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Determination of proteins with Alizarin Red S by Rayleigh light scattering technique
Talanta 62 (2004) 37–42
Determination of proteins with Alizarin Red S by Rayleigh light scattering technique Hui Zhong a , Na Li b , Fenglin Zhao b,∗ , Ke an Li b b
a Department of Chemistry, Huaiyin Normal College, Jiangsu 223001, China Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemistry, Peking University, Beijing 100871, China
Received 27 February 2003; received in revised form 8 June 2003; accepted 9 June 2003
Abstract A new protein determination method by enhanced Rayleigh light scattering (RLS) technique has been developed. In acid condition (pH = 3.60), RLS of 1,2-dihydroxyanthraquinone-3-sulfonate (Alizarin Red S) can be greatly enhanced by addition of proteins, resulting in two characteristic peaks, 360 and 505 nm, respectively. The new protein assay is based on the RLS enhancement and spectrum change. The optimum condition for the reaction was investigated. The linear range is 0.20–24.9 g ml−1 for BSA and 0.20–15.5 g ml−1 for HSA. The detection limits (S/N = 3) are 9.59 ng ml−1 for BSA and 9.51 ng ml−1 for HSA. The results of determination for human serum samples were comparable to those obtained by Bradford method. The binding stoichiometry was determined. © 2003 Elsevier B.V. All rights reserved. Keywords: Rayleigh light scattering (RLS); Alizarin Red S; Protein; Determination
1. Introduction The quantitative analysis of protein is of great importance in biochemistry and clinical application since it can provide information for diagnosis of diseases and measurement of other components. There are many methods for protein determination, such as spectrophotometry [1], fluorometry [2], chemiluminescence [3] and so on. At present, light scattering technique has been developed rapidly, especially in the determination of biological macromolecules, e.g. nucleic acid [4–6], proteins [7–9] and glycogen [10]. Traditionally, Rayleigh light scattering (RLS) is used to obtain size-related information, but now it has been applied extensively in analytical chemistry only in recent years. In 1993, Pasternack et al. [11] established the resonance light scattering in a study of the aggregation of porphyrins assembling on DNA using a common spectrofluorimeter. Huang et al. [12] first developed an assay for determination of nucleic acids with tetra-kis [4-(trimethylammoniumyl) phenyl]porphine. In this paper, 1,2-dihydroxyanthraquinone-3-sulfonate (Alizarin Red S, called ARS for short, see Fig. 1) was used as a probe for the study of its binding with proteins by ∗
Corresponding author. Fax: +86-010-62751708. E-mail address: [email protected] (F. Zhao).
0039-9140/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0039-9140(03)00406-5
RLS technique. It is found that the RLS signals of ARS are strongly enhanced in the presence of proteins. Based on this, a determination method for proteins has been developed. This method is rapid (<2 min), simple (on common spectrofluorimeter) and sensitive (the detection limit for bovine serum albumin, BSA, human serum albumin, HSA, and human immunoglobulin G, IgG all are below 10.0 ng ml−1 ).
2. Experimental 2.1. Apparatus The RLS spectra and the intensities were recorded and measured with 10 mm quartz cells on a Shimadzu RF-540 spectrofluorimeter (Kyoto, Japan), while the absorption spectra were obtained by using a Shimadzu UV-265 spectrophotometer (Kyoto, Japan). A model 821 pH meter (Zhongshan University, China) was used to measure pH of the solutions. 2.2. Reagents All chemicals were of analytical reagent grade or the best grade commercially available in China. Alizarin Red S
38
H. Zhong et al. / Talanta 62 (2004) 37–42
Fig. 1. Structure of Alizarin Red S (ARS).
