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The interaction of hemoglobin with hexadecyltrimethylammonium bromide
International Journal of Biological Macromolecules 37 (2005) 232–238
The interaction of hemoglobin with hexadecyltrimethylammonium bromide Wenjie Liu, Xia Guo, Rong Guo ∗ School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou, Jiangsu 225002, PR China Received 26 June 2005; received in revised form 20 November 2005; accepted 21 November 2005 Available online 28 December 2005
Abstract The interaction of hemoglobin (Hb) with hexadecyltrimethylammonium bromide (CTAB) is investigated by UV–vis absorption spectra and fluorescence spectra method. CTAB monomer can convert methemoglobin (metHb) to hemichrome, and CTAB molecular assemblies, such as micelle, microemulsion and lamellar liquid crystal, can induce heme monomer to leave the hydrophobic cavity of Hb. TEM results show that Hb maintains the spherical structure in CTAB microemulsions while it is unfolded in CTAB lamellar liquid crystals. The existence of proton in the above systems can increase the stability of metHb. © 2005 Elsevier B.V. All rights reserved. Keywords: UV–vis absorption spectra; Fluorescence spectra; Hemoglobin
1. Introduction Hemoglobin (Hb), a kind of respiratory protein of vertebrate erythrocytes, is important in oxygen transport, H2 O2 dispersion and electron transfer to all organs and parts of the body [1]. Now, we have understood its structure, the mechanisms of its oxygen transport function and its electron transfer on electrode surface [2–7], but the research on its interaction with other molecules are almost limited to a few biological molecules, such as bacterial endotoxin [8,9], hydroxyurea [10] and trehalose [11], with the conclusion that some biological molecules can convert oxyhemoglobin (oxyHb) to methemoglobin (metHb) and hemichrome, while some others may slow or reverse the autooxidation process of Hb [8,11]. Since hemichrome accumulation in red cells is typical of some blood diseases [12,13] and aging of the erythrocyte [14], the interrelated study of the conversion between metHb and hemichrome will be worthwhile not only in theory but also in practice. Surfactant–protein interactions are very common in the fields of medicine, chemistry, biology, and so on [15,16], but the reports on the interaction of surfactant with Hb are very few [17,18]. Recently, Venkatesh et al. [19] have studied the release
∗
Corresponding author. E-mail address: [email protected] (R. Guo).
0141-8130/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2005.11.007
of heme in hemoglobin by CTAB and SDS micelle. However, systematic investigation on the interaction of various molecular organized assemblies with Hb has not been reported. Molecular organized assemblies, including micelle, microemulsion, lamellar liquid crystal, etc., have a noticeable application in detergents, cosmetics, material science, energy, biology and medicine [20–23]. And what should be especially noted is that microemulsions and lamellar liquid crystals can be used to simulate some biological systems, and hence, the research on the surfactant–protein interaction carried out in microemulsions and lamellar liquid crystals can offer useful information on life and medicine. Thus, in the present paper, we will investigate the interaction of CTAB with Hb, expatiate upon the influence of CTAB molecular organized assemblies on the above interaction, and discuss the factors that can affect the stability of metHb and the conversion of metHb to hemichrome.
2. Experimental 2.1. Materials Hexadecyltrimethylammonium bromide (CTAB, Aldrich, 99+%), n-pentanol (n-C5 H11 OH, Aldrich, 99+%), hemoglobin (Shanghai Lizhu Dongfeng Biological Technical Co.). Water used was distilled twice.
W. Liu et al. / International Journal of Biological Macromolecules 37 (2005) 232–238
2.2. Methods
(TECNAI 12, Philip Apparatus Co., The Netherlands) after being freeze-fractured (BAF 400 D, Balzers, Germany).
2.2.1. Measurement of the UV–vis absorption spectra Add Hb (0.2 mg/ml, i.e. 12.2 M/heme) into the different CTAB/H2 O systems, CTAB/n-C5 H11 OH/H2 O microemulsions and CTAB/n-C5 H11 OH/H2 O lamellar liquid crystals, respectively, and measure the UV–vis spectra by a Shimadzu UV2501 ultraviolet spectrophotometer at room temperature. A 1 cm × 1 cm (length × width) sample cuvette is chosen and water is used as the reference solution when Hb is dissolved in CTAB aqueous solutions and CTAB/n-C5 H11 OH/H2 O microemulsions, while 1 cm × 0.1 cm (length × width) sample cuvette is chosen when Hb is dissolved in CTAB/nC5 H11 OH/H2 O lamellar liquid crystals, with the corresponding lamellar liquid crystal used as the reference solution. The buffer solution with pH value being 4.0 is prepared by HAc–NaAc and citric acid–Na2 HPO4 , respectively, and that with pH value being 8.0 by NaH2 PO4 –Na2 HPO4 . CTAB/H2 O systems and CTAB/n-C5 H11 OH/H2 O microemulsions with pH values of 4.0 and 8.0 are prepared by the above buffer solutions. Then, Hb is added to the above systems and its UV–vis spectra are measured at room temperature with the corresponding buffer solution as the reference solution. 2.2.2. Measurement of the fluorescence spectra The fluorescence spectra of Hb in different CTAB/H2 O and CTAB/n-C5 H11 OH/H2 O systems were measured with a Shimadzu model RF-5301PC fluorescence spectrophotometer. The excitation wavelength is 280 nm and the excitation and emission slits are 3 nm. 2.2.3. Measurement of the conductance The conductances of CTAB/H2 O systems with and without Hb were measured with a conductor meter (DDS-11A, Shanghai Weiye Apparatus Co., China). 2.2.4. Determination of the diffusion coefficient The voltammetric properties of CTAB were determined by CHI 832 potentiostat (Shanghai Chenhua Apparatus Co., China) and a three-electrode configuration with a platinum foil-working electrode (Model 213, Shanghai Electron Optical Technical Research Center, China), a platinum foil-auxiliary electrode and a SCE reference electrode (Model 232, Shanghai Scientific Apparatus Co., China) was used. The diffusion coefficient (D) of CTAB can be calculated by Eq. (1) [24] ip = 2.69 × 105 n3/2 cD1/2 v1/2 A
