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Rapid and sensitive determination of proteins by enhanced resonance light scattering spectroscopy of sodium lauroyl glutamate
Talanta 71 (2007) 1246–1251
Rapid and sensitive determination of proteins by enhanced resonance light scattering spectroscopy of sodium lauroyl glutamate Zhanguang Chen ∗ , Jinbin Liu, Yali Han Department of Chemistry, Shantou University, Shantou 515063, China Received 15 April 2006; received in revised form 18 June 2006; accepted 18 June 2006 Available online 27 July 2006
Abstract A rapid and sensitive method for the determination of proteins is proposed based on the measurements of the enhanced resonance light scattering (RLS) spectroscopy of sodium lauroyl glutamate (SLG). Under the optimum conditions, the interaction between SLG and proteins occurred rapidly, resulting in greatly enhanced RLS intensity with the maximum peak located at 394 nm. It was found that the enhanced RLS intensities were in proportion to the concentrations of proteins in the range of 0.01–3.1 g ml−1 depending on the kind of proteins. The detection limits were below 6 ng ml−1 . Compared with some other methods for the determination of proteins, this method shows high sensitivity, low detection limit and simplicity. This is an inexpensive, simple and fast one-step procedure which requires only measuring the RLS intensities. Human serum samples were determined with satisfactory results. © 2006 Elsevier B.V. All rights reserved. Keywords: Resonance light scattering (RLS); Sodium lauroyl glutamate (SLG); Acylamino acid-type surfactant; Proteins
1. Introduction The development of novel methods for the determination of proteins is of great importance in a number of areas, such as chemical and biochemical analysis, clinical diagnosis and biotechnology. Spectrophotometric methods, such as Biuret [1], Lowry [2], and Bradford [3], which are based on the distinct color change of dyes on binding proteins, have been widely used for the determination of proteins. However, these methods have their defects in terms of sensitivity, selectivity and simplicity. In order to solve some of these problems, chemiluminescence [4] and fluorescence [5] methods were developed. At present, resonance light scattering (RLS) technique has become the preferred choice for the study and determination of proteins. RLS technique as an analytical method has the distinct advantages of speed, convenience and sensitivity. RLS is an elastic scattering occurs when the incident beam is close in energy to an absorption band. RLS is an extremely sensitive and selective
∗
Corresponding author. Tel.: +86 7542903330. E-mail address: [email protected] (Z. Chen).
0039-9140/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2006.06.025
technique for monitoring molecular assemblies. Since Pasternack et al. established the RLS technique to study biological macromolecules with a common fluorescence spectrometer [6–8], it has been increasingly applied to the study and determination of nucleic acids [9–13], proteins [14–17], amino acid [18], saccharide [19,20], medicines [21,22] and metal ions [23,24]. In this paper, we have developed a novel method for the direct determination of the trace amount of total protein based on the interaction between sodium lauroyl glutamate (SLG) and proteins by RLS technique. This assay is based on the observation that the RLS intensity of SLG is strongly enhanced by proteins. SLG is an acylamino acid-type surfactant that can be easily purchased from the market. The acylamino acid-type surfactants display high foam stability and are less irritating to the skin than many other ionic surfactants [25]. Because of these useful characteristics, they are often combined with other surfactants in many consumer products, such as cosmetics, health care products, and pharmaceuticals. Some anion surfactants have been used for the determination of proteins by RLS technique with good sensitivity [26]. But the use of acylamino acid-type surfactant as the probe for the direct determination of proteins has not yet been reported. Its excellent and special properties would prove SLG
Z. Chen et al. / Talanta 71 (2007) 1246–1251
a much better protein probe than some other anion surfactants. Our experiments showed that this method has the merits of simplicity, high sensitivity and relative absence of interferences. The samples of human serum were analyzed with satisfactory results. The mechanism of the reaction between SLG and proteins was also discussed. 2. Experimental 2.1. Apparatus The RLS spectra and fluorescence spectra were measured with a Perkin-Elmer Model LS-55 spectrofluorometer equipped with a quartz cuvette (1 cm × 1 cm). An SA 720 instrument (Orion Research) was used to measure the pH of the solution. 2.2. Reagents The stock solutions of proteins were prepared by dissolving commercially purchased proteins in doubly distilled water. The concentration of the working solution was 20.0 g ml−1 by diluting the protein stock solutions. Proteins in this work included bovine serum albumin (BSA), human serum albumin (HSA) (Sino-American Biotechnology Company, China) and ␥-globulin (␥-G), lysozyme and hemoglobin (bovine) (Sigma, St. Louis, MO, USA). They were all stored at 0–4 ◦ C. Sodium lauroyl glutamate (SLG) was obtained from Shanghai Leasun Chemical Co. Ltd. (China). Its stock solution was prepared by dissolving the product in the doubly distilled water and the concentration of its working solution was 1.0 × 10−3 mol l−1 . The Britton-Robinson (BR) buffer solution, being made up of 0.04 mol l−1 phosphoric acid, 0.04 mol l−1 acetic acid, 0.04 mol l−1 boric acid, 0.2 mol l−1 sodium hydroxide, was used to control the acidity of the solution. 0.05 mol l−1 NaCl solution was used to adjust the ionic strength of the aqueous solution. Human serum samples were bought from Shantou Central Hospital and diluted with doubly distilled water. All chemicals used were of analytical grade or the best grade commercially available and doubly distilled water was used throughout.