(ARS) was purchased from Beijing Chemical Plant (China) and used as supplied. Coomassie Brilliant Blue G-250 (CBB) was obtained from Fluka. The Clark-Lubs buffer was used to adjust the pH. All stock solutions were prepared in doubly de-ionized water. The following proteins: chymotrypsin, protamine sulfate, hemoglobin (bovine), lysozyme, BSA, HSA, human IgG and egg albumin were obtained from Sigma. The protein concentrations were determined spectrophotometrically at 280 nm using ε1% values: BSA, 6.6 [10]; HSA, 5.3 [11]; ␥-G, 13.8 [1]; lysozyme, 26.04 [1]; and egg albumin, 7.5 [12]. The concentrations of hemoglobin and chymotrypsin were determined as [13]: protein concentration = 144 × (A215 − A225 ), where A215 and A225 were absorbance at 215 and 225 nm measured in 1 cm cell. Fresh human serum was obtained from Peking University Hospital and diluted with doubly de-ionized water. 2.3. Procedures An appropriate volume of working solution of proteins, 2.0 ml of Clark-Lubs buffer solution and an appropriate ARS solution were added into a 10 ml volumetric flask in turn and mixed thoroughly. The mixture was diluted to 10 ml with doubly de-ionized water. The RLS spectra were obtained by scanning simultaneously with same wavelengths of excitation and emission (λex = λem ) from 300 to 700 nm on the spectrofluorimeter (with 5 nm slit-width) and the RLS intensity I for the reaction product and I0 for the reagent blank were measured at the maximum scattered wavelength,
I = I − I0 . The stoichiometry was determined according to the literature [14] using RLS and spectrophotometric methods.
Fig. 2. (a) Absorption spectra of ARS (···); ARS–BSA (—); ARS: 2.0 × 10−4 mol l−1 ; BSA: 5.0 g ml−1 ; pH 3.60. (b) RLS spctra of Alizarin Red S (ARS)–BSA. 1: ARS (2.0 × 10−4 mol l−1 ); 2: ARS (2.0 × 10−4 mol l−1 ) + BSA (5.0 g ml−1 ); 3: ARS (2.0 × 10−4 mol l−1 ) + BSA (10.0 g ml−1 ).
3.2. Stability and addition sequence 3. Results and discussion 3.1. Spectral characteristics The absorption spectra are presented in Fig. 2a. For ARS, the absorption peak is at λmax = 420 nm. While BSA was added, the system showed two absorption peaks at 330 and 530 nm, respectively. It can be concluded that ARS reacts with BSA to form a new red complex. From Fig. 2b, it can be seen that there are two RLS peaks for ARS–BSA system at 360 and 505 nm, which are located at the wavelength of minimum absorption of ARS. As the enhanced signal at 505 nm is greater than that at 360 nm, 505 nm was selected as the determination wavelength.
The reaction between ARS and proteins occurs rapidly (<2 min) and the scattering intensity is stable for at least 3 h at room temperature. Three types of mixing sequences were checked. Different addition orders of reagents have obvious effect on the interaction of ARS and BSA. It is found that if the order of addition reagent is buffer, protein and ARS, both the stability and intensities of RLS signals are the best among the three different mixing sequences. Mixing the buffer and protein first provides positive charged protein for this combination and improve determination sensitivity. When the proteins and ARS are mixed firstly, the pH of solution is nearly neutral, which is not in favor of the combination. At pH 7.0, both
H. Zhong et al. / Talanta 62 (2004) 37–42
39
tion the lower intensity is obtained. At ARS concentration of 2.0 × 10−4 mol l−1 , the system yields a better sensitivity and wider linear range. So 2.0 × 10−4 mol l−1 ARS is selected for the assay. 3.5. Effect of ionic strength
Fig. 3. Effect of pH on RLS intensity of ARS (䊐) and the mixture of ARS and BSA (䊏). BSA: 5.0 g ml−1 ; ARS 2.0 × 10−4 mol l−1 .
