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Characterization of a surfactant–protein complex by small-angle neutron scattering
PCS 1756
Journal of Physics and Chemistry of Solids 60 (1999) 1383–1386
Characterization of a surfactant–protein complex by small-angle neutron scattering Y. Watanabe a,*, T. Otomo b, S. Shimizu c, T. Adachi b, Y. Sano a, M. Furusaka b b
a National Food Research Institute, Ibaraki 305-8642, Japan Neutron Science Laboratory, High Energy Accelerator Research Organization, Ibaraki 305-0801, Japan c College of Science and Technology, Nihon University, Tokyo 101-8306, Japan
Abstract The interior structure of the complex of reduced carboxymethylated lysozyme with dodecyl sulfate was studied by smallangle neutron scattering. The scattering curve of dodecyl sulfate in the complex was similar to that of the micelle formed by pure dodecyl sulfate. The scattering of the protein in the complex was obtained in the D2O buffer solution that matches approximately the scattering length density of perdeuterated dodecyl sulfate. The Kratky plot revealed that the protein polypeptide in the complex is a wormlike chain with a persistence length of 1.6 nm. q 1999 Elsevier Science Ltd. All rights reserved. Keywords: Protein; C. Neutron scattering
1. Introduction The most popular analytical method for estimating the relative molecular weight of a polypeptide chain in every area of biochemistry and molecular biology is by its mobility in polyacrylamide gel electrophoresis in the presence of dodecyl sulfate [1]. The electrophoretic mobility of a protein is determined not only by its molecular weight but also by its net charge and its shape. Proteins that interact in the typical manner with dodecyl sulfate also appear to produce complexes with similar shapes and net charge densities on these chains. The original conformations of the protein are disrupted by the surfactant, but the nature of the conformation they adopt in dodecyl sulfate is not known. A variety of models such as “rodlike-particle model” [2] and “necklace-model” [3] have been proposed and usefully summarized by Ibel et al. [4]. Despite the relatively extensive investigations that have been carried out on the structure of protein–surfactant complexes, definitive results have yet to be obtained. In this study, as a first approach, the perdeuterated dodecyl sulfate in neutron scattering experiments was used to determine separately the configuration of the amphiphilic molecule and that of the proteinpolypeptide derived from reduced carboxymethylated * Corresponding author.
lysozyme in the complex. We confirmed the micelle structure in the complex and found that the protein polypeptide chain in the complex is a wormlike chain with a persistence length of about 1.6 nm.
2. Experimental Sodium dodecyl sulfate (SDS; 99% pure) was purchased from BDH Chemicals (Poole, UK). Deuterated SDS (SDdS; 96% deuterated) was obtained from Matheson Company (Miamisburg, OH, USA). Six times recrystallized and homogeneous lysozyme was obtained from Daiichi Chemicals (Tokyo, Japan). All other chemicals were of reagent grade. Reduced carboxymethylated lysozyme was prepared through cleavage of disulfide linkages with dithiothreitol and carboxymethylation with monoiodoacetate [5]. The product was lyophilized and then stored below 48C. The lyophilized reduced carboxymethylated lysozyme (38 mg) was dissolved in 2 ml of a D2O (99.8%) buffer solution consisting of 10 mM MOPS, pD 7.0, 200 mM LiCl, 125 mM SDS or SDdS and then treated at 908C for 3 min. The protein concentration was determined by measuring the absorbance at 282 nm assuming the specific extinction coefficient to be 2.56 l g 21 cm 21 after correction of molecular
0022-3697/99/$ - see front matter q 1999 Elsevier Science Ltd. All rights reserved. PII: S0022-369 7(99)00124-9
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Fig. 1. Differential neutron scattering curves of protonated dodecyl sulfate against perdeuterated one for the only surfactant (closed circles) and the surfactant–protein complex (open circles). The concentrations of both dodecyl sulfate and the protein were 125 mM (3.5%) and 1.32 mM (1.9%), respectively.