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3. Results and discussion 3.1. Spectral characteristics and reaction The RLS spectra of the reagent blank and the SLG–BSA system under optimum conditions (1.2 × 10−4 mol l−1 SLG; pH 4.0) are shown in Fig. 1. It can be seen that the RLS intensity of the reagent blank is very weak in the wavelength range of 250–700 nm. However, the enhanced RLS intensity can be clearly observed with a maximum peak located at 394 nm when a trace amount of BSA was added. Enhanced RLS shoulder peaks at 454 and 485 nm can also be observed, and moreover, the enhanced RLS intensity increases with the increasing BSA concentration. Therefore, 394 nm was selected as the analytical wavelength. It can be consequently concluded that SLG reacted with BSA and produced a complex of which RLS intensity was much higher than that of BSA or SLG when they existed separately. The fluorescence excitation (line 1) and emission (line 2) spectra of BSA are shown in Fig. 2. It can be seen only one emission peak located at 344 nm in the emission spectra (b) and two excitation peaks located at 224 and 281 nm. Fig. 3a displays the effects of SLG at variable concentrations on the fluorescence
Fig. 1. The RLS spectra of the SLG (line 1) and SLG–BSA system (lines 2–4). Conditions: CSLG = 1.2 × 10−4 mol l−1 , CBSA (g ml−1 ): (1) 0.0; (2) 0.4; (3) 1.0; (4) 2.0; pH 4.0.
2.3. General procedures Into a 10-ml calibrated flask were added 1.0 ml BrittonRobinson buffer, 1.2 ml the working solution of SLG and appropriate proteins (or samples), then diluted to 10.0 ml with doubly distilled water and stirred thoroughly. All RLS spectra were obtained by scanning simultaneously the excitation and emission monochromators (λ = 0.0 nm) from 250.0 to 700.0 nm with the excitation and emission slits 8.0 nm wide. The RLS intensities were measured at 394 nm. The enhanced RLS intensity of the system by proteins was rep0 0 resented as IRLS = IRLS − IRLS (IRLS and IRLS were the RLS intensities of the systems with and without proteins).
Fig. 2. The fluorescence excitation (line 1) and emission (line 2) spectra of BSA. Conditions: CBSA = 1.0 g ml−1 ; pH 4.0.
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Fig. 4. The effect of pH on the RLS intensity. Conditions: CSLG = 1.2 × 10−4 mol l−1 , CBSA = 1.0 g ml−1 .
RLS intensity reached its maximum. This is possibly related to the isoelectric point of proteins. Proteins are positively charged when the pH is lower than their isoelectric points. SLG contains two carboxylic acid groups, which carries strong negative charges. In acidic medium, the electrostatic interaction between SLG and proteins can be very strong, which is favorable for the RLS intensity enhancement. So pH 4.0 was chosen for the determination. 3.3. The effect of SLG concentration (CSLG ) Fig. 3. The effect of SLG at variable concentrations on the fluorescence excitation (a) and emission (b) spectra of BSA. Conditions: CBSA = 1.0 g ml−1 , CSLG (mol l−1 ): (1) 0; (2) 0.4 × 10−4 ; (3) 0.8 × 10−4 ; (4) 1.2 × 10−4 ; (5) 1.6 × 10−4 ; pH 4.0.
excitation spectra of the BSA system (λem = 344 nm). The results show that the intensity of the excitation spectra was affected by the addition of the SLG, together with a gradual red shift. This phenomenon indicated that the interaction between the SLG and the protein occurred. The presence of SLG also modified the wavelength of maximum emission. In particular, as shown in Fig. 3b, it was observed a decrease in the intensity of the fluorescence emission intensity concomitant with a blue shift of the emission maximum in the presence of SLG. Fluorescence quenching has been a powerful tool to reveal the accessibility of fluorophores in the protein matrix to quenchers. Static quenching refers to formation of a nonfluorescent fluorophore–quencher complex [27]. The changes in the emission spectra suggested for the formation of a SLG–protein complex. The association of SLG to proteins with their hydrophobic groups can produce changes in the position or orientation of the amino acid residues, altering their exposure to the solvent water, leading to alterations on the quantum yield, and thus resulting in a quenching of the fluorescence.
As shown in Fig. 5, the concentration of SLG influenced significantly on the RLS intensity of the system. It can be clearly seen from Fig. 5 that SLG concentration in the range of 2.0 × 10−5 mol l−1 to 2.0 × 10−4 mol l−1 had a great influence on the RLS intensity. With increasing SLG concentration, the IRLS increased and reached a maximum at 1.2 × 10−4 mol l−1 . Then an increase of SLG concentration led to a reduction of the RLS intensity. So 1.2 × 10−4 mol l−1 SLG was selected in this assay. Because the critical micelle concentration (CMC) of SLG is 9.44 × 10−4 to 1.02 × 10−3 mol l−1 [28], the interaction between surfactants and proteins can be interpreted in the framework of molecule-to-molecule interaction.
3.2. The effect of pH As Fig. 4 shows, the RLS intensity of the assay system was affected by pH. Variation of pH from 2.1 to 6.0 at the concentration of 1.0 g ml−1 of BSA was studied. At pH 4.0, the enhanced
Fig. 5. The effect of SLG concentration on the RLS intensity. Conditions: CBSA = 1.0 g ml−1 ; pH 4.0.
Z. Chen et al. / Talanta 71 (2007) 1246–1251
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Table 1 Effects of amino acids on the determination of BSA (1.0 g ml−1 ) under the optimum conditions
Fig. 6. Effect of ionic strength on the RLS intensity. Conditions: CSLG = 1.2 × 10−4 mol l−1 , CBSA = 1.0 g ml−1 ; pH 4.0.