the dye and protein are negatively charged and this makes it hard for the two entities to form complex. 3.3. Effect of pH The scattering intensity of the assay system was greatly affected by pH, while the RLS of ARS remained unchanged (Fig. 3). At pH 3.60, the scattering intensity reaches the maximum and so pH 3.60 was selected for the assay. 3.4. Effect of ARS concentration The effect of ARS concentration on the response of the reaction is tested in the range of (0.50–4.0) × 10−4 mol l−1 . When the ARS concentration is less than 1.5 × 10-4 mol l−1 , an increase of ARS concentration will enhance the RLS intensity of the ARS–BSA system. The linear range for the determination widens throughout. But the greater concentra-
The effect of NaCl content on this assay at pH 3.60 is shown in Fig. 4. The RLS intensity of ARS–BSA decreased with increasing ionic strength. It might be that the large amount of Cl− competes with ARS to bind with BSA. The effect of the electrostatic shielding of charges will reduce the binding of the dye to protein and result in a decreased signal. Therefore, the effect of ionic strength of the medium should be considered. 3.6. Interfering substances The influence of coexisting substances, such as amino acids and metal ions are showed in Table 1. It can be seen that amino acids hardly interfere with the determination and most of metal ions and coexisting substances do not affect the determination at the test concentration level. Fig. 5 shows the effects of surfactants. Triton X-100 and -CD almost do not affect the RLS of ARS and ARS–BSA complex. SDBS decreases the scattering intensity of the assay system. Negatively charged SDBS neutralizes the positive charge on the protein, which may reduce the binding of ARS to protein and results in the decrease of scattering intensity. Because CTMAB enhanced the RLS of ARS, the enhancement of RLS intensity in ARS–BSA system could be the result of interaction of CTMAB with excess amount of ARS and also with ARS dissociated from protein as more CTMAB was added. As there were excess CTMAB and also protein, the extent of ARS aggregation on either CTMAB or protein
Fig. 4. Effect of ionic strength on RLS intensity in 2.0 × 10−4 mol l−1 ARS (䊊); a mixture of 2.0 × 10−4 mol l−1 ARS and 5.0 g ml−1 BSA (䊉).
40
H. Zhong et al. / Talanta 62 (2004) 37–42
Table 1 Effect of potentially interfering substances on the reaction of ARS with BSA Interfering substances
Change (%)
Interfering substances
l-Glycine l-Glutamic l-Arginine l-Lysine l-Serine l-Tryptophan l-Cysteine l-Leucine ␣-Alanine -Alanine l-Tyrosine l-Asparagine Cd2+ (nitrate)
−2.1 −1.2 −2.7 −5.3 0.12 −9.9 5.1. 1.1 2.3 5.2 7.7 8.8 1.4
Al3+ (sulfate) Mn2+ (sulfate) Pb2+ (nitrate) Cr3+ (chloride) Cu2+ (nitrate) Co2+ (chloride) Mg2+ (nitrate) Zn2+ (chloride) Ca2+ (nitrate) Fe3+ (nitrate) Ni2+ (chloride) EDTA Ethanol (5%)
Change (%) 6.9 3.2 4.6 −0.12 1.7 2.2 8.9 5.8 0.02 7.2 4.2 −4.7 10.3
Concentration of ARS 2.0 × 10−4 mol l−1 ; pH 3.60; concentration of interfering substances was 20.0 mol l−1 ; BSA: 5.0 g ml−1 .
may be reduced and as a result, the RLS intensity could be reduced. 3.7. Calibration curves and variation between proteins The calibration curve linear regression results for several proteins, such as BSA, HSA, ␥-G, lysozyme, hemoglobin, chymotrypsin, protamine sulfate, and egg albumin are presented in Fig. 6 and in Table 2. It is reported earlier [12] that the intensity of the enhanced RLS signal appeared to depend on the electronic properties of the individual chromophores, the extent of the electric coupling among the chromophores and the size of the aggregate of dyes on macromolecule. Different proteins have different isoelectric points, the sensitivity of RLS for various proteins are different since the size, mass and shape of the molecules of proteins are different.
3.8. Stoichiometry Under experimental conditions, both RLS and spectrophotometric methods [14] were used to measure the stoichiometry for BSA and ARS while the concentration of BSA was kept constant. The results are presented in Fig. 7. Both RLS and spectrophotometric methods gave the same results. The maximum binding number of ARS to BSA was found to be 8. It is obvious that RLS is more sensitive which shows a clearer inflexion. 3.9. Sample determination Under the appropriate condition, six samples were assayed and the results were compared with Coomassie Brilliant Blue (CBB) method using HSA as the standard (see Table 3). It can be clearly shown that the determination of the proteins is reliable, practical and sensitive. 3.10. The discussion of reaction mechanism and the reason of the enhancement of RLS Under the experimental conditions (pH = 3.60), the proteins such as BSA, HSA, and chymotrypsin are macromolecules with positive charges (their isoelectric point pI values are listed in Table 2), while ARS is negatively charged. From Fig. 2, it can be seen that RLS peak of ARS–BSA is close to the absorption peak. This spectral character showed clearly the enhanced resonance RLS mechanism. H2 R− anion interacted with positive charged protein molecules which resulted in the assembling of ARS on the protein. When the dye assembles on the protein, not only does the molecular size increase, but also is the efficient cross-section for light enhanced greatly. Both electrostatic force and the induced chromophore aggregation on the
Fig. 5. Effects of surfactant on the RLS intensity of ARS (···) and mixture of ARS and BSA at pH 3.60 (—). ARS 2.0 × 10−4 mol l−1 ; BSA: 5.0 g ml−1 .