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3. Results and discussion 3.1. Interaction of Hb with CTAB/H2 O systems Fig. 1 shows the plot of the conductance (κ) of CTAB/H2 O systems versus the concentration of CTAB. κ is increased first rapidly and then slowly with the increase of the CTAB concentration. The concentration of CTAB at the change point is 9.3 × 10−4 mol/L without Hb (curve 1) and 1.1 × 10−3 mol/L with the presence of Hb (12.2 M/heme, curve 2). Since the inflexion shows the critical micelle concentration (cmc) of CTAB [26], Fig. 1 indicates that Hb can make CTAB micelles dismissed and increase the cmc of CTAB. Interestingly, Venkatesh et al. [19] have observed a little decrease in the cmc value of CTAB with the addition of HbCO, which could be ascribed to the different conformation of the additional molecules. According to Eqs. (2) and (3) [27], the counter-ion binding degree (K0 ) for micelle can be obtained by Fig. 1 K0 = 1 − α
(2)
α = S2 /S1
(3)
Here, α shows the micelle ionization degree, S1 and S2 indicate the slopes of the straight lines with CTAB concentration smaller and larger than cmc (Fig. 1), respectively. Thus, K0 is 0.710 without Hb and 0.589 with Hb, showing that Hb can decrease the CTAB micelle counter-ion binding degree. Fig. 2 shows the UV–vis spectra of Hb in water and different CTAB/H2 O systems. Curve 1 in Fig. 2 is similar to the characteristic spectra of metHb, which indicates most of Hb is metHb [17] (the effect of other different conformational Hb can be neglected). By the method of Ajloo et al. [18], we can calculate the concentration of metHb (Eq. (4)) according to the absorbance at 540 nm (A540 ), 560 nm (A560 ) and 576 nm (A576 ), and confirm again metHb is the major form [metHb] = (4.5852A540 − 0.8375A560 − 3.7919A576 ) × 10−4 (4)
(1)
where ip is the anodic peak current in ampere, c the concentration of CTAB in mol/cm3 , v the potential sweep rate in V/s, A the total surface area of Pt electrode, D the particle diffusion coefficient in the solution in cm2 /s, and n is the electrons per molecule oxidized or reduced (for the CTAB system, n ≈ 1 [25]). 2.2.5. The measurement of the images of Hb Images of Hb in different CTAB molecular organized assemblies were observed by transmission electronic microscope
Fig. 1. Conductance of CTAB/H2 O systems with CTAB. Concentration of Hb: (1) 0 M/heme and (2) 12.2 M/heme.
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Fig. 2. UV–vis spectra of Hb (12.2 M/heme) in CTAB/H2 O system. Concentration of CTAB: (1) 0 mol/L, (2) 2.0 × 10−4 mol/L, (3) 4.0 × 10−4 mol/L, (4) 1.5 × 10−2 mol/L, and (5) 2.0 × 10−2 mol/L.
Fig. 2 also shows if CTAB with the concentration smaller than cmc is added to the Hb/H2 O system, the Soret band of Hb is shifted from 405 to 411 nm, and the absorption peak at 630 nm disappears while a new one at 537 nm comes into being (curves 2 and 3). Such Q-band of Hb can give rich information on the change of Hb conformation. Further experiments find that NaCl cannot make the absorption peaks of Hb shifted or disappeared (figure not shown). Thus, the different UV–vis spectra of Hb with and without CTAB monomer are not caused by the change of the ionic strength (although sometimes the change of UV–vis spectra of protein may be due to the increase of ionic strength), but by the interaction of CTAB monomer with Hb. The bands at 411 and 537 nm are considered to be characteristic of hemichrome [8]. Thus CTAB can induce metHb to convert to hemichrome. Hb consists of two alpha and two beta subunits, each combining a heme monomer located in the hydrophobic cavity of Hb. Fe3+ ion in heme is coordinated with four N atoms in porphyrins and proximal histidine (His) in the peptide chain. The sixth ligand of Fe3+ ion is H2 O or OH− ion [1]. The appearance of the absorption peaks for hemichrome (411 and 537 nm, curves 2 and 3 in Fig. 2) and the disappearance of those for Hb (405 and 630 nm, curves 2 and 3 in Fig. 2) show that CTAB monomer changes the microenvironment around heme and induces the distal His to replace H2 O or OH− to be the sixth ligand of Fe3+ . Thus, hemichrome is formed. Kaca et al. [8] studied the interaction of free fatty acid with Hb and concluded that the long hydrophobic chain in fatty acid can convert oxyHb to metHb and hemichrome just because of the hydrophobic interaction between the free fatty acid and Hb. Curves 4 and 5 in Fig. 2 show that when the concentration of CTAB in the aqueous solution is higher than cmc, the Soret band of Hb is shifted from 411 to 405 nm, while the absorption peaks at 537 and 565 nm disappear, and a new one at 600 nm comes forth. The absorption peak at 600 nm is considered to be characteristic of heme monomer [28]. Thus, CTAB micelle can induce heme to release from the hydrophobic cavity of Hb, probably because the hydrophobic cavity of CTAB micelle can solubilize heme monomer [28–30]. The solubilization of heme monomer in CTAB micelle can make the micelle volume increase and result in a smaller counter-ion binding degree K0 (shown in the above paragraph) for CTAB micelle with the presence of Hb.