mass increment by carboxymethylation. The weight ratio of dodecyl sulfate to the protein was 1.84 and corresponded to nearly saturation level for dodecyl sulfate binding to the water-soluble protein [6]. Neutron scattering measurements were made at the small and medium angle scattering instrument (WINK) at High Energy Accelerator Research Organization [7]. The solution samples were contained in flat square-shaped quartz cells of
window thickness 1 mm and path length 3 mm. Multiple samples were mounted on a sample changer at 258C. Each measurement was of duration varying in the 4–6 h. The calibration in intensity was based on the scattering from light water. Sample transmission was taken before and after each measurement to check the material conditions and to correct for absorption of the sample. The buffer solution consisting of 99.8% D2O, 10 mM MOPS, pD 7.0,
Fig. 2. Scattering patterns of perdeuterated dodecyl sulfate (open circles) and the surfactant–protein complex (closed circles) in a D2O buffer solution.
Y. Watanabe et al. / Journal of Physics and Chemistry of Solids 60 (1999) 1383–1386
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Fig. 3. The Kratky plot of the protein polypeptide chain in the dodecyl sulfate–protein complex. When the scattering intensity of the perdeuterated dodecyl sulfate was subtracted from that of the surfactant–protein complex, the value of intersection of the straight line Q p was not changed (data not shown).
200 mM LiCl, 3.5 mM (above the critical micelle concentration) SDS or SDdS corresponding to the sample conditions about dodecyl sulfate was used as a reference solution for scattering experiments.
3. Results and discussion Fig. 1 shows differential neutron scattering patterns between protonated dodecyl sulfate and perdeuterated one in D2O buffer solutions containing 0.2 M LiCl. Filled circles are the data of the only surfactant and open circles are the data of the complex between the surfactant and the reduced carboxymethylated lysozyme. The hydrocarbon core of dodecyl sulfate in both cases should dominate the scattering. It was found that both scattering curves show the same features. Since the data in the low-Q region is affected by the depression of the interparticle structure factor below the ideal-solution value unity, it was impossible to discuss quantitatively about the Rg values. A typical amphiphile is a molecule consisting of a hydrophobic hydrocarbon chain and a hydrophilic polar head. This type molecule is considered to form a small spherical micelle above a critical concentration (that is, the critical micelle concentration). Fig. 1 indicates that dodecyl sulfate chains in the surfactantprotein complex are formed as a micelle-like structure. Under the similar condition to that in this study, the aggregation number of the dodecyl sulfate in its micelle was estimated to be 78 [8]. The aggregation number of dodecyl sulfate in the complex can be estimated to be about 97 (for lithium dodecyl sulfate) mole/mole of protein (the molecular weight of lysozyme is 14 300) because the amount of
dodecyl sulfate binding to the protein was estimated to be 1.84 g/g protein [6]. Therefore, on the basis of the aggregation number, the dodecyl sulfate in the surfactant–protein complex will be assembled as a single micelle, and its micelle size may be slightly larger than that of the pure micelle. Moreover, it is noted that the low-Q scattering intensity of dodecyl sulfate in the surfactant–protein complex was larger than that of the pure dodecyl sulfate (Fig. 1). This result supports the above considerations. Closed circles given in Fig. 2 show the scattering curve from the surfactant–protein complex derived from perdeuterated dodecyl sulfate and reduced carboxymethylated lysozyme in a D2O solution. As shown with open circles in the same figure, the solvent matches approximately the scattering length density of the perdeuterated dodecyl sulfate. The closed circles, therefore, can be assigned to the scattering intensity from the protein polypeptide chain in the surfactant–protein complex. Fig. 3 shows the Kratky plot (Q vs. Q 2I) which reveals clearly the transition region. From intersection of the straight line corresponding to a rod conformation, Q p the persistence length Lp may easily be estimated by the equation Lp 6/(p Q p). In this case, since Q p is 1.2 nm 21, Lp is found to be 1.6 nm. The Rg value of the native protein was about 1.5 nm [9] and the content of helix and random coil of lysozyme denatured in dodecyl sulfate was estimated to be about 50% each other from the far-UV region circular dichroism measurements [10]. From these results it is found that the protein polypeptide chain was partially expanded. In the case of lysozyme denatured in dodecyl sulfate, the hydrophobic residues and hydrophilic charged residues, half of which may form helix, are localized in the interior and the interface of the dodecyl sulfate
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micelle, respectively. In conclusion, by using the perdeuterated dodecyl sulfate, we confirmed the micelle structure in the complex and found that the protein polypeptide chain in the complex is a wormlike chain with a persistence length of about 1.6 nm. Further investigations are in progress to characterize the surfactant–protein complex by small-angle neutron and X-ray scattering because the structural model remains to be established. References [1] K.E. van Holde, W.C. Johnson, P.S. Hon, Principles of Physical Biochemistry, Prentice-Hall, Englewood Cliffs, NJ, 1998 p. 222.
