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H2O system
Journal of Colloid and Interface Science 328 (2008) 153–157
Contents lists available at ScienceDirect
Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Distribution and structural behavior of hemoglobin between the two phases in SDS/n-C5 H11 OH/H2 O system Yuan Chen, Rong Guo ∗ School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou, 225002, People’s Republic of China
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 9 May 2008 Accepted 30 August 2008 Available online 4 September 2008
In the sodium dodecyl sulfate (SDS)/n-pentanol (n-C5 H11 OH)/water (H2 O) system, LL is the coexisting region of W/O and O/W or bicontinuous microemulsions. In the LL region, the system separates into two phases (the upper phase and the lower phase) spontaneously. The distribution and structural behavior of hemoglobin (Hb) between the upper and lower phases in SDS/n-C5 H11 OH/H2 O system were studied by UV–vis and circular dichroism (CD) spectroscopy. The results indicate that different structures of the two phases bring the conformational change of Hb. The addition of Hb results in the redistribution of SDS between the upper and lower phases. © 2008 Published by Elsevier Inc.
Keywords: SDS Hemoglobin Circular dichroism Conformational change
1. Introduction It has been well recognized that liquid–liquid interface plays an essential role in biological systems, particularly in biomedical engineering, pharmacology, and food processing [1]. Adsorption and reaction at the liquid/liquid interface have recently become attractive subjects in fundamental studies of solvent extraction of metal ions, and the detection of liquid-membrane separation, ion-selective electrodes, optical sensors and counter current chromatography [2]. Whether from the theory or practice view, the investigation on behavior of amphiphilic or bioactive molecules at liquid–liquid interfaces has been a subject of scientific interest for a long time. Interfaces modified by amphiphilic molecules are of great fundamental and technological relevance in areas including enhanced oil recovery, drug delivery, bio-sensor, extraction, nanoparticle formation, lubrication and in the production of biomimetic materials [3–10]. The liquid–liquid interface formed by the surfactant aggregation, such as the W/O–O/W interface or W/O– BI interface, can be used to mimic biological membrane since it provides a hydrophobic–hydrophilic medium. We have studied the distribution and transfer of l-phenylalanine [11] and l-tryptophan [12] through the water-in-oil (W/O)–oil-in-water (O/W) interface and W/O–BI (bicontinuous) interface in SDS/n-C5 H11 OH/H2 O system by means of UV–vis spectroscopy and AC impedance. The results indicate that the transfer and distribution of biologically active substance between the inside and outside of the membrane can be adjusted by constitutes of the upper and lower phases, and provide theoretical basis for the behavior of the biologically active
*
Corresponding author. Fax: +86 514 7311374. E-mail address: [email protected] (R. Guo).
0021-9797/$ – see front matter doi:10.1016/j.jcis.2008.08.058
©
2008 Published by Elsevier Inc.
substance across membrane transport, and the design of functional biomaterials. Hemoglobin (Hb) has a molecular weight of approximately 67,000 and contains two α and two β subunits, each of which has one redoxiron heme as its prosthetic group; the heme is located in crevices at the exterior of the subunit [13,14]. Proteins interact with the lipid bilayer by binding or adsorption to its surface, insertion into the hydrocarbon region of membrane interior or penetration through the membrane. In this paper, the distribution and structural behavior of Hb between W/O phase (the upper phase) and O/W or bicontinuous phase (the lower phase) in the SDS/n-C5 H11 OH/H2 O system was determined by UV–vis and circular dichroism spectrum. The results will provide some useful information with respect to the penetration and transfer of the biological active substance across the biological membrane interface. 2. Materials and methods 2.1. Reagents Sodium dodecyl sulfate (SDS, Sigma, >98%) was recrystallized twice in ethanol to ensure that its surface tension had no lowest point around the critical micelle concentration (cmc) by the platinum ring method. n-Pentanol (n-C5 H11 OH, Aldrich, 99%), hemoglobin (Sinopharm Chemical Reagent Co., Ltd., China). Double distilled water was used throughout. 2.2. UV–vis spectra measurement A series of SDS/n-C5 H11 OH/Hb/H2 O solutions were prepared kept at 25.0 ± 0.1 ◦ C for 10 h to reach the phase equilibrium, and
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then separated into two phases after the equilibrium. The absorption spectra of the upper and lower phases were recorded using a UV-2550 spectrophotometer (Shimadzu Corporation, Japan) in the range of wavelengths 200-700 nm by using the blank reagent as reference. 2.3. CD spectroscopic study Circular dichroism (CD) measurement was carried out with Jasco-810 spectropolarimeter (Japan). The CD spectra were measured in 190–300 nm at room temperature using 0.1 cm quartz cuvette. Scanning speed 100 nm/min; bandwidth 1 nm; spectral response 0.1 nm. The data points were taken on an average of four. The contents of the α -helicity, β -sheet structures in protein were calculated using the software provided by the manufacturer (Jasco-810 Analytical Manager System) corresponding to standard substances. All the experiments were carried out at 25.0 ± 0.1 ◦ C. In order to attain equilibrium, the samples were placed in a water thermostat at 25.0 ± 0.1 ◦ C for 10 h before the experiments.