3.4. The effect of ionic strength The effect of ionic strength on the RLS intensity of the system is presented clearly in Fig. 6. It can be seen that the RLS intensity of the system SLG–BSA decreased with the increasing of NaCl concentration. The control of ionic strength of the solution is very important, indicating that electrostatic interaction is also one of the main driving forces governing their combination. When the concentration of the NaCl increases, the effects of the electrostatic shielding of charges, including the shielding of protein molecules from SLG or the shielding of SLG from protein molecules, reduce the combination of SLG with protein and result in a decreased RLS signal. 3.5. Incubation time and stability The stability of the SLG–BSA system was studied by measuring the RLS intensity every 2 min for 2 h immediately after mixing. Our results show that the reaction between SLG and proteins occurred rapidly at room temperature (<4 min) and the RLS intensity of the system reached the maximum. Moreover, the RLS intensity remained stable for at least 90 min. Thus, this assay is rapid and does not require crucial timing. 3.6. Interference of foreign substances The effect of the foreign substances on the determination of only 1.0 g ml−1 BSA was tested by pre-mixing BSA with compounds such as common ions, sugars and amino acids under the optimum conditions of the general procedure. The criterion for an interference was an IRLS value varying by more than 5% from the expected value for BSA alone. There was no interference from the following ions: 500 mol l−1 of K+ , 100 mol l−1 of Na+ , 50 mol l−1 of Ca2+ and Mg2+ , 25 mol l−1 of Zn2+ , Al3+ and Fe2+ , 20 mol l−1 of Mn2+ and Cu2+ , 15 mol l−1 of Cr2+ and Co2+ , 10 mol l−1 of Cd2+ , Hg2+ and Pb2+ , 25 mol l−1 of H2 PO4 − , 5.0 mol l−1 of PO4 3− . Especially, glucose, ethanol, and urea could be allowed at very high concentration. On the contrary, some other ions such as I− and Fe3+ only can be tolerated at relative low concentration levels (less than 5.0 mol l−1 ).
Amino acids (50.0 g ml−1 )
Change in IRLS (%)
Amino acids (50.0 g ml−1 )
Change in IRLS (%)
l-Proline l-Cysteine l-Asparagine l-Leucine l-Arginine l-Glutamine
+0.5 +1.1 +0.6 −1.7 +2.8 −1.4
l-Histidine l-Lysine l-Phenylalanine l-Serine l-Tryptophan l-Tyrosine
+0.9 +7.9 +2.3 −5.3 +4.8 −1.2
As shown in Table 1, most of the amino acids also do not interfere with the determination. Dilution with water can minimize all these interferences, which offers this method with the possibility of determination of trace amount of proteins in real samples. 3.7. Calibration curves and sensitivities According to the above standard procedure, the RLS intensities were obtained under the optimum conditions. The analytical parameters of the determination are listed in Table 2. The limits of detection (LOD) are given by 3S0 /S, where 3 is the factor at the 99% confidence level, S0 the standard deviation of the blank measurements (n = 10), and S is the slope of the calibration curve. Satisfactory linear relationship with low detection limits (below 6 ng ml−1 ) and relatively wide linear ranges were obtained. As shown in Table 3, compared with other methods of determination of proteins, this method has the advantages of high sensitivity, low detection limit and fine simplicity. 3.8. Application to sample analysis Table 4 displays the determination results for human serum samples with HSA as the standard. The samples were diluted 2000-fold with doubly distilled water without further purification. The results displayed in Table 4 are in good agreement with the data provided by the hospital. As shown in Table 2, the protein-to-protein variability cannot be strictly avoided with RLS technique, but the responses for some of the proteins such as BSA, HSA and ␥-G, are approximately equal with this method. It is obvious that this method for the determination of the total protein is reliable, sensitive and practical. This measurement system can be encouraged their practical use in chemical and biochemical applications. Table 2 Analytical parameters of this method under the optimum conditions Protein
Linear range (g ml−1 )
Regression equation
Detection limit (ng ml−1 )
Correlation coefficient (r)
Lysozyme BSA HSA ␥-G Hemoglobin
0.08–3.1 0.01–2.7 0.04–2.2 0.08–1.7 0.01–1.3
I = 4.3 + 121.1C I = 2.1 + 134.6C I = 3.7 + 131.2C I = 6.1 + 129.3C I = 1.8 + 109.7C
4.6 1.4 1.7 2.8 5.8
0.9986 0.9991 0.9997 0.9989 0.9981
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Table 3 Comparisons with some other assays of proteins Method
Reagents
Abs Abs Flu Flu RLS RLS RLS RLS RLS RLS RLS RLS
Lowry CBB Chrome azurol S ECRa TPPS4 b Quercetin EBBRc Alizarin Red S Ponceau 4R SDBSd SLSe SLG
a b c d e
Protein
HAS HAS BSA/HSA BSA/HSA BSA/HSA BSA/HSA BSA/HSA BSA/HSA BSA/HSA BSA/HSA
Detection limit (ng ml−1 )
References
1.0 × 105 5000 100 500 18.0/53.5 59.8/44.4 33.0/25.0 9.59/9.51 6.96/5.71 1.8/2.8 12.8/21.6 1.4/1.7
[2] [3] [29] [30] [14] [31] [32] [33] [15] [26] [26] This work
Eriochrome Cyanine R. ␣,,␥,␦-Tetrakis 4-(sulfophenyl) porphine. Eriochrome Blue Black R. Sodium dodecyl benzene sulfonate. Sodium lauryl sulfate.
