Figure 3a represents the fluorescence spectra of myoglobin with increasing concentration of HTAB. The effect of adding HTAB causes a large increase of fluorescence intensity, together with a gradual red shift of maximum from 339 to 347 nm. The rapid increase in intensity is due to strong binding of HTAB with protein by electrostatic interaction between surfactant head groups (CTA+) and oppositely charged amino acids and by hydrophobic interaction between surfactant tails and nonpolar amino acids. The binding of surfactant usually leads to protein unfolding because the hydrophobic amino acids no longer need to reside in the protein core away from water. After 0.6 mM concentration of HTAB, fluorescence intensity attains a limiting value denoting that binding between surfactant and protein becomes saturated at and after this concentration. In the case of the myoglobin−16-2-16 system, fluorescence intensity increases with increasing concentration of surfactant with a gradual red shift of spectra from 339 to 347 nm shown in Figure 3b. Red shifting of spectra denotes the binding. But the rate of increase of intensity is not so rapid like that of HTAB. This implies that the binding of 16-2-16 with myoglobin is not so strong compared to HTAB. At low concentration, intensity remains almost constant signifying that binding does not occur in this region. Since intensity increases linearly, we cannot detect the saturation point. This also supports the weak binding tendency of this gemini surfactant to the protein. In the case of myoglobin−Mega 10 system, fluorescence intensity remains almost constant in the low concentration region of Mega 10 which signifies that at this region, the number of Mega 10 molecules is not sufficient to bind with myoglobin. At this concentration, no shift in spectra is also observed. It also supports that protein does not denature in this concentration. After 4 mM concentration, the spectra are red-

Figure 3. (a) Fluorescence spectra of 0.005 g% myoglobin solution with increasing [HTAB] in phosphate buffer at pH = 7.4. Inset shows fluorescence vs [HTAB] plot of 0.005 g% myoglobin solution in phosphate buffer at pH = 7.4. (b) Dependence of fluorescence spectra of 0.005 g% myoglobin solution at increasing [16-2-16] concentration in phosphate buffer at pH = 7.4. Inset shows fluorescence vs [16-2-16] plot of 0.005 g% myoglobin solution in phosphate buffer at pH = 7.4. (c) Fluorescence vs wavelength plot of 0.005 g% myoglobin solution with increasing [Mega 10] in phosphate buffer at pH = 7.4. Inset shows fluorescence vs [Mega 10] plot of 0.005 g% myoglobin solution in phosphate buffer at pH = 7.4.

shifted from 339 to 345 nm and intensity increases gradually as shown in Figure 3c, indicating that binding between protein and Mega 10 occurs. This binding is also weak, because the saturation point cannot be detected as intensity increases linearly. D

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3.4. Circular Dichroism (CD). The CD spectra are scanned to study the transition in backbone structure of amide linkage of protein. Figure 4a shows the CD spectra of myoglobin in the

of myoglobin decreases as negative ellipticity at 210 and 222 nm decreases. This type of observation is studied for trypsin− SDS,3,29 papain−SDS,3,29 pepsin−HTAB,13 myoglobin−gemini surfactant,9 BSA−gemini surfactant, etc.30 At a very high concentration of HTAB, the peak at 210 nm disappears. This is due to the binding of surfactant on the protein surface which leads the protein to an extended disordered structure with exposed hydrophobic residues and thus initiates unfolding.31−33 At high concentration, HTAB breaks the helical structure of the protein, and a β-sheet and random coil appear. We have calculated the percentage of the helicity of the protein using CDNN software (Table 2). Table 2. Calculation of Helicity of 0.0025 g% Myoglobin in Different Surfactant Concentrations in Phosphate Buffer at pH = 7.4a HTAB [surfactant] (mM)

α-helix (%)

β-sheet (%)

random coil (%)

0.0 0.03 0.09 0.19 0.29 0.59

81.90 75.02 68.27 56.17 54.33 36.38

10.60 13.96 18.52 25.32 26.19 33.83

7.50 11.02 13.21 18.51 19.48 29.79

[surfactant] (mM)

α-helix (%)

β-sheet (%)

random coil (%)

0.0 0.002 0.009 0.028 0.045 0.061 0.069

81.90 79.98 79.48 75.27 73.14 67.60 65.79

10.60 11.74 11.92 14.13 14.80 17.45 18.67

7.50 8.28 8.60 10.60 12.06 14.95 15.54

[surfactant] (mM)

α-helix (%)

β-sheet (%)

random coil (%)

0.0 0.15 0.29 1.76

81.90 81.35 80.95 20.79

10.60 11.10 11.35 35.93

7.50 7.55 7.70 43.28

16-2-16

Mega 10

a

Uncertainty in result is ±1%.

