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Comparison of the Different Types of Surfactants for the Effect on Activity and Structure of Soybean Peroxidase Weican Zhang, Xuhui Dai, Yue Zhao, Xuemei Lu,* and Peiji Gao State Key Laboratory of Microbial Technology, Shandong UniVersity, Jinan, Shandong, China, 250100 ReceiVed October 3, 2008. ReVised Manuscript ReceiVed NoVember 24, 2008 In the pH 2.6 and 5.2 systems, soybean peroxidase (SBP) (isoelectric point, pI 3.9) has positive and negative charge, respectively. In order to acquire detailed knowledge on the role played by electrostatics in the denaturation of proteins, a comparison of anionic surfactant sodium dodecyl sulfate (SDS), nonionic surfactant nonaethylene glycol monododecyl ether [C12H25O(CH2CH2O)9H] (AEO9), and cationic surfactant cetyltrimethylammonium bromide (CTAB) for the influences on the activity and structure of soybean peroxidase (SBP) was carried out by measuring the activity, far-UV circular dichrosm, fluorescence, and electronic absorption spectra of SBP in the pH 2.6 and 5.2 systems at 30 °C. In the pH 2.6 systems, the interaction of SDS with SBP results in an increase in the fluorescence intensity with a red shift of the emission maximum of the tryptophan fluorescence and a blue shift of the Soret band. In the meantime, the R-helix of SBP is unfolded and the activity of SBP is lost irreversibly. In pH 5.2 systems, the fluorescence spectra features of SBP are similar to those in pH 2.6 systems with increasing SDS concentration, but a red shift of Soret band as well as an alteration of the tertiary structure of SBP occurs, and the lost activity is recoverable. The electrostatic interactions between SBP and SDS play an important role in the denaturation of SBP. The effects of AEO9 and CTAB in pH 2.6 and 5.2 systems on the activity and spectral features of SBP are similar to that of SDS in pH 5.2 systems, but AEO9 is prone to unfold the β-sheet of SBP in pH 2.6 systems. The electrostatic interactions of CTAB with SBP are not the primary elements for denaturation of SBP, which distinctly differ from those of SDS. These results can be useful with respect to wide applications of the surfactants in the separation and purification of proteins.
Introduction The interactions of surfactants and proteins have been studied extensively due to their importance in many biological, pharmaceutical, and industrial systems.1-7 Anionic surfactants, such as sodium n-dodecyl sulfate, are widely used for the solubilization of membrane proteins and for size separation by electrophoresis or by molecular-sieve chromatography. The nonionic surfactant is often added to a protein solution to prevent aggregation and unwanted adsorption during purification, filtration, transportation, freeze-drying, spray-drying, and storage.5,8-11 In these systems, the protein-surfactant interactions will alter the functional properties of the proteins. In some cases, the interactions may be advantageous, whereas in others they may have a damaging effect on the protein stability or enzyme activity.12-18 An * To whom correspondence should be addressed. E-mail: luxuemei@ sdu.edu.cn. (1) Sahu, K.; Roy, D.; Mondal, S. K.; Karmakar, R.; Bhattacharyya, K. Chem. Phys. Lett. 2005, 404, 341–345. (2) Lu, R. C.; Xiao, J. X.; Cao, A. N.; Lai, L. H.; Zhu, B. Y.; Zhao, G. X. Biochim. Biophys. Acta 2005, 1722, 271–281. (3) Mackie, A.; Wilde, P. AdV. Colloid Interface Sci. 2005, 117, 3–13. (4) Seddon, A. M.; Curnow1, P.; Booth, P. J. Biochim. Biophys. Acta 2004, 1666, 105–117. (5) Chi, E. Y.; Krishnan, S.; Randolph, T. W.; Carpenter, J. F. Pharm. Res. 2003, 20, 1325–1336. (6) Booth, P. J. Biochim. Biophys. Acta 2003, 1610, 51–56. (7) Kiefer, H. Biochim. Biophys. Acta 2003, 1610, 57–62. (8) Blanco, E.; Ruso, J. M.; Prieto, G.; Sarmiento, F. J. Colloid Interface Sci. 2007, 316, 37–42. (9) Haitsma, J. J.; Lachmann, U.; Lachmann, B. AdV. Drug DeliVery ReV. 2001, 47, 197–207. (10) Tani, H.; Kamidate, T.; Watanabe, H. J. Chromatogr., A 1997, 780, 229– 241. (11) Wittenberg, C.; Triplett, E. L. J. Biol. Chem. 1985, 260, 12535–12541. (12) Roy, S.; Dasgupta, A.; Das, P. K. Langmuir 2006, 22, 4567–4573. (13) Ternstro¨m, T.; Svendsen, A.; Akke, M.; Adlercreutz, P. Biochim. Biophys. Acta 2005, 1748, 74–83. (14) Nielsen, A. D.; Arleth, L.; Westh, P. Biochim. Biophys. Acta 2005, 1752, 124–132. (15) Prieto, T.; Nascimento, O. R.; Tersariol, I. L. S.; Faljoni-Alario, A.; Nantes, I. L. J. Phys. Chem. B 2004, 108, 11124–11132.