H. Zhong et al. / Talanta 62 (2004) 37–42
41
Fig. 6. RLS response for various proteins. BSA ( ); HSA (夹); ␥-G (䉱); hemoglobin (䊉); protamin sulfate (䉬); lysozyme (䊏); egg albumin ( ); chymotrypsin (䉲). ARS 2.0 × 10−4 mol l−1 , pH 3.60.
Table 2 Standard regression equation of proteinsa Proteins
Regression equation
BSA HSA Protamine sulfate ␥-G Hemoglobin Chymotrypsin Egg albumin
I I I I I I I I I
Lysozyme a b
= 5.14 + 2.49c = 5.73 + 2.51c = 3.62 + 0.60c = 4.98 + 3.31c = 4.27 + 4.31c = 4.08 + 0.59c = 6.64 + 1.83c = 14.49 + 0.63c = 1.43 + 0.65c × 10−4
Correlation coefficient
Linear range (g ml−1 )
Determination limitb (ng ml−1 )
pI
0.9995 0.9994 0.9987 0.9992 0.9994 0.9985 0.9996 0.9993 0.9971
0.2–24.9 0.2–15.5 0.3–17.1 0.2–28.0 0.3–18.4 0.3–18.0 0.7–6.8 6.8–34.8 0.5–9.3
9.59 9.51 39.8 7.21 5.54 40.5 13.0 37.9 36.72
4.8–4.9 4.7 10–12 5.8–6.6 6.9 1.0 4.6–4.7
mol l−1 ;
Concentration of ARS 2.0 pH 3.60. Calculated from three times the S.D. of 11 blank measurements.
Fig. 7. The determination of stoichiometry by two methods. RLS method (䊊); spectrophotometric method (×). BSA: 5.0 mg ml−1 .
11.0–11.2
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H. Zhong et al. / Talanta 62 (2004) 37–42
Table 3 Analytical results for protein in human seruma Sample
This method (mg ml−1 )
R.S.D.
CBB method (mg ml−1 )
1 2 3 4 5 6
90.1 83.2 76.8 88.1 81.6 92.7
1.6 1.3 2.2 3.4 2.8 4.7
89.7 83.6 76.7 87.6 81.9 93.8
parable with CBB method. Under the same conditions, the stoichiometry was determined by both RLS and spectrophotometric methods. The maximum binding number of ARS on BSA is 8.
References
R.S.D.: relative standard deviation. a Each result was the average of seven measurements.
[1] [2] [3] [4] [5]
biological macromolecule lead to the significant enhancement of RLS.
[6] [7]
4. Conclusion A new method for the determination of proteins was developed based on the fact that the weak RLS intensity of ARS is greatly enhanced by the addition of proteins. The method is sensitive, simple and repeatable. It is applied to determination of human serum and the results were com-
[8] [9] [10] [11] [12] [13] [14]
C.Q. Ma, K.A. Li, S.Y. Tong, Anal. Lett. 31 (1998) 1021. N. Li, K.A. Li, S.Y. Tong, Anal. Lett. 28 (1995) 1763. Z.P. Li, K.A. Li, S.Y. Tong, Microchem. J. 60 (1998) 217. R.F. Pasternack, P.J. Collings, Science 269 (1995) 935. Y. Liu, C.Q. Ma, K.A. Li, S.Y. Tong, Anal. Biochem. 268 (1999) 187. W. Lu, C.Z. Huang, Y.F. Li, Analyst 127 (2002) 1392. Y.T. Wang, F.L. Zhao, K.A. Li, S.Y. Tong, Fr. J. Anal. Chem. 364 (1999) 560. S.P. Liu, Q. Liu, Anal. Sci. 17 (2001) 239. Y.K. Zhao, W.B. Chang, Y.X. Ci, Talanta 59 (2003) 477. S.Z. Zhang, F.L. Zhao, K.A. Li, S.Y. Tong, Anal. Chim. Acta 431 (2001) 133. R.F. Pasternack, C. Bustamante, P.J. Colling, A. Giannetto, E.J. Gibbs, J. Ann. Chem. Soc. 115 (1993) 5393. C.Z. Huang, K.A. Li, S.Y. Tong, Anal. Chem. 69 (1996) 514. S.G. Stanton, R. Pecora, B.S. Hudson, J. Chem. Phys. 75 (1981) 5615. N. Li, Ph.D. Thesis, Peking University, China, 1995.