In the case of the 16-2-16/myoglobin system, with increasing surfactant concentration, α-helicity of myoglobin decreases as negative ellipticity at 210 and 222 nm decreases, but the peak at 210 nm does not disappear (dissimilar to the myoglobin− HTAB system) shown in Figure 4b. From this observation, it is concluded that the binding of gemini surfactant on protein surface leading to unfolding of proteins is not as strong as that of HTAB. In the case of the myoglobin−Mega 10 system up to 0.3 mM concentration of Mega 10, the CD spectra have no significant difference from that of the myoglobin alone. This indicates that no change occurs in the secondary structure of myoglobin as well as no interaction between Mega 10 and myoglobin. With the further addition of Mega 10, the peak at 210 nm completely disappears and proteins are converted mostly into β-sheet and random coil structure shown in Figure 4c. This signifies that at concentrations higher than 0.3 mM Mega 10 binds strongly with myoglobin which leads to the unfolding of the protein structure.

Figure 4. CD spectra of 0.0025 g% myoglobin solution in phosphate buffer at pH = 7.4 in concentration (mM) of (a) HTAB of (1) 0.0, (2) 0.03, (3) 0.09, (4) 0.19 (5) 0.29, (6) 0.59; (b) 16-2-16 of (1) 0.0, (2) 0.002, (3) 0.009, (4) 0.028, (5) 0.045, (6) 0.061, (7) 0.069; (c) Mega 10 of (1) 0.0, (2) 0.15, (3) 0.29, (4) 1.76.

presence of HTAB in phosphate buffer. The spectra exhibit two negative peaks at 210 and 222 nm which are characteristic of αhelical structure of protein.5,7,10,28,29 The spectra are scanned in the 200−250 nm wavelength regions which is the far-UV region. We have analyzed these spectra quantitatively using CDNN software. Up to 0.03 mM concentration of HTAB, the CD spectra have no significant difference from the myoglobin alone. At this concentration, myoglobin retains its natural secondary structure. Then with increasing [HTAB], α-helicity E

DOI: 10.1021/acs.jced.5b00858 J. Chem. Eng. Data XXXX, XXX, XXX−XXX

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3.5. Differential Scanning Calorimetry (DSC). DSC is primarily used to determine the energetics of phase transitions, conformational changes, and quantification of temperature dependence of protein. Here, all DSC experiments were performed at low and narrow range of concentrations of HTAB. In this range, the peak corresponding to the transition appears which signifies that the binding of the monomeric HTA+ on the oppositely charged myoglobin backbone occurs. The concentration of protein used was 0.05 g% in phosphate buffer medium of pH 7.4. The DSC data were fitted to a simple 2-state unfolding model to give thermodynamic data for the thermally induced transition. By this model, the enthalpy of unfolding and also the peak of the transition are estimated (Figure 5a).34,35

There are two different zones in the temperature of transition versus concentration of HTAB plot (shown in Figure 5a). Zone 1 is ranging from 0 to 0.15 mM and zone 2 is ranging from 0.15 to 0.4 mM. In the first zone, the protein is in the native state. Here, the Tm does not change much. In the second zone, the Tm drops almost 10 °C which implies that the protein is in a partially unfolded state. At higher concentrations (>0.4 mM) (data not shown), the protein is in an unfolded state. The transition is around of the concentration of 0.15 mM of HTAB. The enthalpy value (ΔH) determined is close to 1.2 kcal/mol in absence of HTAB, whereas that in the presence of 0.4 mM of HTAB is 0.45 kcal/mol. Table 3 shows ΔH values of the system in detail indicating the decreasing trend of ΔH with increasing concentration of HTAB. Table 3. ΔH Values of 0.05 g% Myoglobin in Different Surfactant Concentrations in Phosphate Buffer at pH = 7.4a HTAB