[2] J.A. Reynolds, C. Tanford, J. Biol. Chem. 245 (1970) 5161. [3] K. Shirahama, K. Tsujii, T. Takagi, J. Biochem. 75 (1974) 309. [4] K. Ibel, R.P. May, K. Kirschner, H. Szadkowski, E. Mascher, P. Lundahl, Eur, J. Biochem. 190 (1990) 311. [5] C.H.W. Hirs, Methods in Enzymol. 11 (1967) 199. [6] P.F. Rao, T. Takagi, Anal. Biochem. 174 (1988) 251. [7] M. Furusaka, K. Suzuya, N. Watanabe, M. Osawa, I. Fujikawa, S. Satoh, KENS Report 9 (1993) 25. [8] D. Bandedouch, S.H. Chen, W.C. Koehier, J. Phys. Chem. 87 (1983) 153. [9] D.I. Svergun, S. Richard, H.J. Koch, Z. Sayers, S. Kuprin, G. Zaccai, Proc. Natl. Acad. Sci. USA 95 (1998) 2267. [10] W. Mattice, J.M. Riser, D.S. Clark, Biochem. 15 (1976) 4264.
Journal of Physics and Chemistry of Solids 60 (1999) 1383–1386
Characterization of a surfactant–protein complex by small-angle neutron scattering Y. Watanabe a,*, T. Otomo b, S. Shimizu c, T. Adachi b, Y. Sano a, M. Furusaka b b
a National Food Research Institute, Ibaraki 305-8642, Japan Neutron Science Laboratory, High Energy Accelerator Research Organization, Ibaraki 305-0801, Japan c College of Science and Technology, Nihon University, Tokyo 101-8306, Japan
Abstract The interior structure of the complex of reduced carboxymethylated lysozyme with dodecyl sulfate was studied by smallangle neutron scattering. The scattering curve of dodecyl sulfate in the complex was similar to that of the micelle formed by pure dodecyl sulfate. The scattering of the protein in the complex was obtained in the D2O buffer solution that matches approximately the scattering length density of perdeuterated dodecyl sulfate. The Kratky plot revealed that the protein polypeptide in the complex is a wormlike chain with a persistence length of 1.6 nm. q 1999 Elsevier Science Ltd. All rights reserved. Keywords: Protein; C. Neutron scattering
1. Introduction The most popular analytical method for estimating the relative molecular weight of a polypeptide chain in every area of biochemistry and molecular biology is by its mobility in polyacrylamide gel electrophoresis in the presence of dodecyl sulfate [1]. The electrophoretic mobility of a protein is determined not only by its molecular weight but also by its net charge and its shape. Proteins that interact in the typical manner with dodecyl sulfate also appear to produce complexes with similar shapes and net charge densities on these chains. The original conformations of the protein are disrupted by the surfactant, but the nature of the conformation they adopt in dodecyl sulfate is not known. A variety of models such as “rodlike-particle model” [2] and “necklace-model” [3] have been proposed and usefully summarized by Ibel et al. [4]. Despite the relatively extensive investigations that have been carried out on the structure of protein–surfactant complexes, definitive results have yet to be obtained. In this study, as a first approach, the perdeuterated dodecyl sulfate in neutron scattering experiments was used to determine separately the configuration of the amphiphilic molecule and that of the proteinpolypeptide derived from reduced carboxymethylated * Corresponding author.
lysozyme in the complex. We confirmed the micelle structure in the complex and found that the protein polypeptide chain in the complex is a wormlike chain with a persistence length of about 1.6 nm.