3.9. Mechanism of the reaction Proteins and surfactants share the property of having both charged groups and hydrophobic portions. This implies that the interactions between surfactants and proteins are intrinsically complex processes, involving different types of intermolecular forces. In order to clarify the mechanism of the interaction between them, dependence of the RLS intensity on pH was carefully studied (see Fig. 4). The results show that the RLS intensity was sensitive to the solution pH. It is particularly relevant to the characteristics of both SLG and proteins. In pH < 2.9, both of the two carboxylic acid groups of SLG would be protonated, so the RLS intensity was relatively small. Whereas, in pH > 4.6, proteins were negatively charged. In pH 3.8–4.2, SLG carried strong negative charges, and proteins carried positive charges. So the RLS intensity was strong. The presence of two negative charged hydrophilic groups in the same SLG molecule leads to the better combination with the proteins by their electrostatic interactions. The dependence of the RLS intensity on the pH and the ionic strength is fully compatible with this interpretation. Besides the properties mentioned above, SLG possesses the acylamino group, which can easily form strong hydrogen bonding with proteins [25]. It thus performed better as protein RLS probe than other anion surfactants.
Table 4 Analytical results of human serum samplesa Samples (no.)
This method (n = 6, mg ml−1 )
R.S.D. (%)
Clinical datab (mg ml−1 )
Serum 1 Serum 2 Serum 3
69.4 ± 0.76 67.9 ± 0.61 84.1 ± 1.7
1.1 0.9 2.1
71.1 ± 1.4 69.8 ± 0.94 81.3 ± 1.5
Data shown are average ± S.D. from six independent experiments. Obtained from the Shantou Central Hospital, average from three measurements. a
b
As evidenced from the fluorescence excitation and emission spectra changing with the variable SLG concentrations, it can be concluded that the change of the fluorescence spectra resulted from the changes in the orientation of the protein structures [34]. The change of the protein structures would lead to a better combination with their hydrophobic groups. The data obtained in the present work support a SLG–protein interaction in which hydrophobic forces also play an important role in the RLS intensity enhancement. The surfactant SLG with special characteristics favors better interaction, thus SLG was shown better performance over other surfactants for the determination of proteins. 4. Conclusion The study of the interaction between surfactants and proteins is of significant scientific and technological importance. The acylamino acid-type surfactants with special properties and advantages are attractive molecules to probe proteins by RLS technique. The results of this study show that SLG played an effective role as a protein probe in the determination of trace amount of proteins using RLS technique. This is an inexpensive, simple and fast one-step procedure which requires only measuring the RLS intensities. It was applied to determine human serum samples satisfactorily. Therefore, it is a useful method to be used in practical applications. Acknowledgements All authors express their sincere thanks for the support from the National Natural Science Foundation of China (no. 30271033) and the Municipal Science Foundation of Shantou (no. S02023). References [1] L.X. Zhang, G.L. Wu, Advanced Biochemical Experiments, Advanced Education Press, Beijing, 1989, p. 192. [2] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193 (1951) 265. [3] M.M. Bradford, Anal. Biochem. 72 (1976) 248. [4] T. Hara, M. Toriyama, K. Tsukagoshi, Bull. Chem. Soc. Jpn. 61 (1988) 2779. [5] N. Li, K.A. Li, S.Y. Tong, Anal. Biochem. 233 (1996) 151. [6] R.F. Pasternack, C. Bustamante, P.J. Collings, A. Giannetto, E.J. Gibbs, J. Am. Chem. Soc. 115 (1993) 5393. [7] R.F. Pasternack, P.J. Collings, Science 269 (1993) 935. [8] R.F. Pasternack, K.F. Schaefer, P. Hambright, Inorg. Chem. 33 (1994) 2062. [9] C.Z. Huang, K.A. Li, S.Y. Tong, Anal. Chem. 68 (1996) 2259. [10] Z.P. Li, K.A. Li, S.Y. Tong, Talanta 55 (2001) 669. [11] Z.G. Chen, W.F. Ding, F.L. Ren, J.B. Liu, Y.Z. Liang, Anal. Chim. Acta 550 (2005) 204. [12] Z.G. Chen, W.F. Ding, F.L. Ren, Y.Z. Liang, J.B. Liu, Microchim. Acta 150 (2005) 34. [13] Z.G. Chen, W.F. Ding, F.L. Ren, Y.L. Han, J.B. Liu, Anal. Lett. 38 (2005) 2301. [14] R.P. Jia, L.J. Dong, Q.F. Li, X.G. Chen, Z.D. Hu, Talanta 57 (2002) 693. [15] H. Zhong, J.J. Xu, H.Y. Chen, Talanta 67 (2005) 749. [16] Z.G. Chen, T.Y. Zhang, F.L. Ren, W.F. Ding, Microchim. Acta 153 (2006) 65.
Z. Chen et al. / Talanta 71 (2007) 1246–1251 [17] Z.G. Chen, W.F. Ding, F.L. Ren, J.B. Liu, Y.L. Han, Can. J. Anal. Sci. Spect. 50 (2005) 54. [18] Z.G. Chen, J.B. Liu, Y.L. Han, L. Zhu, Anal. Chim. Acta 570 (2006) 109. [19] S.Z. Zhang, F.L. Zhao, K.A. Li, S.Y. Tong, Anal. Chim. Acta 431 (2001) 133. [20] S.Z. Zhang, F.L. Zhao, K.A. Li, S.Y. Tong, Talanta 54 (2001) 333. [21] C.Z. Huang, X.B. Pang, Y.F. Li, Y.J. Long, Talanta 69 (2006) 180. [22] P. Feng, W.Q. Shu, C.Z. Huang, Y.F. Li, Anal. Chem. 73 (2001) 4307. [23] S.P. Liu, Q. Liu, Z.F. Liu, M. Li, C.Z. Huang, Anal. Chim. Acta 379 (1999) 53. [24] X.F. Long, S.P. Bi, H.Y. Ni, X.C. Tao, N. Gan, Anal. Chim. Acta 501 (2004) 89. [25] C. Liu, X.M. Jiang, B.Z. Li, W.F. Zhang, Application Encyclopedia of Surfactants, Beijing Industry University Press, Beijing, 1992, p. 43.