Mega 10

[surfactant]/mM

ΔH kcal/mol

[surfactant]/mM

ΔH kcal/mol

0.008 0.03 0.040 0.050 0.065 0.085 0.100 0.120 0.140 0.160 0.180 0.200 0.220 0.240

1.08 1.04 0.98 0.99 0.99 0.96 0.91 0.91 0.84 0.82 0.80 0.76 0.69 0.66

0.24 0.49 0.73 0.98 1.40 1.63 1.95 2.44 2.76 2.90 3.40 3.60 3.90 4.22

98.0 107.0 79.0 89.0 90.0 91.0 110.0 88.0 88.6 95.0 78.0 82.0 35.0 48.0

In the case of 16-2-16, ΔH does not change, and so data for 16-2-16 is not shown. Standard deviation of ΔH is ±0.005 kcal/mol.

a

There is no significant interaction between Myoglobin and 16-2-16. The Tm does not change too much. Probably, under this situation, the state of protein is similar to the native one. In the case of Mega 10, up to 1 mM concentration of it, there is no significant change in transition temperature; so the protein remains in the native state in this region (shown in Figure 5c). After that concentration, transition temperature of the solution drops and it continues up to 4 mM concentration of Mega 10 (5 °C) signifying that protein is partially unfolded. After 4 mM concentration, the transition peak disappears and therefore, no Tm is observed, so the protein becomes completely unfolded in this region (not shown in figure). The ΔH value in the presence of 4 mM of Mega 10 is close to 37 kcal/mol.

4. CONCLUSION The comparative interaction of myoglobin with two cationic and one nonionic amphiphiles has been investigated here. Myoglobin develops conformational changes in the presence of surfactants that are explained in terms of protein unfolding. It has surface activity. The UV−vis spectra give the information about the heme environment of the myoglobin. With increasing concentration of HTAB, the metal ion of the heme group changes its spin states, but in 16-2-16 and Mega 10 media, spin does not change. Fluorescence intensity increases with

Figure 5. (a) Plot of temperature of transition of 0.05 g% myoglobin as a function of concentration of HTAB in phosphate buffer at pH = 7.4. (b) Plot of temperature of transition of 0.05 g% myoglobin as a function of concentration of 16-2-16 in phosphate buffer at pH = 7.4. (c) Plot of temperature of transition of 0.05 g% myoglobin as a function of concentration of Mega 10 in phosphate buffer at pH = 7.4. F