2. Experimental Sodium dodecyl sulfate (SDS; 99% pure) was purchased from BDH Chemicals (Poole, UK). Deuterated SDS (SDdS; 96% deuterated) was obtained from Matheson Company (Miamisburg, OH, USA). Six times recrystallized and homogeneous lysozyme was obtained from Daiichi Chemicals (Tokyo, Japan). All other chemicals were of reagent grade. Reduced carboxymethylated lysozyme was prepared through cleavage of disulfide linkages with dithiothreitol and carboxymethylation with monoiodoacetate [5]. The product was lyophilized and then stored below 48C. The lyophilized reduced carboxymethylated lysozyme (38 mg) was dissolved in 2 ml of a D2O (99.8%) buffer solution consisting of 10 mM MOPS, pD 7.0, 200 mM LiCl, 125 mM SDS or SDdS and then treated at 908C for 3 min. The protein concentration was determined by measuring the absorbance at 282 nm assuming the specific extinction coefficient to be 2.56 l g 21 cm 21 after correction of molecular
0022-3697/99/$ - see front matter q 1999 Elsevier Science Ltd. All rights reserved. PII: S0022-369 7(99)00124-9
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Y. Watanabe et al. / Journal of Physics and Chemistry of Solids 60 (1999) 1383–1386
Fig. 1. Differential neutron scattering curves of protonated dodecyl sulfate against perdeuterated one for the only surfactant (closed circles) and the surfactant–protein complex (open circles). The concentrations of both dodecyl sulfate and the protein were 125 mM (3.5%) and 1.32 mM (1.9%), respectively.
mass increment by carboxymethylation. The weight ratio of dodecyl sulfate to the protein was 1.84 and corresponded to nearly saturation level for dodecyl sulfate binding to the water-soluble protein [6]. Neutron scattering measurements were made at the small and medium angle scattering instrument (WINK) at High Energy Accelerator Research Organization [7]. The solution samples were contained in flat square-shaped quartz cells of
window thickness 1 mm and path length 3 mm. Multiple samples were mounted on a sample changer at 258C. Each measurement was of duration varying in the 4–6 h. The calibration in intensity was based on the scattering from light water. Sample transmission was taken before and after each measurement to check the material conditions and to correct for absorption of the sample. The buffer solution consisting of 99.8% D2O, 10 mM MOPS, pD 7.0,
Fig. 2. Scattering patterns of perdeuterated dodecyl sulfate (open circles) and the surfactant–protein complex (closed circles) in a D2O buffer solution.
Y. Watanabe et al. / Journal of Physics and Chemistry of Solids 60 (1999) 1383–1386
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Fig. 3. The Kratky plot of the protein polypeptide chain in the dodecyl sulfate–protein complex. When the scattering intensity of the perdeuterated dodecyl sulfate was subtracted from that of the surfactant–protein complex, the value of intersection of the straight line Q p was not changed (data not shown).
200 mM LiCl, 3.5 mM (above the critical micelle concentration) SDS or SDdS corresponding to the sample conditions about dodecyl sulfate was used as a reference solution for scattering experiments.