Fig. 1. Partial phase diagram of the SDS/n-C5 H11 OH/H2 O system at 25 ◦ C. Total SDS content: a–a : 1.0%; b–b : 2.0%; c–c : 3.0%; d–d : 4.0%; e–e : 5.0%.
3. Results and discussion 3.1. Distribution of SDS between the upper and lower phases The distribution constant ( K SDS ) of SDS between the upper and lower phases in the SDS/n-C5 H11 OH/H2 O system can be expressed as follows: w SDS-up K= , (1) w SDS-down where w SDS-up and w SDS-down are qualitative percent of SDS in the upper and lower phases, respectively. As shown in the partial phase diagram of the SDS/n-C5 H11 OH/ H2 O system (Fig. 1) prepared in our previous study [15], there are several isotropic regions with different structures. L1 and L2 represent the oil-in-water (O/W) and water-in-oil (W/O) microemulsion regions, respectively; the narrow strip connecting L1 and L2 regions is the oil–water bicontinuous (the mixed region of O/W and W/O) region (BI); and LL is the coexisting region of W/O and O/W or BI microemulsions, which is of great interest in the present study. In the LL region, the system of SDS/n-C5 H11 OH/H2 O separates into two phases (the upper phase and the lower phase) spontaneously. With the constant weight ratio of n-C5 H11 OH/H2 O at 50/50 and the rise in the total content of SDS in the system, both the SDS contents in the upper and lower phases increase, but the increase in the lower phase is much more rapid (Fig. 2a). So the distribution coefficient of SDS ( K SDS ) between the upper and lower phases decreases (Fig. 2b). When the total SDS content is less than 3.0% (wt), the upper phase is W/O microemulsion, and the lower phase is O/W microemulsion. When the total SDS content is greater than 3.0%, the upper phase is still W/O microemulsion, while the lower phase turns into bicontinuous microemulsion that presents a network structure composed of O/W and W/O structures [11,12]. The addition of Hb has an effect on the distribution of SDS between the upper and lower phases (the SDS contents were determined by sodium-ion-selective electrode). As shown in Fig. 3a, with the increase of the total Hb concentration at a constant total SDS content, the SDS content increases slightly in the upper phase and decreases appreciably in the lower phase, namely the distribution coefficient of SDS ( K SDS ) between the upper and lower phases increases with the increase of the total Hb concentration at a constant total SDS content (Fig. 3b), which indicates that a small amount of SDS transfers from the lower to the upper phase with the addition of Hb.
(a)
(b) Fig. 2. (a) Variation of the SDS content in the upper and lower phases with the total SDS content (The X -axis represents the total SDS content, and the Y -axis represents the SDS content in respective phase.); (b) variation of the distribution coefficient K SDS between the upper and lower phases with the total SDS content.
Y. Chen, R. Guo / Journal of Colloid and Interface Science 328 (2008) 153–157
155
(a) (a)
(b) (b) Fig. 3. (a) Variation of the SDS contents in the upper and lower phases, and (b) variation of the distribution coefficient K SDS between the upper and lower phases with the total Hb concentration at constant total content of SDS: (!) 1.0% SDS, (") 4.0% SDS.