understanding of the mechanisms involved in proteinsurfactant interactions will provide a basis for rational strategies to optimize these applications. Surfactants are amphiphilic molecules composed of both hydrophilic and hydrophobic groups. Their molecular structures play an important role in protein-surfactant interactions.19-22 It is commonly believed that the binding forces in the surfactant-protein complex may have both electrostatic and hydrophobic nature, which gives rise to the deactivation or unfolding of the proteins. But the mechanism by which the surfactants influence protein structure and function is still not well defined. Thus, the aim of this paper is to increase knowledge about the physical interactions that take place in these systems. Soybean peroxidase (SBP) is an excellent model for studying the mechanism involved in protein-surfactant interactions due to its great stability and activity under wide pH conditions and at elevated temperatures.23-26 It contains 326 amino acids with a mean molecular mass of ∼37 kDa and belongs to the class III of the plant peroxidase superfamily.27,28 Recently, the crystal structures of SBP were determined. Its common features include (16) Chen, J.; Xia, C.; Niu, J.; Li, S. Biochem. Biophys. Res. Commun. 2001, 282, 1220–1223. (17) Jones, M. N. Biochim. Biophys. Acta 1977, 491, 121–128. (18) Moore, B. M.; Flurkey, W. H. J. Biol. Chem. 1990, 265, 4982–4988. (19) Lu, R. C.; Cao, A. N.; Lai, L. H.; Xiao, J. X. Colloids Surf., B 2008, 64, 98–103. (20) Chodankar, S.; Aswal, V. K.; Hassan, P. A.; Wagh, A. G. Phys. B. 2007, 398, 112–117. (21) Khodarahmi, R.; Yazdanparast, R. Int. J. Biol. Macromol 2005, 36, 191– 197. (22) Viseu, M. I.; Carvalho, T. I.; Costa, S. M. Biophys. J. 2004, 86, 2392– 2402. (23) Kamal, J. K. A.; Behere, D. V. Biochemistry 2002, 41, 9034–9042. (24) Geng, Z.; Rao, K. J.; Bassi, A. S.; Gijzen, M.; Krishnamoorthy, N. Catal. Today 2001, 64, 233–238. (25) Anicell, J.; Wright, H. Enzyme Microb. Technol. 1997, 21, 302–310. (26) McEldoon, J. P.; Dordick, J. S. Biotechnol. Prog. 1996, 12, 555–558. (27) Nissum, M.; Feis, A.; Smulevich, G. Biospectroscopy 1998, 4, 355–364. (28) Gijzen, M. Plant J. 1997, 12, 991–998.
10.1021/la803240x CCC: $40.75 2009 American Chemical Society Published on Web 01/22/2009
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Fe(III) protoporphyrin IX as the prosthetic group, a single tryptophan (Trp117), four disulfide bonds, two Ca2+ binding sites located distal and proximal to heme, and eight glycans.>29,30 In this work, three different surfactants, anionic surfactant sodium dodecyl sulfate (SDS), nonionic surfactant nonaethylene glycol monododecyl ether (C12H25O(CH2CH2O)9H) (AEO9), and cationic surfactant cetyltrimethylammonium bromide (CTAB), have been chosen to allow us to characterize the interactions as a function of the counterion effect. The effect of them on the activity and structure of soybean peroxidase have been compared in pH 2.6 and 5.2 buffers in which SBP (isoelectric point, pI 3.9) has positive and negative charge, respectively. In this way, we can acquire detailed knowledge on the role that electrostatics play in the denaturation of proteins. The results can be useful with respect to wide applications of the surfactants in the separation and purification of proteins.