Fig. 3. Fluorescence spectra of Hb (12.2 M/heme). From bottom to top, the concentration of CTAB: 0, 2.0 × 10−4 , 6.0 × 10−4 , 1.0 × 10−3 , 1.0 × 10−2 mol/L. Inset in figure shows the fluorescence intensity of Hb (12.2 M/heme) (λem ≈ 334 nm) with CTAB.
Fig. 3 shows the fluorescence spectra of Hb in water and different CTAB/H2 O systems. When CTAB concentration is smaller than cmc (1.1 × 10−3 ), the fluorescence intensity of Hb is increased gradually with increasing CTAB concentration. The fluorescence of Hb is mainly due to tryptophan (Trp) and the efficient energy transfer from Trp to heme significantly quenches the protein fluorescence [31]. As a result, the fluorescence intensity of Hb in water is very low. However, the hydrophobic chain in CTAB molecule may penetrate inside the hydrophobic cavity of heme and make heme exposed with the presence of CTAB monomers. Thus, the quenching reaction of Trp with heme is depressed and the fluorescence intensity of Hb is obviously increased when CTAB monomer exists. Fig. 3 also shows that the fluorescence intensity of Hb increases slowly first and then remains unchanged with increasing CTAB concentration when CTAB concentration is higher than cmc. The former may be due to the solubilization of heme in micelle, which results in a further reduced rate for the fluorescence quenching of Trp by
Fig. 4. The diffusion coefficient D of CTAB. Concentration of CTAB: (1) 2.0 × 10−4 mol/L and (2) 2.0 × 10−2 mol/L.
W. Liu et al. / International Journal of Biological Macromolecules 37 (2005) 232–238
Fig. 5. Schematic diagram for the interaction of CTAB monomers (
heme (compared with the case in which CTAB concentration is smaller than cmc), and the latter may indicate the solubilization of heme in micelle is complete. It is noted that CTAB can induce denaturalization of Hb, which can also increase Hb fluorescence intensity. However, both the effects co-exist under the present condition. It is very difficult to tell which of the effects contributes to the increase of fluorescence intensity. Further studies are needed. Curve 1 in Fig. 4 shows that the diffusion coefficient (D) of CTAB with concentration smaller than cmc is decreased with
235
) with ␣ chains in Hb molecules.
the addition of Hb. Just as discussed above, the hydrophobic chain in CTAB molecule can penetrate inside the hydrophobic cavity of heme. Thus, the apparent volume of CTAB molecule is increased and as a result, the value of D is decreased with Hb. Curve 2 in Fig. 4 shows the effect of Hb on the diffusion coefficient of CTAB micelles. Compared with the case for CTAB monomer (curve 1 in Fig. 4), the value D of CTAB micelle is decreased slowly with the addition of Hb. This is because heme is very small, and its solubilization in micelle can have only a little influence on the diffusion coefficient of CTAB micelle.
Fig. 6. Freeze-fractured images of CTAB/H2 O/Hb (12.2 M/heme) systems. Concentration of CTAB: (a) 0 mol/L, (b) 2.0 × 10−4 mol/L, (c) 1.0 × 10−2 mol/L, (d) 4.0 × 10−2 mol/L. Inset in Fig. 9b shows the dwindled image.
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Fig. 7. UV–vis spectra of Hb (12.2 M/heme) weight ratio: CTAB/nC5 H11 OH = 3/5, H2 O (wt.%) = (1) 97.0, (2) 40.0, (3) 20.0; CTAB/H2 O = 3/2, n-C5 H11 OH (wt.%) = (4) 27.0.
Fig. 5 shows the schematic diagram for the interaction of CTAB monomers. It indicates the hydrophobic tail in CTAB molecules penetrates inside heme. Freeze-fractured images of Hb with CTAB monomer explain this conclusion again. Hb is spherical in water (Fig. 6a) and remains folded in the aqueous solution of CTAB monomer (Fig. 6b). But if we compare Fig. 6b with a, it should be noted that Hb molecules in Fig. 6b are obviously aggregated, implying that there still exits an electrostatic interaction between Hb and CTAB [18] besides the hydrophobic interaction (Fig. 5). As a result, Hb molecules are aggregated. Fig. 6c and d show the freeze-fractured images of Hb in CTAB micelles. Hb remains folded in this case, but the structure of Hb is looser in CTAB micelle than in water as observed in Fig. 6a, c and d, which confirms again that heme can release from the hydrophobic cavity of Hb in CTAB micelle. 3.2. The interaction of Hb with CTAB/n-C5 H11 OH/H2 O systems Fig. 7 shows the UV–vis spectra of Hb in CTAB/nC5 H11 OH/H2 O systems. According to the phase diagram of CTAB/n-C5 H11 OH/H2 O system [32], CTAB/n-C5 H11 OH/H2 O systems used for curves 1–4 in this figure show oil in water (O/W), bicontinuous (BI), water in oil (W/O) and lamellar liq-
Fig. 9. UV–vis spectra of Hb in CTAB/H2 O system with pH value being 4.0 (curves 1 and 2) and 8.0 (curves 3 and 4). Concentration of CTAB: 2.0 × 10−4 mol/L (curves 1 and 3) and 1.0 × 10−2 mol/L (curves 2 and 4).