3.2. Distribution and conformational change of Hb in the upper and lower phases Fig. 4a shows that when the total concentration of Hb is 5.0 × 10−6 mol/L, with an increase of the total SDS content, the absorbance of Hb (415 nm) increases in the lower phase and decreases in the upper phase, namely the distribution coefficient of Hb ( K Hb ) between the upper and lower phases decreases (Fig. 4b), suggesting that Hb transfers from the upper phase to the lower phase with the addition of SDS. The possible reason is that the increase of SDS content in the lower phase is relatively greater and the interaction between SDS and Hb is stronger. As seen from Figs. 5a and b, the absorbance of Hb in the upper and lower phases increase with the increase of the total Hb concentration when the total SDS content is 1.0% or 4.0%. The distribution coefficient of Hb ( K Hb ) between the upper and lower phases decreases with the increase of the total Hb concentration (Fig. 5c), which indicates that the increase of Hb concentration in the lower phase is larger than that in the upper phase. In addition, compared with the decrease of K Hb at 1.0% total SDS content, the decrease of K Hb at 4.0% total SDS content slows down obviously, which is related to the transformation of the lower phase from O/W microemulsion to BI microemulsion.
Fig. 4. (a) Variation of the absorbance of Hb in the upper and lower phases with the total SDS content; (b) variation of the distribution coefficient K Hb between the upper and lower phases with the total SDS content.
Fig. 6a shows the effect of SDS content on the CD spectra of Hb in the lower phase (the CD spectra of Hb in the upper phase are not shown because the spectra nearly have no change). All α -proteins show two strong negative ellipticity at 222 (due to n → π ∗ ) and 208–210 nm (due to π → π ∗ ) and a strong maximum at 191–193 nm (due to π → π ∗ ) (Fig. 6a), which are characteristic of α -helix. The intensity of the three CD bands reflects the amount of helicity in protein. When SDS is added in the system, the secondary structure of Hb is changed. The content of α -helix decreases, but the content of β -sheet increases (Fig. 6b), which indicates unambiguously that α -helicity of hemoglobin subunits [16] are gradually lost. The formerly compact structure becomes relax, so we conclude that the presence of SDS results in the loss or breakage of heme because heme plays a key role in the maintaining the conformation of Hb. The UV–vis spectra of Hb is intimately related to the environmental change of hydrophobic cavity of heme in its interior [17,18]. As shown in Fig. 7, the peak position in the upper phase has no any significant shift (∼402 nm), and the Soret band of Hb in the lower phase is gradually shifted from 412 to 403 nm around, which indicate that it mostly exists as heme monomer in the upper phase and hemichrome induced from SDS is partly converted to heme in the lower phase. With the increase of the SDS concentration, heme monomer appears in the solution. Owing to the increase of the content of microemulsion droplets, the amount of the solubilized
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Y. Chen, R. Guo / Journal of Colloid and Interface Science 328 (2008) 153–157
(a) (a)
(b) (b) Fig. 6. Effects of SDS on (a) CD spectrum and (b) secondary structure parameters of Hb in the lower phase. (!) α -Helix, (") β -sheet.
(c) Fig. 5. Variation of the absorbance of Hb in the upper and lower phases at 1.0% (a) and 4.0% (b) total SDS content with the total Hb concentration; (c) variation of the distribution coefficient K Hb between the upper and lower phases with the total Hb concentration at constant total content of SDS: (!) 1.0% SDS, (") 4.0% SDS.
heme monomer increases too. The electrostatic attraction between heme and the peptide chains plays a key role in maintaining the position of heme in Hb [19,20]. SDS is an amphiphilic molecule and its hydrophobic group can insert in the inner of hydropho-
Fig. 7. Variation of peak position of Hb with the total SDS content (total Hb concentration is fixed at 5.0 × 10−6 mol/L).
bic core. The negative charge of hydrophobic group neutralizes the positive charge of His and Lys of the peptide chains, which reduces the electrostatic attraction between His, Lys and the side chain of metacetonic acid group. Thus heme releases from the hydrophobic cavity of Hb.