Experimental Section Materials. SDS, AEO9, CTAB, and o-methoxyphenol were purchased from Sigma. SBP (RZ > 3) was purified as previously described.31 Other reagents were analytic grade. Sample Preparation. The samples of SBP (0.25 mg/mL) in pH 2.6 (20 mM citric acid-Na2HPO4) and pH 5.2 (50 mM NaAc-HAc) buffer solutions that had different surfactant concentrations were incubated for 2 h at 30 °C. Control Sample Preparation. The compositions of the control sample were the same as the corresponding sample except for the absence of SBP. Measurement of Effects of Surfactant Concentrations on SBP Activity. SBP is able to catalyze oxidation of o-methoxyphenol by H2O2 to form o-methoxyphenol alliance. The product has an apparent absorbance in the range 400-500 nm. This property has been used to measure the activity of SBP.24 The sample obtained (see Sample Preparation) was diluted with the control sample solution to 2.5 × 10-3 µg/mL SBP. These diluted solutions were used as the stock enzyme solution for the measurement of SBP activity. Reactions were started by the addition of 0.1 mL of the stock enzyme solution to 3.0 mL of control sample solution, which contained 0.5 mM hydrogen peroxide and 8.0 mM omethoxyphenol at 30 °C. The linear absorbance change of assay mixture at 470 nm was recorded with a Shimadzu UV-3100 spectrophotometer. The activity was determined by measuring the rate of oxidation of o-methoxyphenol. Measurement of Recovery of SBP Activity. The sample SBP obtained (see Sample Preparation) was diluted with the corresponding pH value buffer to 2.5 × 10-3 µg/mL SBP. These diluted solutions were used as the stock enzyme solution for the measurement of SBP activity. Reactions were started by the addition of 0.1 mL of the stock enzyme solution to 3.0 mL of the corresponding pH buffers, which contained 0.5 mM hydrogen peroxide and 8.0 mM omethoxyphenol at 30 °C. The activity was determined by the same method as above (see Measurement of Effects of Surfactant Concentrations on SBP Activity). Effects of Surfactant Concentrations on Contents of r-Helix and β-Sheet of SBP. The far-UV CD data of the SBP samples (see Sample Preparation) were recorded on a JASCO 810 spectropolarimeter in the 190-260 nm region using a rectangular cuvette of 1 mm path length at 30 °C. The R-helix and β-sheet contents of SBP were computed with Protein Secondary Structure Estimation Program attached to this instrument. In the measurements, the control sample was used. (29) Welinder, K. G.; Larsen, Y. B. Biochim. Biophys. Acta 2004, 1698, 121– 126. (30) Henriksen, A.; Mirza, O.; Indiani, C.; Teilum, K.; Smulevich, G.; Welinder, K. G.; Gajhede, M. Protein Sci. 2001, 10, 108–115. (31) Liu, W.; Fang, J.; Gao, P. J. Chin. Biochem. Mol. Biol. 1998, 14, 577– 582.
Figure 1. Effects of SDS (A), AEO9 (B), and CTAB (C) concentrations on the SBP relative activity in pH 2.6 and pH 5.2 buffers at 30 °C.
Effects of Surfactant Concentrations on Fluorescence Spectra of SBP. Measurements of steady-state fluorescence were performed on a JASCO FP-6500 spectrofluorimeter at 30 °C. The SBP samples obtained (see Sample Preparation) were placed in a 3 mm path length cylindrical cuvette, and the emission spectra in the range of 300-400 nm were recorded with a fixed excitation wavelength at 295 nm. In the measurements, the control sample was used. Effects of Surfactant Concentrations on Soret Band of SBP. The Soret bands of the SBP samples (see Sample Preparation) were recorded on a Shimadzu UV-3100 spectrophotometer using a 1 cm path length quartz cuvette at 30 °C. In the measurements, the control sample was used.