uid crystal structures, respectively. Since the absorption peak at 600 nm is characteristic of heme monomer [28], Fig. 7 indicates heme monomer releases from the hydrophobic cavity of Hb in the O/W, BI, W/O microemulsions and lamellar liquid crystal. Just as discussed above, the fluorescence intensity of Hb should be increased once heme monomer escapes from the cavity of Hb. Thus, the much larger fluorescence intensity of Hb in CTAB/nC5 H11 OH/H2 O systems than in water (Table 1) also proves that heme releases from the hydrophobic cavity of Hb in CTAB/nC5 H11 OH/H2 O systems. Freeze-fractured images (Fig. 8) show that Hb remains folded in microemulsions whereas it is unfolded in lamellar liquid crystal. 3.3. The effect of pH on the interaction of CTAB with Hb Fig. 9 shows the UV–vis spectra of Hb in CTAB aqueous solutions with pH values being 4.0 and 8.0, respectively. According to Eq. (4), we can calculate the concentration of metHb in these systems. Fig. 10 shows that the concentration of metHb decreases in water and in the buffer solution with pH value being 8.0, while it increases in the buffer solution with pH value being 4.0 with the increase of CTAB concentration. Thus, proton can depress the interaction of CTAB with Hb, and increase the stability of metHb. Fig. 9 also indicates that heme does not release from the hydrophobic cavity of Hb in acidic CTAB micelle since
Fig. 8. Freeze-fractured images of Hb (12.2 M/heme) in: (a) O/W microemulsion (CTAB/n-C5 H11 OH = 3/5, H2 O (wt.%) = 97.0) and (b) lamellar liquid crystal (CTAB/H2 O = 3/2, n-C5 H11 OH (wt.%) = 27.0). Inset in Fig. 11b shows the image of the lamellar liquid crystal without Hb.
W. Liu et al. / International Journal of Biological Macromolecules 37 (2005) 232–238
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Table 1 The fluorescence intensity of Hb in CTAB/n-C5 H11 OH/H2 O systems Medium
Structure
If
Composition OHa ,
CTAB/n-C5 H11 (n-C5 H11 OH/H2 O)b CTAB/n-C5 H11 OH/H2 O microemulsion
H2 O a b
O/W
3/5
W/O
(4/1)
–
–
H2
Oa ,
(CTAB)b
90.0%
347.0
92.5% 95.0% (20.0%) (30.0%) (40.0%) –
368.0 368.0 456.0 454.0 432.0 51.0
Weight ratio or weight percent in O/W microemulsion. Weight ratio or weight percent in W/O microemulsion.
and 4 shows more Hb molecules exist in metHb form in acidic medium. Thus, proton can block the conversion of metHb to hemichrome and retain met-form of Hb both in CTAB/H2 O and CTAB/n-C5 H11 OH/H2 O systems. 4. Conclusions CTAB monomer in Hb aqueous solution can covert metHb to hemichrome, while CTAB micelles, microemulsions and lamellar liquid crystals can make heme release from Hb because heme monomer can be solubilized in the CTAB/n-C5 H11 OH/H2 O systems. Proton can depress the interaction of CTAB with Hb, so more Hb molecules can exist in metHb form in acidic environment. Fig. 10. The concentration of metHb with CTAB. Solvent: H2 O (curve 1), buffer solution with pH being 8.0 (curve 2) and 4.0 (curve 3).
there is no absorbance at 600 nm (curve 2 in Fig. 9). The reason may be that proton can combine with the anion of the hydrophilic groups in Hb and reduce the electrostatic interaction of CTAB with Hb, which makes Hb structure tighter than that in other cases and results in the difficult release of heme from Hb. Fig. 11 shows the UV–vis spectra of Hb in CTAB/nC5 H11 OH/H2 O O/W microemulsions with different pH values. The existence of the absorption peak at 630 nm in curves 3
Fig. 11. UV–vis spectra of Hb (12.2 M/heme) in CTAB/n-C5 H11 OH/H2 O O/W microemulsion (CTAB/n-C5 H11 OH = 3/5, H2 O (wt.%) = 97.0). pH 7.0 (curve 1), 8.0 (curve 2), 4.0 (curves 3 and 4, prepared by HAc–NaAc and citric acid–Na2 HPO4 , respectively).
Acknowledgement This work is supported by the National Natural Scientific Foundation of China (No. 20233010). References [1] J. Baldwin, C. Chothia, J. Mol. Biol. 129 (1979) 175–220. [2] D.J. Wang, X.J. Zhao, T.J. Shen, C. Ho, T.G. Spiro, J. Am. Chem. Soc. 121 (1999) 11197–11203. [3] M. Nagai, M. Aki, R. Li, Y. Jin, H. Sakai, S. Nagatomo, Biochemistry 39 (2000) 13093–13105. [4] M.F. Perutz, Nature 228 (1970) 726–734. [5] L.W. Wang, N.F. Hu, Bioelectrochem. Bioenerg. 53 (2001) 205–212. [6] H.Y. Gu, A.M. Yu, H.Y. Chen, J. Electroanal. Chem. 516 (2001) 119–126. [7] C.H. Fan, H.Y. Wang, S. Sun, D.X. Zhu, G. Wagner, G.X. Li, Anal. Chem. 73 (2001) 2850–2854. [8] W. Kaca, R.I. Roth, K.D. Vandegriff, G.C. Chen, F.A. Kuypers, R.M. Winslow, J. Levin, Biochemistry 34 (1995) 11176–11185. [9] W. Kaca, R.I. Roth, J. Levin, J. Biol. Chem. 269 (1994) 25078– 25084. [10] J.W. Rupon, S.R. Domingo, S.V. Smith, B.K. Gummadi, H. Shields, S.K. Ballas, S.B. King, D.B. Kim-Shapiro, Biophys. Chem. 84 (2000) 1–11. [11] A. Ray, B.A. Friedman, J.M. Friedman, J. Am. Chem. Soc. 124 (2002) 7270–7271. [12] E.A. Rachmilewitz, J. Peisach, T.B. Bradley, W.E. Blumberg, Nature 222 (1969) 248–250.