Y. Chen, R. Guo / Journal of Colloid and Interface Science 328 (2008) 153–157
4. Conclusions From CD results, we know that the structural changes of Hb in the upper and lower phases are different. In the upper phase, it mostly exists as heme monomer; in the lower phase, hemichrome induced from SDS is partly converted to heme. Furthermore, the addition of Hb results in the redistribution of SDS between the upper and lower phases. At the weight ratio of n-C5 H11 OH/H2 O = 50/50, with the increase of the total concentration of Hb in the system, a few SDS molecules transfer from the lower phase to the upper phase. In addition, the distribution of Hb between the two phases can be adjusted by changing the total SDS content. Acknowledgments This work was supported by the National Nature Science Foundation of China (Nos. 20633010 and 20773106). References [1] A.G. Volkov, D.W. Deamer, Liquid–Liquid Interfaces: Theory and Methods, CRC Press, Boca Raton, FL, 1996.
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[2] J. Gracia-Fadrique, Langmuir 15 (1999) 3279. [3] P. Pandit, S. Basu, J. Colloid Interface Sci. 245 (2002) 208. [4] B. Su, J.P. Abid, D.J. Fermin, H.H. Girault, H. Hoffmannová, P. Krtil, Z. Samec, J. Am. Chem. Soc. 126 (2004) 915. [5] F. Kanoufi, C. Cannes, Y.B. Zu, A.J. Bard, J. Phys. Chem. B 105 (2001) 8951. [6] Y.T. Kong, S. Imabayashi, T. Kakiuchi, J. Am. Chem. Soc. 122 (2000) 8215. [7] G. Mériguet, E. Dubois, R. Perzynski, J. Colloid Interface Sci. 267 (2003) 78. [8] C. Forssten, J. Strutwolf, D.E. Williams, Electrochem. Commun. 3 (2001) 619. [9] A. Mälkiä, P. Liljeroth, A.-K. Kontturi, K. Kontturi, J. Phys. Chem. B 105 (2001) 10884. [10] M. Adachi, Y. Murata, K. Sago, K. Nakagawa, Langmuir 20 (2004) 5965. [11] Y. Chen, R. Guo, J. Dispersion Sci. Technol. 27 (2006) 635. [12] Y. Chen, R. Guo, J. Colloid Interface Sci. 307 (2007) 488. [13] C.X. Chen, J. Chen. Anal. Biochem. 325 (2004) 285. [14] X.F. Yang, X.Q. Guo, H. Li, Talanta 61 (2003) 439. [15] T.Q. Liu, X. Wei, R. Guo, Acta Chim. Sinica 60 (2002) 633. [16] G. Geraci, L.J. Parkhurst, Methods Enzymol. 76 (1981) 262. [17] Apurba Kumar Sau, Douglas Currell, Shyamalava Mazumdar, Biophys. Chem. 98 (2002) 267. [18] D. Ajloo, A.A. Moosavi-Movahedi, G.H. Hakimelahi, Colloids Surf. B 26 (2002) 185. [19] Y.F. Dai, P. Chang, G.L. Li, Y.Q. Lin, J. Hyg. Res. 28 (1999) 30. [20] J.C. Lin, Blood Biochemistry, People’s Medical Publishing House, Beijing, 1988, p. 231.
Contents lists available at ScienceDirect
Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Distribution and structural behavior of hemoglobin between the two phases in SDS/n-C5 H11 OH/H2 O system Yuan Chen, Rong Guo ∗ School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou, 225002, People’s Republic of China
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 9 May 2008 Accepted 30 August 2008 Available online 4 September 2008
In the sodium dodecyl sulfate (SDS)/n-pentanol (n-C5 H11 OH)/water (H2 O) system, LL is the coexisting region of W/O and O/W or bicontinuous microemulsions. In the LL region, the system separates into two phases (the upper phase and the lower phase) spontaneously. The distribution and structural behavior of hemoglobin (Hb) between the upper and lower phases in SDS/n-C5 H11 OH/H2 O system were studied by UV–vis and circular dichroism (CD) spectroscopy. The results indicate that different structures of the two phases bring the conformational change of Hb. The addition of Hb results in the redistribution of SDS between the upper and lower phases. © 2008 Published by Elsevier Inc.