Determination of proteins with Alizarin Red S by Rayleigh light scattering technique Hui Zhong a , Na Li b , Fenglin Zhao b,∗ , Ke an Li b b
a Department of Chemistry, Huaiyin Normal College, Jiangsu 223001, China Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemistry, Peking University, Beijing 100871, China
Received 27 February 2003; received in revised form 8 June 2003; accepted 9 June 2003
Abstract A new protein determination method by enhanced Rayleigh light scattering (RLS) technique has been developed. In acid condition (pH = 3.60), RLS of 1,2-dihydroxyanthraquinone-3-sulfonate (Alizarin Red S) can be greatly enhanced by addition of proteins, resulting in two characteristic peaks, 360 and 505 nm, respectively. The new protein assay is based on the RLS enhancement and spectrum change. The optimum condition for the reaction was investigated. The linear range is 0.20–24.9 g ml−1 for BSA and 0.20–15.5 g ml−1 for HSA. The detection limits (S/N = 3) are 9.59 ng ml−1 for BSA and 9.51 ng ml−1 for HSA. The results of determination for human serum samples were comparable to those obtained by Bradford method. The binding stoichiometry was determined. © 2003 Elsevier B.V. All rights reserved. Keywords: Rayleigh light scattering (RLS); Alizarin Red S; Protein; Determination
1. Introduction The quantitative analysis of protein is of great importance in biochemistry and clinical application since it can provide information for diagnosis of diseases and measurement of other components. There are many methods for protein determination, such as spectrophotometry [1], fluorometry [2], chemiluminescence [3] and so on. At present, light scattering technique has been developed rapidly, especially in the determination of biological macromolecules, e.g. nucleic acid [4–6], proteins [7–9] and glycogen [10]. Traditionally, Rayleigh light scattering (RLS) is used to obtain size-related information, but now it has been applied extensively in analytical chemistry only in recent years. In 1993, Pasternack et al. [11] established the resonance light scattering in a study of the aggregation of porphyrins assembling on DNA using a common spectrofluorimeter. Huang et al. [12] first developed an assay for determination of nucleic acids with tetra-kis [4-(trimethylammoniumyl) phenyl]porphine. In this paper, 1,2-dihydroxyanthraquinone-3-sulfonate (Alizarin Red S, called ARS for short, see Fig. 1) was used as a probe for the study of its binding with proteins by ∗
Corresponding author. Fax: +86-010-62751708. E-mail address: [email protected] (F. Zhao).
0039-9140/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0039-9140(03)00406-5
RLS technique. It is found that the RLS signals of ARS are strongly enhanced in the presence of proteins. Based on this, a determination method for proteins has been developed. This method is rapid (<2 min), simple (on common spectrofluorimeter) and sensitive (the detection limit for bovine serum albumin, BSA, human serum albumin, HSA, and human immunoglobulin G, IgG all are below 10.0 ng ml−1 ).
2. Experimental 2.1. Apparatus The RLS spectra and the intensities were recorded and measured with 10 mm quartz cells on a Shimadzu RF-540 spectrofluorimeter (Kyoto, Japan), while the absorption spectra were obtained by using a Shimadzu UV-265 spectrophotometer (Kyoto, Japan). A model 821 pH meter (Zhongshan University, China) was used to measure pH of the solutions. 2.2. Reagents All chemicals were of analytical reagent grade or the best grade commercially available in China. Alizarin Red S
38
H. Zhong et al. / Talanta 62 (2004) 37–42
Fig. 1. Structure of Alizarin Red S (ARS).