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[13] M.H. Sadrzadeh, E. Graf, S.S. Panter, P.E. Hallaway, J.W. Eaton, J. Biol. Chem. 259 (1984) 14354–14356. [14] D.A. Rigas, R.D. Koler, Blood 18 (1961) 1. [15] R. Guo, G.K. Fan, T.Q. Liu, X.A. Jiao, Acta Phys.-Chim. Sin. 17 (2001) 185–188. [16] G. Parthasarathi, N. Udupan, G.K. Pillai, Indian J. Pharm. Sci. 56 (1994) 90–94. [17] A.K. Sau, D. Currell, S. Mazumdar, S. Mitra, Biophys. Chem. 98 (2002) 267–273. [18] D. Ajloo, A.A. Moosavi-Movahedi, G.H. Hakimelahi, A.A. Saboury, H. Gharibi, Colloids Surf. B: Biointerf. 26 (2002) 185–196. [19] B. Venkatesh, S. Venkateshrao, S. Jayadevan, J.M. Rifkind, P.T. Manoharan, Pept. Sci. 80 (2005) 18–25. [20] M.J. Schwuger, K. Stickdorn, R. Schomaecker, Chem. Rev. 95 (1995) 849–864. [21] V. Pillai, P. Kumar, M.J. Hou, P. Ayyub, D.D. Shah, Adv. Colloid Interf. Sci. 55 (1995) 241–269.
[22] M.P. Pileni, J. Phys. Chem. 97 (1993) 6961–6973. [23] K. Holmberg, Adv. Colloid Interf. Sci. 51 (1994) 137–174. [24] A.J. Bard, L.R. Faulker, Electrochemical Methods, John Wiley and Sons, New York, 1998. [25] R. Guo, Y. Ji, Q.Q. Zhang, X.H. Zhang, Acta Phys.-Chim. Sin. 18 (2002) 50–54. [26] M. Miura, M. Kodama, Bull. Chem. Soc. Jap. 45 (1972) 428–431. [27] R. Zana, A. Djavanbakht, Tenside Surfact. Deterg. 26 (1989) 227– 230. [28] A. Boffi, T.K. Das, S.D. Longa, C. Spagnuolo, D.L. Rousseau, Biophys. J. 77 (1999) 1143–1149. [29] T.K. Das, S. Mazumdar, S. Mitra, J. Chem. Soc., Chem. Commun. 18 (1993) 1447–1448. [30] J. Simplicio, Biochemistry 11 (1972) 2525–2528. [31] S.C.M. Agustinho, M.H. Tinto, J.R. Perussi, M. Tabak, H. Imasato, Comp. Biochem. Physiol. 118A (1997) 171–181. [32] W.L. Yu, R. Guo, J. Dispersion Sic. Technol. 20 (1999) 1359–1387.
The interaction of hemoglobin with hexadecyltrimethylammonium bromide Wenjie Liu, Xia Guo, Rong Guo ∗ School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou, Jiangsu 225002, PR China Received 26 June 2005; received in revised form 20 November 2005; accepted 21 November 2005 Available online 28 December 2005
Abstract The interaction of hemoglobin (Hb) with hexadecyltrimethylammonium bromide (CTAB) is investigated by UV–vis absorption spectra and fluorescence spectra method. CTAB monomer can convert methemoglobin (metHb) to hemichrome, and CTAB molecular assemblies, such as micelle, microemulsion and lamellar liquid crystal, can induce heme monomer to leave the hydrophobic cavity of Hb. TEM results show that Hb maintains the spherical structure in CTAB microemulsions while it is unfolded in CTAB lamellar liquid crystals. The existence of proton in the above systems can increase the stability of metHb. © 2005 Elsevier B.V. All rights reserved. Keywords: UV–vis absorption spectra; Fluorescence spectra; Hemoglobin
1. Introduction Hemoglobin (Hb), a kind of respiratory protein of vertebrate erythrocytes, is important in oxygen transport, H2 O2 dispersion and electron transfer to all organs and parts of the body [1]. Now, we have understood its structure, the mechanisms of its oxygen transport function and its electron transfer on electrode surface [2–7], but the research on its interaction with other molecules are almost limited to a few biological molecules, such as bacterial endotoxin [8,9], hydroxyurea [10] and trehalose [11], with the conclusion that some biological molecules can convert oxyhemoglobin (oxyHb) to methemoglobin (metHb) and hemichrome, while some others may slow or reverse the autooxidation process of Hb [8,11]. Since hemichrome accumulation in red cells is typical of some blood diseases [12,13] and aging of the erythrocyte [14], the interrelated study of the conversion between metHb and hemichrome will be worthwhile not only in theory but also in practice. Surfactant–protein interactions are very common in the fields of medicine, chemistry, biology, and so on [15,16], but the reports on the interaction of surfactant with Hb are very few [17,18]. Recently, Venkatesh et al. [19] have studied the release
∗
Corresponding author. E-mail address: [email protected] (R. Guo).
0141-8130/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2005.11.007
of heme in hemoglobin by CTAB and SDS micelle. However, systematic investigation on the interaction of various molecular organized assemblies with Hb has not been reported. Molecular organized assemblies, including micelle, microemulsion, lamellar liquid crystal, etc., have a noticeable application in detergents, cosmetics, material science, energy, biology and medicine [20–23]. And what should be especially noted is that microemulsions and lamellar liquid crystals can be used to simulate some biological systems, and hence, the research on the surfactant–protein interaction carried out in microemulsions and lamellar liquid crystals can offer useful information on life and medicine. Thus, in the present paper, we will investigate the interaction of CTAB with Hb, expatiate upon the influence of CTAB molecular organized assemblies on the above interaction, and discuss the factors that can affect the stability of metHb and the conversion of metHb to hemichrome.
2. Experimental 2.1. Materials Hexadecyltrimethylammonium bromide (CTAB, Aldrich, 99+%), n-pentanol (n-C5 H11 OH, Aldrich, 99+%), hemoglobin (Shanghai Lizhu Dongfeng Biological Technical Co.). Water used was distilled twice.