Keywords: SDS Hemoglobin Circular dichroism Conformational change
1. Introduction It has been well recognized that liquid–liquid interface plays an essential role in biological systems, particularly in biomedical engineering, pharmacology, and food processing [1]. Adsorption and reaction at the liquid/liquid interface have recently become attractive subjects in fundamental studies of solvent extraction of metal ions, and the detection of liquid-membrane separation, ion-selective electrodes, optical sensors and counter current chromatography [2]. Whether from the theory or practice view, the investigation on behavior of amphiphilic or bioactive molecules at liquid–liquid interfaces has been a subject of scientific interest for a long time. Interfaces modified by amphiphilic molecules are of great fundamental and technological relevance in areas including enhanced oil recovery, drug delivery, bio-sensor, extraction, nanoparticle formation, lubrication and in the production of biomimetic materials [3–10]. The liquid–liquid interface formed by the surfactant aggregation, such as the W/O–O/W interface or W/O– BI interface, can be used to mimic biological membrane since it provides a hydrophobic–hydrophilic medium. We have studied the distribution and transfer of l-phenylalanine [11] and l-tryptophan [12] through the water-in-oil (W/O)–oil-in-water (O/W) interface and W/O–BI (bicontinuous) interface in SDS/n-C5 H11 OH/H2 O system by means of UV–vis spectroscopy and AC impedance. The results indicate that the transfer and distribution of biologically active substance between the inside and outside of the membrane can be adjusted by constitutes of the upper and lower phases, and provide theoretical basis for the behavior of the biologically active
*
Corresponding author. Fax: +86 514 7311374. E-mail address: [email protected] (R. Guo).
0021-9797/$ – see front matter doi:10.1016/j.jcis.2008.08.058
©
2008 Published by Elsevier Inc.
substance across membrane transport, and the design of functional biomaterials. Hemoglobin (Hb) has a molecular weight of approximately 67,000 and contains two α and two β subunits, each of which has one redoxiron heme as its prosthetic group; the heme is located in crevices at the exterior of the subunit [13,14]. Proteins interact with the lipid bilayer by binding or adsorption to its surface, insertion into the hydrocarbon region of membrane interior or penetration through the membrane. In this paper, the distribution and structural behavior of Hb between W/O phase (the upper phase) and O/W or bicontinuous phase (the lower phase) in the SDS/n-C5 H11 OH/H2 O system was determined by UV–vis and circular dichroism spectrum. The results will provide some useful information with respect to the penetration and transfer of the biological active substance across the biological membrane interface. 2. Materials and methods 2.1. Reagents Sodium dodecyl sulfate (SDS, Sigma, >98%) was recrystallized twice in ethanol to ensure that its surface tension had no lowest point around the critical micelle concentration (cmc) by the platinum ring method. n-Pentanol (n-C5 H11 OH, Aldrich, 99%), hemoglobin (Sinopharm Chemical Reagent Co., Ltd., China). Double distilled water was used throughout. 2.2. UV–vis spectra measurement A series of SDS/n-C5 H11 OH/Hb/H2 O solutions were prepared kept at 25.0 ± 0.1 ◦ C for 10 h to reach the phase equilibrium, and
154
Y. Chen, R. Guo / Journal of Colloid and Interface Science 328 (2008) 153–157
then separated into two phases after the equilibrium. The absorption spectra of the upper and lower phases were recorded using a UV-2550 spectrophotometer (Shimadzu Corporation, Japan) in the range of wavelengths 200-700 nm by using the blank reagent as reference. 2.3. CD spectroscopic study Circular dichroism (CD) measurement was carried out with Jasco-810 spectropolarimeter (Japan). The CD spectra were measured in 190–300 nm at room temperature using 0.1 cm quartz cuvette. Scanning speed 100 nm/min; bandwidth 1 nm; spectral response 0.1 nm. The data points were taken on an average of four. The contents of the α -helicity, β -sheet structures in protein were calculated using the software provided by the manufacturer (Jasco-810 Analytical Manager System) corresponding to standard substances. All the experiments were carried out at 25.0 ± 0.1 ◦ C. In order to attain equilibrium, the samples were placed in a water thermostat at 25.0 ± 0.1 ◦ C for 10 h before the experiments.