3. Results and discussion Fig. 1 shows differential neutron scattering patterns between protonated dodecyl sulfate and perdeuterated one in D2O buffer solutions containing 0.2 M LiCl. Filled circles are the data of the only surfactant and open circles are the data of the complex between the surfactant and the reduced carboxymethylated lysozyme. The hydrocarbon core of dodecyl sulfate in both cases should dominate the scattering. It was found that both scattering curves show the same features. Since the data in the low-Q region is affected by the depression of the interparticle structure factor below the ideal-solution value unity, it was impossible to discuss quantitatively about the Rg values. A typical amphiphile is a molecule consisting of a hydrophobic hydrocarbon chain and a hydrophilic polar head. This type molecule is considered to form a small spherical micelle above a critical concentration (that is, the critical micelle concentration). Fig. 1 indicates that dodecyl sulfate chains in the surfactantprotein complex are formed as a micelle-like structure. Under the similar condition to that in this study, the aggregation number of the dodecyl sulfate in its micelle was estimated to be 78 [8]. The aggregation number of dodecyl sulfate in the complex can be estimated to be about 97 (for lithium dodecyl sulfate) mole/mole of protein (the molecular weight of lysozyme is 14 300) because the amount of
dodecyl sulfate binding to the protein was estimated to be 1.84 g/g protein [6]. Therefore, on the basis of the aggregation number, the dodecyl sulfate in the surfactant–protein complex will be assembled as a single micelle, and its micelle size may be slightly larger than that of the pure micelle. Moreover, it is noted that the low-Q scattering intensity of dodecyl sulfate in the surfactant–protein complex was larger than that of the pure dodecyl sulfate (Fig. 1). This result supports the above considerations. Closed circles given in Fig. 2 show the scattering curve from the surfactant–protein complex derived from perdeuterated dodecyl sulfate and reduced carboxymethylated lysozyme in a D2O solution. As shown with open circles in the same figure, the solvent matches approximately the scattering length density of the perdeuterated dodecyl sulfate. The closed circles, therefore, can be assigned to the scattering intensity from the protein polypeptide chain in the surfactant–protein complex. Fig. 3 shows the Kratky plot (Q vs. Q 2I) which reveals clearly the transition region. From intersection of the straight line corresponding to a rod conformation, Q p the persistence length Lp may easily be estimated by the equation Lp 6/(p Q p). In this case, since Q p is 1.2 nm 21, Lp is found to be 1.6 nm. The Rg value of the native protein was about 1.5 nm [9] and the content of helix and random coil of lysozyme denatured in dodecyl sulfate was estimated to be about 50% each other from the far-UV region circular dichroism measurements [10]. From these results it is found that the protein polypeptide chain was partially expanded. In the case of lysozyme denatured in dodecyl sulfate, the hydrophobic residues and hydrophilic charged residues, half of which may form helix, are localized in the interior and the interface of the dodecyl sulfate
1386
Y. Watanabe et al. / Journal of Physics and Chemistry of Solids 60 (1999) 1383–1386
micelle, respectively. In conclusion, by using the perdeuterated dodecyl sulfate, we confirmed the micelle structure in the complex and found that the protein polypeptide chain in the complex is a wormlike chain with a persistence length of about 1.6 nm. Further investigations are in progress to characterize the surfactant–protein complex by small-angle neutron and X-ray scattering because the structural model remains to be established. References [1] K.E. van Holde, W.C. Johnson, P.S. Hon, Principles of Physical Biochemistry, Prentice-Hall, Englewood Cliffs, NJ, 1998 p. 222.
[2] J.A. Reynolds, C. Tanford, J. Biol. Chem. 245 (1970) 5161. [3] K. Shirahama, K. Tsujii, T. Takagi, J. Biochem. 75 (1974) 309. [4] K. Ibel, R.P. May, K. Kirschner, H. Szadkowski, E. Mascher, P. Lundahl, Eur, J. Biochem. 190 (1990) 311. [5] C.H.W. Hirs, Methods in Enzymol. 11 (1967) 199. [6] P.F. Rao, T. Takagi, Anal. Biochem. 174 (1988) 251. [7] M. Furusaka, K. Suzuya, N. Watanabe, M. Osawa, I. Fujikawa, S. Satoh, KENS Report 9 (1993) 25. [8] D. Bandedouch, S.H. Chen, W.C. Koehier, J. Phys. Chem. 87 (1983) 153. [9] D.I. Svergun, S. Richard, H.J. Koch, Z. Sayers, S. Kuprin, G. Zaccai, Proc. Natl. Acad. Sci. USA 95 (1998) 2267. [10] W. Mattice, J.M. Riser, D.S. Clark, Biochem. 15 (1976) 4264.