W. Liu et al. / International Journal of Biological Macromolecules 37 (2005) 232–238
2.2. Methods
(TECNAI 12, Philip Apparatus Co., The Netherlands) after being freeze-fractured (BAF 400 D, Balzers, Germany).
2.2.1. Measurement of the UV–vis absorption spectra Add Hb (0.2 mg/ml, i.e. 12.2 M/heme) into the different CTAB/H2 O systems, CTAB/n-C5 H11 OH/H2 O microemulsions and CTAB/n-C5 H11 OH/H2 O lamellar liquid crystals, respectively, and measure the UV–vis spectra by a Shimadzu UV2501 ultraviolet spectrophotometer at room temperature. A 1 cm × 1 cm (length × width) sample cuvette is chosen and water is used as the reference solution when Hb is dissolved in CTAB aqueous solutions and CTAB/n-C5 H11 OH/H2 O microemulsions, while 1 cm × 0.1 cm (length × width) sample cuvette is chosen when Hb is dissolved in CTAB/nC5 H11 OH/H2 O lamellar liquid crystals, with the corresponding lamellar liquid crystal used as the reference solution. The buffer solution with pH value being 4.0 is prepared by HAc–NaAc and citric acid–Na2 HPO4 , respectively, and that with pH value being 8.0 by NaH2 PO4 –Na2 HPO4 . CTAB/H2 O systems and CTAB/n-C5 H11 OH/H2 O microemulsions with pH values of 4.0 and 8.0 are prepared by the above buffer solutions. Then, Hb is added to the above systems and its UV–vis spectra are measured at room temperature with the corresponding buffer solution as the reference solution. 2.2.2. Measurement of the fluorescence spectra The fluorescence spectra of Hb in different CTAB/H2 O and CTAB/n-C5 H11 OH/H2 O systems were measured with a Shimadzu model RF-5301PC fluorescence spectrophotometer. The excitation wavelength is 280 nm and the excitation and emission slits are 3 nm. 2.2.3. Measurement of the conductance The conductances of CTAB/H2 O systems with and without Hb were measured with a conductor meter (DDS-11A, Shanghai Weiye Apparatus Co., China). 2.2.4. Determination of the diffusion coefficient The voltammetric properties of CTAB were determined by CHI 832 potentiostat (Shanghai Chenhua Apparatus Co., China) and a three-electrode configuration with a platinum foil-working electrode (Model 213, Shanghai Electron Optical Technical Research Center, China), a platinum foil-auxiliary electrode and a SCE reference electrode (Model 232, Shanghai Scientific Apparatus Co., China) was used. The diffusion coefficient (D) of CTAB can be calculated by Eq. (1) [24] ip = 2.69 × 105 n3/2 cD1/2 v1/2 A
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3. Results and discussion 3.1. Interaction of Hb with CTAB/H2 O systems Fig. 1 shows the plot of the conductance (κ) of CTAB/H2 O systems versus the concentration of CTAB. κ is increased first rapidly and then slowly with the increase of the CTAB concentration. The concentration of CTAB at the change point is 9.3 × 10−4 mol/L without Hb (curve 1) and 1.1 × 10−3 mol/L with the presence of Hb (12.2 M/heme, curve 2). Since the inflexion shows the critical micelle concentration (cmc) of CTAB [26], Fig. 1 indicates that Hb can make CTAB micelles dismissed and increase the cmc of CTAB. Interestingly, Venkatesh et al. [19] have observed a little decrease in the cmc value of CTAB with the addition of HbCO, which could be ascribed to the different conformation of the additional molecules. According to Eqs. (2) and (3) [27], the counter-ion binding degree (K0 ) for micelle can be obtained by Fig. 1 K0 = 1 − α
(2)
α = S2 /S1
(3)
Here, α shows the micelle ionization degree, S1 and S2 indicate the slopes of the straight lines with CTAB concentration smaller and larger than cmc (Fig. 1), respectively. Thus, K0 is 0.710 without Hb and 0.589 with Hb, showing that Hb can decrease the CTAB micelle counter-ion binding degree. Fig. 2 shows the UV–vis spectra of Hb in water and different CTAB/H2 O systems. Curve 1 in Fig. 2 is similar to the characteristic spectra of metHb, which indicates most of Hb is metHb [17] (the effect of other different conformational Hb can be neglected). By the method of Ajloo et al. [18], we can calculate the concentration of metHb (Eq. (4)) according to the absorbance at 540 nm (A540 ), 560 nm (A560 ) and 576 nm (A576 ), and confirm again metHb is the major form [metHb] = (4.5852A540 − 0.8375A560 − 3.7919A576 ) × 10−4 (4)
(1)
where ip is the anodic peak current in ampere, c the concentration of CTAB in mol/cm3 , v the potential sweep rate in V/s, A the total surface area of Pt electrode, D the particle diffusion coefficient in the solution in cm2 /s, and n is the electrons per molecule oxidized or reduced (for the CTAB system, n ≈ 1 [25]). 2.2.5. The measurement of the images of Hb Images of Hb in different CTAB molecular organized assemblies were observed by transmission electronic microscope
Fig. 1. Conductance of CTAB/H2 O systems with CTAB. Concentration of Hb: (1) 0 M/heme and (2) 12.2 M/heme.
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Fig. 2. UV–vis spectra of Hb (12.2 M/heme) in CTAB/H2 O system. Concentration of CTAB: (1) 0 mol/L, (2) 2.0 × 10−4 mol/L, (3) 4.0 × 10−4 mol/L, (4) 1.5 × 10−2 mol/L, and (5) 2.0 × 10−2 mol/L.