Fig. 1. Partial phase diagram of the SDS/n-C5 H11 OH/H2 O system at 25 ◦ C. Total SDS content: a–a : 1.0%; b–b : 2.0%; c–c : 3.0%; d–d : 4.0%; e–e : 5.0%.
3. Results and discussion 3.1. Distribution of SDS between the upper and lower phases The distribution constant ( K SDS ) of SDS between the upper and lower phases in the SDS/n-C5 H11 OH/H2 O system can be expressed as follows: w SDS-up K= , (1) w SDS-down where w SDS-up and w SDS-down are qualitative percent of SDS in the upper and lower phases, respectively. As shown in the partial phase diagram of the SDS/n-C5 H11 OH/ H2 O system (Fig. 1) prepared in our previous study [15], there are several isotropic regions with different structures. L1 and L2 represent the oil-in-water (O/W) and water-in-oil (W/O) microemulsion regions, respectively; the narrow strip connecting L1 and L2 regions is the oil–water bicontinuous (the mixed region of O/W and W/O) region (BI); and LL is the coexisting region of W/O and O/W or BI microemulsions, which is of great interest in the present study. In the LL region, the system of SDS/n-C5 H11 OH/H2 O separates into two phases (the upper phase and the lower phase) spontaneously. With the constant weight ratio of n-C5 H11 OH/H2 O at 50/50 and the rise in the total content of SDS in the system, both the SDS contents in the upper and lower phases increase, but the increase in the lower phase is much more rapid (Fig. 2a). So the distribution coefficient of SDS ( K SDS ) between the upper and lower phases decreases (Fig. 2b). When the total SDS content is less than 3.0% (wt), the upper phase is W/O microemulsion, and the lower phase is O/W microemulsion. When the total SDS content is greater than 3.0%, the upper phase is still W/O microemulsion, while the lower phase turns into bicontinuous microemulsion that presents a network structure composed of O/W and W/O structures [11,12]. The addition of Hb has an effect on the distribution of SDS between the upper and lower phases (the SDS contents were determined by sodium-ion-selective electrode). As shown in Fig. 3a, with the increase of the total Hb concentration at a constant total SDS content, the SDS content increases slightly in the upper phase and decreases appreciably in the lower phase, namely the distribution coefficient of SDS ( K SDS ) between the upper and lower phases increases with the increase of the total Hb concentration at a constant total SDS content (Fig. 3b), which indicates that a small amount of SDS transfers from the lower to the upper phase with the addition of Hb.
(a)
(b) Fig. 2. (a) Variation of the SDS content in the upper and lower phases with the total SDS content (The X -axis represents the total SDS content, and the Y -axis represents the SDS content in respective phase.); (b) variation of the distribution coefficient K SDS between the upper and lower phases with the total SDS content.
Y. Chen, R. Guo / Journal of Colloid and Interface Science 328 (2008) 153–157
155
(a) (a)
(b) (b) Fig. 3. (a) Variation of the SDS contents in the upper and lower phases, and (b) variation of the distribution coefficient K SDS between the upper and lower phases with the total Hb concentration at constant total content of SDS: (!) 1.0% SDS, (") 4.0% SDS.
3.2. Distribution and conformational change of Hb in the upper and lower phases Fig. 4a shows that when the total concentration of Hb is 5.0 × 10−6 mol/L, with an increase of the total SDS content, the absorbance of Hb (415 nm) increases in the lower phase and decreases in the upper phase, namely the distribution coefficient of Hb ( K Hb ) between the upper and lower phases decreases (Fig. 4b), suggesting that Hb transfers from the upper phase to the lower phase with the addition of SDS. The possible reason is that the increase of SDS content in the lower phase is relatively greater and the interaction between SDS and Hb is stronger. As seen from Figs. 5a and b, the absorbance of Hb in the upper and lower phases increase with the increase of the total Hb concentration when the total SDS content is 1.0% or 4.0%. The distribution coefficient of Hb ( K Hb ) between the upper and lower phases decreases with the increase of the total Hb concentration (Fig. 5c), which indicates that the increase of Hb concentration in the lower phase is larger than that in the upper phase. In addition, compared with the decrease of K Hb at 1.0% total SDS content, the decrease of K Hb at 4.0% total SDS content slows down obviously, which is related to the transformation of the lower phase from O/W microemulsion to BI microemulsion.