Results Effects of SDS, CTAB, and AEO9 Concentrations on the SBP Relative Activity. The effects of SDS, CTAB, AEO9 concentrations on the relative activity of SBP in pH 2.6 and 5.2 systems are showed in Figure 1. In pH 2.6 systems (Figure 1A), the activity of SBP sharply looses on addition of a small amount of SDS such that no activity can be detected above 0.5 mM SDS. In these cases, SBP possesses the opposite charge of SDS. However, even up to 80 mM SDS, 20% of enzyme activity is still observed in pH 5.2 systems in which SBP has the same charge as SDS. The contrast between the pH 2.6 system and pH 5.2 systems is very marked and clearly illustrates the importance of the electrostatic charges of SBP in the deactivation process of SBP. In both the pH 2.6 and 5.2 systems, the influences of AEO9 on the relative activity of SBP are almost the same, as shown in Figure 1B. AEO9 does not deactivate SBP over the concentration range 0-15 mM; with increasing AEO9 concentration, the activity decreases gradually. The effect of CTAB on the relative activity of SBP (Figure 1C) is distinctly different from that of SDS, though it is also an ionic surfactant. For CTAB concentration in the 0.1-60 mM range, the activity decreases significantly with increasing CTAB concentration in both pH 2.6 and 5.2 systems. The SBP inactivation is not distinctly correlative with the electrostatic charges of SBP, indicating that the inactivation mechanism of SBP by CTAB is different from that by SDS. Recovery of SBP Activity. The data of the recovery of SBP activity are not showed. The results show that the enzyme activity of SBP is irreversibly lost with SDS treatments in pH 2.6 systems. By contrast, the lost activity with the addition of SDS is entirely recovered in pH 5.2 systems. In both pH 2.6 and 5.2 systems, the lost activity with the addition of AEO9 and CTAB is also
Effect of Surfactant on Soybean Peroxidase
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Table 1. Effects of the SDS Concentrations on the Contents of r-Helix and β-Sheet of SBP in pH 2.6 and 5.2 Systems at 30 °C pH 2.6 [SDS] (mM) R-helix (%) β-sheet (%) rms pH 5.2 [SDS] (mM) R-helix (%) β-sheet (%) rms
0 21.4 25.5 6.02 0 19.8 24.3 5.93
0.1 22.0 21.4 5.10 2.5 19.5 25.3 6.99
0.25 21.3 24.4 7.00 5.0 19.7 24.5 5.38
0.5 24.1 25.3 5.55 10.0 19.1 27.0 5.24
1.0 21.2 26.3 6.48 20.0 19.3 24.2 6.44
2.5 20.5 28.4 7.15 40.0 20.5 23.4 5.92
5.0 17.2 33.1 7.89 80.0 19.9 24.8 5.56
entirely recoverable. In the measurements of recovery of SBP activity, the surfactant concentrations in the assay mixtures are so low as to have no influence on the SBP activity. The above results indicate that the mechanism of deactivation is probably different in these cases. In order to elucidate the above observations, we measured the effects of SDS, AEO9, and CTAB concentrations on the structure of SBP by CD spectroscopy, tryptophan fluorescence, and Soret absorption. Effects of SDS and AEO9 Concentrations on the Contents of r-Helix and β-Sheet of SBP. The effects of SDS and AEO9 concentrations on the contents of R-helix and β-sheet of SBP molecule in pH 2.6 and 5.2 systems is shown in the Tables 1 and 2, respectively. The data for CTAB has not been obtained, because of the intense absorption of CTAB in the far-UV region. In pH 2.6 systems (see Table 1), the secondary structure of SBP remains almost unchanged at low concentrations of SDS ( SDS (pH 5.2) > AEO9 > CTAB (pH 2.6) > CTAB (pH 5.2), when their concentrations are equal. This result as well as the CD data and Soret band suggests that the intensity of interactions of SBP with SDS, AEO9, and CTAB follows the same series. Acknowledgment. This project was financially supported by Shandong Natural Science Foundation (No. y2005B24, y2007D35), Sci-Tech Development Project of Shandong Province (2007GG30002020), and National Natural Science Foundation (No. 30670063). LA803240X