Fig. 2 also shows if CTAB with the concentration smaller than cmc is added to the Hb/H2 O system, the Soret band of Hb is shifted from 405 to 411 nm, and the absorption peak at 630 nm disappears while a new one at 537 nm comes into being (curves 2 and 3). Such Q-band of Hb can give rich information on the change of Hb conformation. Further experiments find that NaCl cannot make the absorption peaks of Hb shifted or disappeared (figure not shown). Thus, the different UV–vis spectra of Hb with and without CTAB monomer are not caused by the change of the ionic strength (although sometimes the change of UV–vis spectra of protein may be due to the increase of ionic strength), but by the interaction of CTAB monomer with Hb. The bands at 411 and 537 nm are considered to be characteristic of hemichrome [8]. Thus CTAB can induce metHb to convert to hemichrome. Hb consists of two alpha and two beta subunits, each combining a heme monomer located in the hydrophobic cavity of Hb. Fe3+ ion in heme is coordinated with four N atoms in porphyrins and proximal histidine (His) in the peptide chain. The sixth ligand of Fe3+ ion is H2 O or OH− ion [1]. The appearance of the absorption peaks for hemichrome (411 and 537 nm, curves 2 and 3 in Fig. 2) and the disappearance of those for Hb (405 and 630 nm, curves 2 and 3 in Fig. 2) show that CTAB monomer changes the microenvironment around heme and induces the distal His to replace H2 O or OH− to be the sixth ligand of Fe3+ . Thus, hemichrome is formed. Kaca et al. [8] studied the interaction of free fatty acid with Hb and concluded that the long hydrophobic chain in fatty acid can convert oxyHb to metHb and hemichrome just because of the hydrophobic interaction between the free fatty acid and Hb. Curves 4 and 5 in Fig. 2 show that when the concentration of CTAB in the aqueous solution is higher than cmc, the Soret band of Hb is shifted from 411 to 405 nm, while the absorption peaks at 537 and 565 nm disappear, and a new one at 600 nm comes forth. The absorption peak at 600 nm is considered to be characteristic of heme monomer [28]. Thus, CTAB micelle can induce heme to release from the hydrophobic cavity of Hb, probably because the hydrophobic cavity of CTAB micelle can solubilize heme monomer [28–30]. The solubilization of heme monomer in CTAB micelle can make the micelle volume increase and result in a smaller counter-ion binding degree K0 (shown in the above paragraph) for CTAB micelle with the presence of Hb.
Fig. 3. Fluorescence spectra of Hb (12.2 M/heme). From bottom to top, the concentration of CTAB: 0, 2.0 × 10−4 , 6.0 × 10−4 , 1.0 × 10−3 , 1.0 × 10−2 mol/L. Inset in figure shows the fluorescence intensity of Hb (12.2 M/heme) (λem ≈ 334 nm) with CTAB.
Fig. 3 shows the fluorescence spectra of Hb in water and different CTAB/H2 O systems. When CTAB concentration is smaller than cmc (1.1 × 10−3 ), the fluorescence intensity of Hb is increased gradually with increasing CTAB concentration. The fluorescence of Hb is mainly due to tryptophan (Trp) and the efficient energy transfer from Trp to heme significantly quenches the protein fluorescence [31]. As a result, the fluorescence intensity of Hb in water is very low. However, the hydrophobic chain in CTAB molecule may penetrate inside the hydrophobic cavity of heme and make heme exposed with the presence of CTAB monomers. Thus, the quenching reaction of Trp with heme is depressed and the fluorescence intensity of Hb is obviously increased when CTAB monomer exists. Fig. 3 also shows that the fluorescence intensity of Hb increases slowly first and then remains unchanged with increasing CTAB concentration when CTAB concentration is higher than cmc. The former may be due to the solubilization of heme in micelle, which results in a further reduced rate for the fluorescence quenching of Trp by
Fig. 4. The diffusion coefficient D of CTAB. Concentration of CTAB: (1) 2.0 × 10−4 mol/L and (2) 2.0 × 10−2 mol/L.
W. Liu et al. / International Journal of Biological Macromolecules 37 (2005) 232–238
Fig. 5. Schematic diagram for the interaction of CTAB monomers (
heme (compared with the case in which CTAB concentration is smaller than cmc), and the latter may indicate the solubilization of heme in micelle is complete. It is noted that CTAB can induce denaturalization of Hb, which can also increase Hb fluorescence intensity. However, both the effects co-exist under the present condition. It is very difficult to tell which of the effects contributes to the increase of fluorescence intensity. Further studies are needed. Curve 1 in Fig. 4 shows that the diffusion coefficient (D) of CTAB with concentration smaller than cmc is decreased with
235
) with ␣ chains in Hb molecules.
the addition of Hb. Just as discussed above, the hydrophobic chain in CTAB molecule can penetrate inside the hydrophobic cavity of heme. Thus, the apparent volume of CTAB molecule is increased and as a result, the value of D is decreased with Hb. Curve 2 in Fig. 4 shows the effect of Hb on the diffusion coefficient of CTAB micelles. Compared with the case for CTAB monomer (curve 1 in Fig. 4), the value D of CTAB micelle is decreased slowly with the addition of Hb. This is because heme is very small, and its solubilization in micelle can have only a little influence on the diffusion coefficient of CTAB micelle.
Fig. 6. Freeze-fractured images of CTAB/H2 O/Hb (12.2 M/heme) systems. Concentration of CTAB: (a) 0 mol/L, (b) 2.0 × 10−4 mol/L, (c) 1.0 × 10−2 mol/L, (d) 4.0 × 10−2 mol/L. Inset in Fig. 9b shows the dwindled image.
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Fig. 7. UV–vis spectra of Hb (12.2 M/heme) weight ratio: CTAB/nC5 H11 OH = 3/5, H2 O (wt.%) = (1) 97.0, (2) 40.0, (3) 20.0; CTAB/H2 O = 3/2, n-C5 H11 OH (wt.%) = (4) 27.0.