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[26] R.T. Liu, J.H. Yang, C.X. Sun, X. Wu, L. Li, Z.M. Li, Anal. Bioanal. Chem. 377 (2003) 375. [27] E.L. Gelamo, M. Tabak, Spectrochim. Acta A 56 (2000) 2255. [28] X.L. Wei, B.L. Yin, Q. Sang, H.B. Zhang, Liaocheng Shiyuan Xuebao 14 (2001) 66. [29] Y. Saito, Y. Inden-Okazaki, S. Wada-Yano, A. Kanetsuna, K. Miyazaki, M. Mifune, Y. Tanaka, Anal. Chim. Acta 178 (1985) 337. [30] Y.X. Ci, L. Chen, Analyst 113 (1988) 679. [31] P. Feng, X.L. Hu, C.Z. Huang, Anal. Lett. 32 (1999) 1323. [32] Y.K. Zhao, W.B. Chang, Y.X. Ci, Talanta 59 (2003) 477. [33] H. Zhong, N. Li, F.L. Zhao, K.A. Li, Talanta 62 (2004) 37. [34] G.V. Lubarsky, M.M. Browne, S.A. Mitchell, M.R. Davidson, R.H. Bradley, Colloid Surf. B 44 (2005) 56.
Rapid and sensitive determination of proteins by enhanced resonance light scattering spectroscopy of sodium lauroyl glutamate Zhanguang Chen ∗ , Jinbin Liu, Yali Han Department of Chemistry, Shantou University, Shantou 515063, China Received 15 April 2006; received in revised form 18 June 2006; accepted 18 June 2006 Available online 27 July 2006
Abstract A rapid and sensitive method for the determination of proteins is proposed based on the measurements of the enhanced resonance light scattering (RLS) spectroscopy of sodium lauroyl glutamate (SLG). Under the optimum conditions, the interaction between SLG and proteins occurred rapidly, resulting in greatly enhanced RLS intensity with the maximum peak located at 394 nm. It was found that the enhanced RLS intensities were in proportion to the concentrations of proteins in the range of 0.01–3.1 g ml−1 depending on the kind of proteins. The detection limits were below 6 ng ml−1 . Compared with some other methods for the determination of proteins, this method shows high sensitivity, low detection limit and simplicity. This is an inexpensive, simple and fast one-step procedure which requires only measuring the RLS intensities. Human serum samples were determined with satisfactory results. © 2006 Elsevier B.V. All rights reserved. Keywords: Resonance light scattering (RLS); Sodium lauroyl glutamate (SLG); Acylamino acid-type surfactant; Proteins
1. Introduction The development of novel methods for the determination of proteins is of great importance in a number of areas, such as chemical and biochemical analysis, clinical diagnosis and biotechnology. Spectrophotometric methods, such as Biuret [1], Lowry [2], and Bradford [3], which are based on the distinct color change of dyes on binding proteins, have been widely used for the determination of proteins. However, these methods have their defects in terms of sensitivity, selectivity and simplicity. In order to solve some of these problems, chemiluminescence [4] and fluorescence [5] methods were developed. At present, resonance light scattering (RLS) technique has become the preferred choice for the study and determination of proteins. RLS technique as an analytical method has the distinct advantages of speed, convenience and sensitivity. RLS is an elastic scattering occurs when the incident beam is close in energy to an absorption band. RLS is an extremely sensitive and selective
∗
Corresponding author. Tel.: +86 7542903330. E-mail address: [email protected] (Z. Chen).
0039-9140/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2006.06.025
technique for monitoring molecular assemblies. Since Pasternack et al. established the RLS technique to study biological macromolecules with a common fluorescence spectrometer [6–8], it has been increasingly applied to the study and determination of nucleic acids [9–13], proteins [14–17], amino acid [18], saccharide [19,20], medicines [21,22] and metal ions [23,24]. In this paper, we have developed a novel method for the direct determination of the trace amount of total protein based on the interaction between sodium lauroyl glutamate (SLG) and proteins by RLS technique. This assay is based on the observation that the RLS intensity of SLG is strongly enhanced by proteins. SLG is an acylamino acid-type surfactant that can be easily purchased from the market. The acylamino acid-type surfactants display high foam stability and are less irritating to the skin than many other ionic surfactants [25]. Because of these useful characteristics, they are often combined with other surfactants in many consumer products, such as cosmetics, health care products, and pharmaceuticals. Some anion surfactants have been used for the determination of proteins by RLS technique with good sensitivity [26]. But the use of acylamino acid-type surfactant as the probe for the direct determination of proteins has not yet been reported. Its excellent and special properties would prove SLG
Z. Chen et al. / Talanta 71 (2007) 1246–1251
a much better protein probe than some other anion surfactants. Our experiments showed that this method has the merits of simplicity, high sensitivity and relative absence of interferences. The samples of human serum were analyzed with satisfactory results. The mechanism of the reaction between SLG and proteins was also discussed. 2. Experimental 2.1. Apparatus The RLS spectra and fluorescence spectra were measured with a Perkin-Elmer Model LS-55 spectrofluorometer equipped with a quartz cuvette (1 cm × 1 cm). An SA 720 instrument (Orion Research) was used to measure the pH of the solution. 2.2. Reagents The stock solutions of proteins were prepared by dissolving commercially purchased proteins in doubly distilled water. The concentration of the working solution was 20.0 g ml−1 by diluting the protein stock solutions. Proteins in this work included bovine serum albumin (BSA), human serum albumin (HSA) (Sino-American Biotechnology Company, China) and ␥-globulin (␥-G), lysozyme and hemoglobin (bovine) (Sigma, St. Louis, MO, USA). They were all stored at 0–4 ◦ C. Sodium lauroyl glutamate (SLG) was obtained from Shanghai Leasun Chemical Co. Ltd. (China). Its stock solution was prepared by dissolving the product in the doubly distilled water and the concentration of its working solution was 1.0 × 10−3 mol l−1 . The Britton-Robinson (BR) buffer solution, being made up of 0.04 mol l−1 phosphoric acid, 0.04 mol l−1 acetic acid, 0.04 mol l−1 boric acid, 0.2 mol l−1 sodium hydroxide, was used to control the acidity of the solution. 0.05 mol l−1 NaCl solution was used to adjust the ionic strength of the aqueous solution. Human serum samples were bought from Shantou Central Hospital and diluted with doubly distilled water. All chemicals used were of analytical grade or the best grade commercially available and doubly distilled water was used throughout.