(ARS) was purchased from Beijing Chemical Plant (China) and used as supplied. Coomassie Brilliant Blue G-250 (CBB) was obtained from Fluka. The Clark-Lubs buffer was used to adjust the pH. All stock solutions were prepared in doubly de-ionized water. The following proteins: chymotrypsin, protamine sulfate, hemoglobin (bovine), lysozyme, BSA, HSA, human IgG and egg albumin were obtained from Sigma. The protein concentrations were determined spectrophotometrically at 280 nm using ε1% values: BSA, 6.6 [10]; HSA, 5.3 [11]; ␥-G, 13.8 [1]; lysozyme, 26.04 [1]; and egg albumin, 7.5 [12]. The concentrations of hemoglobin and chymotrypsin were determined as [13]: protein concentration = 144 × (A215 − A225 ), where A215 and A225 were absorbance at 215 and 225 nm measured in 1 cm cell. Fresh human serum was obtained from Peking University Hospital and diluted with doubly de-ionized water. 2.3. Procedures An appropriate volume of working solution of proteins, 2.0 ml of Clark-Lubs buffer solution and an appropriate ARS solution were added into a 10 ml volumetric flask in turn and mixed thoroughly. The mixture was diluted to 10 ml with doubly de-ionized water. The RLS spectra were obtained by scanning simultaneously with same wavelengths of excitation and emission (λex = λem ) from 300 to 700 nm on the spectrofluorimeter (with 5 nm slit-width) and the RLS intensity I for the reaction product and I0 for the reagent blank were measured at the maximum scattered wavelength,
I = I − I0 . The stoichiometry was determined according to the literature [14] using RLS and spectrophotometric methods.
Fig. 2. (a) Absorption spectra of ARS (···); ARS–BSA (—); ARS: 2.0 × 10−4 mol l−1 ; BSA: 5.0 g ml−1 ; pH 3.60. (b) RLS spctra of Alizarin Red S (ARS)–BSA. 1: ARS (2.0 × 10−4 mol l−1 ); 2: ARS (2.0 × 10−4 mol l−1 ) + BSA (5.0 g ml−1 ); 3: ARS (2.0 × 10−4 mol l−1 ) + BSA (10.0 g ml−1 ).
3.2. Stability and addition sequence 3. Results and discussion 3.1. Spectral characteristics The absorption spectra are presented in Fig. 2a. For ARS, the absorption peak is at λmax = 420 nm. While BSA was added, the system showed two absorption peaks at 330 and 530 nm, respectively. It can be concluded that ARS reacts with BSA to form a new red complex. From Fig. 2b, it can be seen that there are two RLS peaks for ARS–BSA system at 360 and 505 nm, which are located at the wavelength of minimum absorption of ARS. As the enhanced signal at 505 nm is greater than that at 360 nm, 505 nm was selected as the determination wavelength.
The reaction between ARS and proteins occurs rapidly (<2 min) and the scattering intensity is stable for at least 3 h at room temperature. Three types of mixing sequences were checked. Different addition orders of reagents have obvious effect on the interaction of ARS and BSA. It is found that if the order of addition reagent is buffer, protein and ARS, both the stability and intensities of RLS signals are the best among the three different mixing sequences. Mixing the buffer and protein first provides positive charged protein for this combination and improve determination sensitivity. When the proteins and ARS are mixed firstly, the pH of solution is nearly neutral, which is not in favor of the combination. At pH 7.0, both
H. Zhong et al. / Talanta 62 (2004) 37–42
39
tion the lower intensity is obtained. At ARS concentration of 2.0 × 10−4 mol l−1 , the system yields a better sensitivity and wider linear range. So 2.0 × 10−4 mol l−1 ARS is selected for the assay. 3.5. Effect of ionic strength
Fig. 3. Effect of pH on RLS intensity of ARS (䊐) and the mixture of ARS and BSA (䊏). BSA: 5.0 g ml−1 ; ARS 2.0 × 10−4 mol l−1 .