Fig. 5 shows the schematic diagram for the interaction of CTAB monomers. It indicates the hydrophobic tail in CTAB molecules penetrates inside heme. Freeze-fractured images of Hb with CTAB monomer explain this conclusion again. Hb is spherical in water (Fig. 6a) and remains folded in the aqueous solution of CTAB monomer (Fig. 6b). But if we compare Fig. 6b with a, it should be noted that Hb molecules in Fig. 6b are obviously aggregated, implying that there still exits an electrostatic interaction between Hb and CTAB [18] besides the hydrophobic interaction (Fig. 5). As a result, Hb molecules are aggregated. Fig. 6c and d show the freeze-fractured images of Hb in CTAB micelles. Hb remains folded in this case, but the structure of Hb is looser in CTAB micelle than in water as observed in Fig. 6a, c and d, which confirms again that heme can release from the hydrophobic cavity of Hb in CTAB micelle. 3.2. The interaction of Hb with CTAB/n-C5 H11 OH/H2 O systems Fig. 7 shows the UV–vis spectra of Hb in CTAB/nC5 H11 OH/H2 O systems. According to the phase diagram of CTAB/n-C5 H11 OH/H2 O system [32], CTAB/n-C5 H11 OH/H2 O systems used for curves 1–4 in this figure show oil in water (O/W), bicontinuous (BI), water in oil (W/O) and lamellar liq-
Fig. 9. UV–vis spectra of Hb in CTAB/H2 O system with pH value being 4.0 (curves 1 and 2) and 8.0 (curves 3 and 4). Concentration of CTAB: 2.0 × 10−4 mol/L (curves 1 and 3) and 1.0 × 10−2 mol/L (curves 2 and 4).
uid crystal structures, respectively. Since the absorption peak at 600 nm is characteristic of heme monomer [28], Fig. 7 indicates heme monomer releases from the hydrophobic cavity of Hb in the O/W, BI, W/O microemulsions and lamellar liquid crystal. Just as discussed above, the fluorescence intensity of Hb should be increased once heme monomer escapes from the cavity of Hb. Thus, the much larger fluorescence intensity of Hb in CTAB/nC5 H11 OH/H2 O systems than in water (Table 1) also proves that heme releases from the hydrophobic cavity of Hb in CTAB/nC5 H11 OH/H2 O systems. Freeze-fractured images (Fig. 8) show that Hb remains folded in microemulsions whereas it is unfolded in lamellar liquid crystal. 3.3. The effect of pH on the interaction of CTAB with Hb Fig. 9 shows the UV–vis spectra of Hb in CTAB aqueous solutions with pH values being 4.0 and 8.0, respectively. According to Eq. (4), we can calculate the concentration of metHb in these systems. Fig. 10 shows that the concentration of metHb decreases in water and in the buffer solution with pH value being 8.0, while it increases in the buffer solution with pH value being 4.0 with the increase of CTAB concentration. Thus, proton can depress the interaction of CTAB with Hb, and increase the stability of metHb. Fig. 9 also indicates that heme does not release from the hydrophobic cavity of Hb in acidic CTAB micelle since
Fig. 8. Freeze-fractured images of Hb (12.2 M/heme) in: (a) O/W microemulsion (CTAB/n-C5 H11 OH = 3/5, H2 O (wt.%) = 97.0) and (b) lamellar liquid crystal (CTAB/H2 O = 3/2, n-C5 H11 OH (wt.%) = 27.0). Inset in Fig. 11b shows the image of the lamellar liquid crystal without Hb.
W. Liu et al. / International Journal of Biological Macromolecules 37 (2005) 232–238
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Table 1 The fluorescence intensity of Hb in CTAB/n-C5 H11 OH/H2 O systems Medium
Structure
If
Composition OHa ,
CTAB/n-C5 H11 (n-C5 H11 OH/H2 O)b CTAB/n-C5 H11 OH/H2 O microemulsion
H2 O a b
O/W
3/5
W/O
(4/1)
–
–
H2
Oa ,
(CTAB)b
90.0%
347.0
92.5% 95.0% (20.0%) (30.0%) (40.0%) –
368.0 368.0 456.0 454.0 432.0 51.0
Weight ratio or weight percent in O/W microemulsion. Weight ratio or weight percent in W/O microemulsion.
and 4 shows more Hb molecules exist in metHb form in acidic medium. Thus, proton can block the conversion of metHb to hemichrome and retain met-form of Hb both in CTAB/H2 O and CTAB/n-C5 H11 OH/H2 O systems. 4. Conclusions CTAB monomer in Hb aqueous solution can covert metHb to hemichrome, while CTAB micelles, microemulsions and lamellar liquid crystals can make heme release from Hb because heme monomer can be solubilized in the CTAB/n-C5 H11 OH/H2 O systems. Proton can depress the interaction of CTAB with Hb, so more Hb molecules can exist in metHb form in acidic environment. Fig. 10. The concentration of metHb with CTAB. Solvent: H2 O (curve 1), buffer solution with pH being 8.0 (curve 2) and 4.0 (curve 3).
there is no absorbance at 600 nm (curve 2 in Fig. 9). The reason may be that proton can combine with the anion of the hydrophilic groups in Hb and reduce the electrostatic interaction of CTAB with Hb, which makes Hb structure tighter than that in other cases and results in the difficult release of heme from Hb. Fig. 11 shows the UV–vis spectra of Hb in CTAB/nC5 H11 OH/H2 O O/W microemulsions with different pH values. The existence of the absorption peak at 630 nm in curves 3
Fig. 11. UV–vis spectra of Hb (12.2 M/heme) in CTAB/n-C5 H11 OH/H2 O O/W microemulsion (CTAB/n-C5 H11 OH = 3/5, H2 O (wt.%) = 97.0). pH 7.0 (curve 1), 8.0 (curve 2), 4.0 (curves 3 and 4, prepared by HAc–NaAc and citric acid–Na2 HPO4 , respectively).
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