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3. Results and discussion 3.1. Spectral characteristics and reaction The RLS spectra of the reagent blank and the SLG–BSA system under optimum conditions (1.2 × 10−4 mol l−1 SLG; pH 4.0) are shown in Fig. 1. It can be seen that the RLS intensity of the reagent blank is very weak in the wavelength range of 250–700 nm. However, the enhanced RLS intensity can be clearly observed with a maximum peak located at 394 nm when a trace amount of BSA was added. Enhanced RLS shoulder peaks at 454 and 485 nm can also be observed, and moreover, the enhanced RLS intensity increases with the increasing BSA concentration. Therefore, 394 nm was selected as the analytical wavelength. It can be consequently concluded that SLG reacted with BSA and produced a complex of which RLS intensity was much higher than that of BSA or SLG when they existed separately. The fluorescence excitation (line 1) and emission (line 2) spectra of BSA are shown in Fig. 2. It can be seen only one emission peak located at 344 nm in the emission spectra (b) and two excitation peaks located at 224 and 281 nm. Fig. 3a displays the effects of SLG at variable concentrations on the fluorescence
Fig. 1. The RLS spectra of the SLG (line 1) and SLG–BSA system (lines 2–4). Conditions: CSLG = 1.2 × 10−4 mol l−1 , CBSA (g ml−1 ): (1) 0.0; (2) 0.4; (3) 1.0; (4) 2.0; pH 4.0.
2.3. General procedures Into a 10-ml calibrated flask were added 1.0 ml BrittonRobinson buffer, 1.2 ml the working solution of SLG and appropriate proteins (or samples), then diluted to 10.0 ml with doubly distilled water and stirred thoroughly. All RLS spectra were obtained by scanning simultaneously the excitation and emission monochromators (λ = 0.0 nm) from 250.0 to 700.0 nm with the excitation and emission slits 8.0 nm wide. The RLS intensities were measured at 394 nm. The enhanced RLS intensity of the system by proteins was rep0 0 resented as IRLS = IRLS − IRLS (IRLS and IRLS were the RLS intensities of the systems with and without proteins).
Fig. 2. The fluorescence excitation (line 1) and emission (line 2) spectra of BSA. Conditions: CBSA = 1.0 g ml−1 ; pH 4.0.
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Fig. 4. The effect of pH on the RLS intensity. Conditions: CSLG = 1.2 × 10−4 mol l−1 , CBSA = 1.0 g ml−1 .
RLS intensity reached its maximum. This is possibly related to the isoelectric point of proteins. Proteins are positively charged when the pH is lower than their isoelectric points. SLG contains two carboxylic acid groups, which carries strong negative charges. In acidic medium, the electrostatic interaction between SLG and proteins can be very strong, which is favorable for the RLS intensity enhancement. So pH 4.0 was chosen for the determination. 3.3. The effect of SLG concentration (CSLG ) Fig. 3. The effect of SLG at variable concentrations on the fluorescence excitation (a) and emission (b) spectra of BSA. Conditions: CBSA = 1.0 g ml−1 , CSLG (mol l−1 ): (1) 0; (2) 0.4 × 10−4 ; (3) 0.8 × 10−4 ; (4) 1.2 × 10−4 ; (5) 1.6 × 10−4 ; pH 4.0.
excitation spectra of the BSA system (λem = 344 nm). The results show that the intensity of the excitation spectra was affected by the addition of the SLG, together with a gradual red shift. This phenomenon indicated that the interaction between the SLG and the protein occurred. The presence of SLG also modified the wavelength of maximum emission. In particular, as shown in Fig. 3b, it was observed a decrease in the intensity of the fluorescence emission intensity concomitant with a blue shift of the emission maximum in the presence of SLG. Fluorescence quenching has been a powerful tool to reveal the accessibility of fluorophores in the protein matrix to quenchers. Static quenching refers to formation of a nonfluorescent fluorophore–quencher complex [27]. The changes in the emission spectra suggested for the formation of a SLG–protein complex. The association of SLG to proteins with their hydrophobic groups can produce changes in the position or orientation of the amino acid residues, altering their exposure to the solvent water, leading to alterations on the quantum yield, and thus resulting in a quenching of the fluorescence.
As shown in Fig. 5, the concentration of SLG influenced significantly on the RLS intensity of the system. It can be clearly seen from Fig. 5 that SLG concentration in the range of 2.0 × 10−5 mol l−1 to 2.0 × 10−4 mol l−1 had a great influence on the RLS intensity. With increasing SLG concentration, the IRLS increased and reached a maximum at 1.2 × 10−4 mol l−1 . Then an increase of SLG concentration led to a reduction of the RLS intensity. So 1.2 × 10−4 mol l−1 SLG was selected in this assay. Because the critical micelle concentration (CMC) of SLG is 9.44 × 10−4 to 1.02 × 10−3 mol l−1 [28], the interaction between surfactants and proteins can be interpreted in the framework of molecule-to-molecule interaction.