Fig. 4. (a) Variation of the absorbance of Hb in the upper and lower phases with the total SDS content; (b) variation of the distribution coefficient K Hb between the upper and lower phases with the total SDS content.
Fig. 6a shows the effect of SDS content on the CD spectra of Hb in the lower phase (the CD spectra of Hb in the upper phase are not shown because the spectra nearly have no change). All α -proteins show two strong negative ellipticity at 222 (due to n → π ∗ ) and 208–210 nm (due to π → π ∗ ) and a strong maximum at 191–193 nm (due to π → π ∗ ) (Fig. 6a), which are characteristic of α -helix. The intensity of the three CD bands reflects the amount of helicity in protein. When SDS is added in the system, the secondary structure of Hb is changed. The content of α -helix decreases, but the content of β -sheet increases (Fig. 6b), which indicates unambiguously that α -helicity of hemoglobin subunits [16] are gradually lost. The formerly compact structure becomes relax, so we conclude that the presence of SDS results in the loss or breakage of heme because heme plays a key role in the maintaining the conformation of Hb. The UV–vis spectra of Hb is intimately related to the environmental change of hydrophobic cavity of heme in its interior [17,18]. As shown in Fig. 7, the peak position in the upper phase has no any significant shift (∼402 nm), and the Soret band of Hb in the lower phase is gradually shifted from 412 to 403 nm around, which indicate that it mostly exists as heme monomer in the upper phase and hemichrome induced from SDS is partly converted to heme in the lower phase. With the increase of the SDS concentration, heme monomer appears in the solution. Owing to the increase of the content of microemulsion droplets, the amount of the solubilized
156
Y. Chen, R. Guo / Journal of Colloid and Interface Science 328 (2008) 153–157
(a) (a)
(b) (b) Fig. 6. Effects of SDS on (a) CD spectrum and (b) secondary structure parameters of Hb in the lower phase. (!) α -Helix, (") β -sheet.
(c) Fig. 5. Variation of the absorbance of Hb in the upper and lower phases at 1.0% (a) and 4.0% (b) total SDS content with the total Hb concentration; (c) variation of the distribution coefficient K Hb between the upper and lower phases with the total Hb concentration at constant total content of SDS: (!) 1.0% SDS, (") 4.0% SDS.
heme monomer increases too. The electrostatic attraction between heme and the peptide chains plays a key role in maintaining the position of heme in Hb [19,20]. SDS is an amphiphilic molecule and its hydrophobic group can insert in the inner of hydropho-
Fig. 7. Variation of peak position of Hb with the total SDS content (total Hb concentration is fixed at 5.0 × 10−6 mol/L).
bic core. The negative charge of hydrophobic group neutralizes the positive charge of His and Lys of the peptide chains, which reduces the electrostatic attraction between His, Lys and the side chain of metacetonic acid group. Thus heme releases from the hydrophobic cavity of Hb.
Y. Chen, R. Guo / Journal of Colloid and Interface Science 328 (2008) 153–157
4. Conclusions From CD results, we know that the structural changes of Hb in the upper and lower phases are different. In the upper phase, it mostly exists as heme monomer; in the lower phase, hemichrome induced from SDS is partly converted to heme. Furthermore, the addition of Hb results in the redistribution of SDS between the upper and lower phases. At the weight ratio of n-C5 H11 OH/H2 O = 50/50, with the increase of the total concentration of Hb in the system, a few SDS molecules transfer from the lower phase to the upper phase. In addition, the distribution of Hb between the two phases can be adjusted by changing the total SDS content. Acknowledgments This work was supported by the National Nature Science Foundation of China (Nos. 20633010 and 20773106). References [1] A.G. Volkov, D.W. Deamer, Liquid–Liquid Interfaces: Theory and Methods, CRC Press, Boca Raton, FL, 1996.
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