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alkyltrimethylammonium bromides: A detailed physicochemical study with a critical analysis. J. Colloid Interface Sci. 2007, 307, 543− 553. (13) Chakraborty, T.; Chakraborty, I.; Moulik, S. P.; Ghosh, S. Physicochemical studies on pepsin-CTAB interaction: energetics and structural changes. J. Phys. Chem. B 2007, 111, 2736−2746. (14) Chakraborty, T.; Chakraborty, I.; Ghosh, S. Sodium carboxymethylcellulose-CTAB interaction: a detailed thermodynamic study of polymer-surfactant interaction with opposite charges. Langmuir 2006, 22, 9905−9913. (15) Green, R. J.; Su, T. J.; Joy, H.; Lu, J. R. Interaction of lysozyme and sodium dodecyl sulfate at the air-liquid interface. Langmuir 2000, 16, 5797−5805. (16) James, J.; Ramalechume, C.; Mandal, A. B. Self-diffusion studies on PEO−PPO−PEO triblock copolymer micelles in SDS micelles and vice versa using cyclic voltammetry. Chem. Phys. Lett. 2005, 405, 84− 89. (17) James, J.; Vellaichami, S.; Krishnan, R. S. G.; Samikannu, S.; Mandal, A. B. Interaction of poly (ethylene oxide)−poly (propylene oxide)−poly (ethylene oxide) triblock copolymer of molecular weight 2800 with sodium dodecylsulfate (SDS) micelles: some physicochemical studies. Chem. Phys. 2005, 312, 275−287. (18) Mandal, A. B.; Ramesh, D. V.; Dhar, S. C. Physico-chemical studies of micelle formation on sepia cartilage collagen solutions in acetate buffer and its interaction with ionic and nonionic micelles. Hydrodynamic and thermodynamic studies. Eur. J. Biochem. 1987, 169, 617−628. (19) Rose, C.; Mandal, A. B. The interaction of sodium dodecyl sulfate and urea with cat-fish collagen solutions in acetate buffer: hydrodynamic and thermodynamic studies. Int. J. Biol. Macromol. 1996, 18, 41−53. (20) Jayakumar, R.; Mandal, A. B.; Manoharan, P. T. Micelle formation of Boc-Val-Val-lle-OMe tripeptide in chloroform and its conformational Analysis. J. Chem. Soc., Chem. Commun. 1993, 853− 855. (21) Mandal, A. B.; Jayakumar, R. Aggregation, hydrogen bonding and thermodynamic studies on tetrapeptide micelles. J. Chem. Soc., Faraday Trans. 1994, 90, 161−165. (22) James, J.; Mandal, A. B. Micelle formation of Tyr-Phe dipeptide and Val-Tyr-Val tripeptide in aqueous solution and their influence on the aggregation of SDS and PEO−PPO−PEO copolymer micelles. Colloids Surf., B 2011, 84, 172−180. (23) James, J.; Ramalechume, C.; Mandal, A. B. Two-dimensional surface properties of PEO−PPO−PEO triblock copolymer film at the air/water interface in the absence and presence of Tyr-Phe dipeptide, Val-Tyr-Val tripeptide, SDS and stearic acid. Colloids Surf., B 2011, 82, 345−353. (24) Devi, L. B.; Mandal, A. B. BSA can form micelle in aqueous solution. J. Surface Sci. Technol. 2015, 31, 21−29. (25) Chakraborty, T.; Chakraborty, I.; Moulik, S. P.; Ghosh, S. Physicochemical and conformational studies on BSA-surfactant interaction in aqueous medium. Langmuir 2009, 25, 3062−3074. (26) Eaton, W. A.; Hochstrasser, R. M. Single-crystal spectra of ferrimyoglobin complexes in polarized light. J. Chem. Phys. 1968, 49, 985−995. (27) Dautrevaux, M.; Boulanger, Y.; Han, K.; Biserte, G. Structure covalente de la myoglobine de cheval. Eur. J. Biochem. 1969, 11, 267− 277. (28) Adler, A. J. Methods in Enzymology; Academic Press: New York, 1973; Vol. 27. (29) Ghosh, S. Interaction of trypsin with sodium dodecyl sulfate in aqueous medium: a conformational view. Colloids Surf., B 2008, 66, 178−186. (30) Li, Y.; Wang, X.; Wang, Y. Comparative studies on interactions of bovine serum albumin with cationic Gemini and single chain surfactants. J. Phys. Chem. B 2006, 110, 8499−8505. (31) Sun, C.; Yang, J.; Wu, X.; Huang, X.; Wang, F.; Liuh, S. Unfolding and refolding of bovine serum albumin induced by cetylpyridinium bromide. Biophys. J. 2005, 88, 3518−3524.

increasing surfactant concentration. Fluorescence spectra clearly denote the unfolding process in HTAB, but in gemini and Mega 10, it cannot detect the saturation point. The CD spectra give information about the helical structure of the protein. With increasing concentration of surfactant, α-helicity of myoglobin decreases and β-sheet and random coil appear. In the case of HTAB, the helicity change is more than that of 162-16 and Mega 10. DSC data show that protein is partially unfolded in the presence of HTAB and Mega 10. Thus, we conclude that a cationic surfactant has a stronger interaction with myoglobin than their corresponding gemini surfactant. The nonionic surfactant has a weak interaction with myoglobin.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Funding

S.M. acknowledges Senior Research Fellowship from UGC and Dr. Suman Das for giving technical support of Circular Dichroism. S.G. thanks India-Pein Programme for pursuing this work. G.P. and M.L.R. thank the Spanish “Ministerio de Economı ́a y Competitividad” (Project MAT2011-26330) and the “European Regional Development Fund (ERDF)” for financial support. Notes

The authors declare no competing financial interest.

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DOI: 10.1021/acs.jced.5b00858 J. Chem. Eng. Data XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jced.5b00858 J. Chem. Eng. Data XXXX, XXX, XXX−XXX