3.2. The effect of pH As Fig. 4 shows, the RLS intensity of the assay system was affected by pH. Variation of pH from 2.1 to 6.0 at the concentration of 1.0 g ml−1 of BSA was studied. At pH 4.0, the enhanced
Fig. 5. The effect of SLG concentration on the RLS intensity. Conditions: CBSA = 1.0 g ml−1 ; pH 4.0.
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Table 1 Effects of amino acids on the determination of BSA (1.0 g ml−1 ) under the optimum conditions
Fig. 6. Effect of ionic strength on the RLS intensity. Conditions: CSLG = 1.2 × 10−4 mol l−1 , CBSA = 1.0 g ml−1 ; pH 4.0.
3.4. The effect of ionic strength The effect of ionic strength on the RLS intensity of the system is presented clearly in Fig. 6. It can be seen that the RLS intensity of the system SLG–BSA decreased with the increasing of NaCl concentration. The control of ionic strength of the solution is very important, indicating that electrostatic interaction is also one of the main driving forces governing their combination. When the concentration of the NaCl increases, the effects of the electrostatic shielding of charges, including the shielding of protein molecules from SLG or the shielding of SLG from protein molecules, reduce the combination of SLG with protein and result in a decreased RLS signal. 3.5. Incubation time and stability The stability of the SLG–BSA system was studied by measuring the RLS intensity every 2 min for 2 h immediately after mixing. Our results show that the reaction between SLG and proteins occurred rapidly at room temperature (<4 min) and the RLS intensity of the system reached the maximum. Moreover, the RLS intensity remained stable for at least 90 min. Thus, this assay is rapid and does not require crucial timing. 3.6. Interference of foreign substances The effect of the foreign substances on the determination of only 1.0 g ml−1 BSA was tested by pre-mixing BSA with compounds such as common ions, sugars and amino acids under the optimum conditions of the general procedure. The criterion for an interference was an IRLS value varying by more than 5% from the expected value for BSA alone. There was no interference from the following ions: 500 mol l−1 of K+ , 100 mol l−1 of Na+ , 50 mol l−1 of Ca2+ and Mg2+ , 25 mol l−1 of Zn2+ , Al3+ and Fe2+ , 20 mol l−1 of Mn2+ and Cu2+ , 15 mol l−1 of Cr2+ and Co2+ , 10 mol l−1 of Cd2+ , Hg2+ and Pb2+ , 25 mol l−1 of H2 PO4 − , 5.0 mol l−1 of PO4 3− . Especially, glucose, ethanol, and urea could be allowed at very high concentration. On the contrary, some other ions such as I− and Fe3+ only can be tolerated at relative low concentration levels (less than 5.0 mol l−1 ).
Amino acids (50.0 g ml−1 )
Change in IRLS (%)
Amino acids (50.0 g ml−1 )
Change in IRLS (%)
l-Proline l-Cysteine l-Asparagine l-Leucine l-Arginine l-Glutamine
+0.5 +1.1 +0.6 −1.7 +2.8 −1.4
l-Histidine l-Lysine l-Phenylalanine l-Serine l-Tryptophan l-Tyrosine
+0.9 +7.9 +2.3 −5.3 +4.8 −1.2
As shown in Table 1, most of the amino acids also do not interfere with the determination. Dilution with water can minimize all these interferences, which offers this method with the possibility of determination of trace amount of proteins in real samples. 3.7. Calibration curves and sensitivities According to the above standard procedure, the RLS intensities were obtained under the optimum conditions. The analytical parameters of the determination are listed in Table 2. The limits of detection (LOD) are given by 3S0 /S, where 3 is the factor at the 99% confidence level, S0 the standard deviation of the blank measurements (n = 10), and S is the slope of the calibration curve. Satisfactory linear relationship with low detection limits (below 6 ng ml−1 ) and relatively wide linear ranges were obtained. As shown in Table 3, compared with other methods of determination of proteins, this method has the advantages of high sensitivity, low detection limit and fine simplicity. 3.8. Application to sample analysis Table 4 displays the determination results for human serum samples with HSA as the standard. The samples were diluted 2000-fold with doubly distilled water without further purification. The results displayed in Table 4 are in good agreement with the data provided by the hospital. As shown in Table 2, the protein-to-protein variability cannot be strictly avoided with RLS technique, but the responses for some of the proteins such as BSA, HSA and ␥-G, are approximately equal with this method. It is obvious that this method for the determination of the total protein is reliable, sensitive and practical. This measurement system can be encouraged their practical use in chemical and biochemical applications. Table 2 Analytical parameters of this method under the optimum conditions Protein
Linear range (g ml−1 )
Regression equation
Detection limit (ng ml−1 )
Correlation coefficient (r)
Lysozyme BSA HSA ␥-G Hemoglobin
0.08–3.1 0.01–2.7 0.04–2.2 0.08–1.7 0.01–1.3
I = 4.3 + 121.1C I = 2.1 + 134.6C I = 3.7 + 131.2C I = 6.1 + 129.3C I = 1.8 + 109.7C
4.6 1.4 1.7 2.8 5.8
0.9986 0.9991 0.9997 0.9989 0.9981
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Table 3 Comparisons with some other assays of proteins Method
Reagents
Abs Abs Flu Flu RLS RLS RLS RLS RLS RLS RLS RLS
Lowry CBB Chrome azurol S ECRa TPPS4 b Quercetin EBBRc Alizarin Red S Ponceau 4R SDBSd SLSe SLG
a b c d e
Protein
HAS HAS BSA/HSA BSA/HSA BSA/HSA BSA/HSA BSA/HSA BSA/HSA BSA/HSA BSA/HSA
Detection limit (ng ml−1 )
References
1.0 × 105 5000 100 500 18.0/53.5 59.8/44.4 33.0/25.0 9.59/9.51 6.96/5.71 1.8/2.8 12.8/21.6 1.4/1.7
[2] [3] [29] [30] [14] [31] [32] [33] [15] [26] [26] This work
Eriochrome Cyanine R. ␣,,␥,␦-Tetrakis 4-(sulfophenyl) porphine. Eriochrome Blue Black R. Sodium dodecyl benzene sulfonate. Sodium lauryl sulfate.
3.9. Mechanism of the reaction Proteins and surfactants share the property of having both charged groups and hydrophobic portions. This implies that the interactions between surfactants and proteins are intrinsically complex processes, involving different types of intermolecular forces. In order to clarify the mechanism of the interaction between them, dependence of the RLS intensity on pH was carefully studied (see Fig. 4). The results show that the RLS intensity was sensitive to the solution pH. It is particularly relevant to the characteristics of both SLG and proteins. In pH < 2.9, both of the two carboxylic acid groups of SLG would be protonated, so the RLS intensity was relatively small. Whereas, in pH > 4.6, proteins were negatively charged. In pH 3.8–4.2, SLG carried strong negative charges, and proteins carried positive charges. So the RLS intensity was strong. The presence of two negative charged hydrophilic groups in the same SLG molecule leads to the better combination with the proteins by their electrostatic interactions. The dependence of the RLS intensity on the pH and the ionic strength is fully compatible with this interpretation. Besides the properties mentioned above, SLG possesses the acylamino group, which can easily form strong hydrogen bonding with proteins [25]. It thus performed better as protein RLS probe than other anion surfactants.
Table 4 Analytical results of human serum samplesa Samples (no.)
This method (n = 6, mg ml−1 )
R.S.D. (%)
Clinical datab (mg ml−1 )
Serum 1 Serum 2 Serum 3
69.4 ± 0.76 67.9 ± 0.61 84.1 ± 1.7
1.1 0.9 2.1
71.1 ± 1.4 69.8 ± 0.94 81.3 ± 1.5
Data shown are average ± S.D. from six independent experiments. Obtained from the Shantou Central Hospital, average from three measurements. a
b
As evidenced from the fluorescence excitation and emission spectra changing with the variable SLG concentrations, it can be concluded that the change of the fluorescence spectra resulted from the changes in the orientation of the protein structures [34]. The change of the protein structures would lead to a better combination with their hydrophobic groups. The data obtained in the present work support a SLG–protein interaction in which hydrophobic forces also play an important role in the RLS intensity enhancement. The surfactant SLG with special characteristics favors better interaction, thus SLG was shown better performance over other surfactants for the determination of proteins. 4. Conclusion The study of the interaction between surfactants and proteins is of significant scientific and technological importance. The acylamino acid-type surfactants with special properties and advantages are attractive molecules to probe proteins by RLS technique. The results of this study show that SLG played an effective role as a protein probe in the determination of trace amount of proteins using RLS technique. This is an inexpensive, simple and fast one-step procedure which requires only measuring the RLS intensities. It was applied to determine human serum samples satisfactorily. Therefore, it is a useful method to be used in practical applications. Acknowledgements All authors express their sincere thanks for the support from the National Natural Science Foundation of China (no. 30271033) and the Municipal Science Foundation of Shantou (no. S02023). References [1] L.X. Zhang, G.L. Wu, Advanced Biochemical Experiments, Advanced Education Press, Beijing, 1989, p. 192. [2] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193 (1951) 265. [3] M.M. Bradford, Anal. Biochem. 72 (1976) 248. [4] T. Hara, M. Toriyama, K. Tsukagoshi, Bull. Chem. Soc. Jpn. 61 (1988) 2779. [5] N. Li, K.A. Li, S.Y. Tong, Anal. Biochem. 233 (1996) 151. [6] R.F. Pasternack, C. Bustamante, P.J. Collings, A. Giannetto, E.J. Gibbs, J. Am. Chem. Soc. 115 (1993) 5393. [7] R.F. Pasternack, P.J. Collings, Science 269 (1993) 935. [8] R.F. Pasternack, K.F. Schaefer, P. Hambright, Inorg. Chem. 33 (1994) 2062. [9] C.Z. Huang, K.A. Li, S.Y. Tong, Anal. Chem. 68 (1996) 2259. [10] Z.P. Li, K.A. Li, S.Y. Tong, Talanta 55 (2001) 669. [11] Z.G. Chen, W.F. Ding, F.L. Ren, J.B. Liu, Y.Z. Liang, Anal. Chim. Acta 550 (2005) 204. [12] Z.G. Chen, W.F. Ding, F.L. Ren, Y.Z. Liang, J.B. Liu, Microchim. Acta 150 (2005) 34. [13] Z.G. Chen, W.F. Ding, F.L. Ren, Y.L. Han, J.B. Liu, Anal. Lett. 38 (2005) 2301. [14] R.P. Jia, L.J. Dong, Q.F. Li, X.G. Chen, Z.D. Hu, Talanta 57 (2002) 693. [15] H. Zhong, J.J. Xu, H.Y. Chen, Talanta 67 (2005) 749. [16] Z.G. Chen, T.Y. Zhang, F.L. Ren, W.F. Ding, Microchim. Acta 153 (2006) 65.
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