the dye and protein are negatively charged and this makes it hard for the two entities to form complex. 3.3. Effect of pH The scattering intensity of the assay system was greatly affected by pH, while the RLS of ARS remained unchanged (Fig. 3). At pH 3.60, the scattering intensity reaches the maximum and so pH 3.60 was selected for the assay. 3.4. Effect of ARS concentration The effect of ARS concentration on the response of the reaction is tested in the range of (0.50–4.0) × 10−4 mol l−1 . When the ARS concentration is less than 1.5 × 10-4 mol l−1 , an increase of ARS concentration will enhance the RLS intensity of the ARS–BSA system. The linear range for the determination widens throughout. But the greater concentra-
The effect of NaCl content on this assay at pH 3.60 is shown in Fig. 4. The RLS intensity of ARS–BSA decreased with increasing ionic strength. It might be that the large amount of Cl− competes with ARS to bind with BSA. The effect of the electrostatic shielding of charges will reduce the binding of the dye to protein and result in a decreased signal. Therefore, the effect of ionic strength of the medium should be considered. 3.6. Interfering substances The influence of coexisting substances, such as amino acids and metal ions are showed in Table 1. It can be seen that amino acids hardly interfere with the determination and most of metal ions and coexisting substances do not affect the determination at the test concentration level. Fig. 5 shows the effects of surfactants. Triton X-100 and -CD almost do not affect the RLS of ARS and ARS–BSA complex. SDBS decreases the scattering intensity of the assay system. Negatively charged SDBS neutralizes the positive charge on the protein, which may reduce the binding of ARS to protein and results in the decrease of scattering intensity. Because CTMAB enhanced the RLS of ARS, the enhancement of RLS intensity in ARS–BSA system could be the result of interaction of CTMAB with excess amount of ARS and also with ARS dissociated from protein as more CTMAB was added. As there were excess CTMAB and also protein, the extent of ARS aggregation on either CTMAB or protein
Fig. 4. Effect of ionic strength on RLS intensity in 2.0 × 10−4 mol l−1 ARS (䊊); a mixture of 2.0 × 10−4 mol l−1 ARS and 5.0 g ml−1 BSA (䊉).
40
H. Zhong et al. / Talanta 62 (2004) 37–42
Table 1 Effect of potentially interfering substances on the reaction of ARS with BSA Interfering substances
Change (%)
Interfering substances
l-Glycine l-Glutamic l-Arginine l-Lysine l-Serine l-Tryptophan l-Cysteine l-Leucine ␣-Alanine -Alanine l-Tyrosine l-Asparagine Cd2+ (nitrate)
−2.1 −1.2 −2.7 −5.3 0.12 −9.9 5.1. 1.1 2.3 5.2 7.7 8.8 1.4
Al3+ (sulfate) Mn2+ (sulfate) Pb2+ (nitrate) Cr3+ (chloride) Cu2+ (nitrate) Co2+ (chloride) Mg2+ (nitrate) Zn2+ (chloride) Ca2+ (nitrate) Fe3+ (nitrate) Ni2+ (chloride) EDTA Ethanol (5%)
Change (%) 6.9 3.2 4.6 −0.12 1.7 2.2 8.9 5.8 0.02 7.2 4.2 −4.7 10.3
Concentration of ARS 2.0 × 10−4 mol l−1 ; pH 3.60; concentration of interfering substances was 20.0 mol l−1 ; BSA: 5.0 g ml−1 .
may be reduced and as a result, the RLS intensity could be reduced. 3.7. Calibration curves and variation between proteins The calibration curve linear regression results for several proteins, such as BSA, HSA, ␥-G, lysozyme, hemoglobin, chymotrypsin, protamine sulfate, and egg albumin are presented in Fig. 6 and in Table 2. It is reported earlier [12] that the intensity of the enhanced RLS signal appeared to depend on the electronic properties of the individual chromophores, the extent of the electric coupling among the chromophores and the size of the aggregate of dyes on macromolecule. Different proteins have different isoelectric points, the sensitivity of RLS for various proteins are different since the size, mass and shape of the molecules of proteins are different.
3.8. Stoichiometry Under experimental conditions, both RLS and spectrophotometric methods [14] were used to measure the stoichiometry for BSA and ARS while the concentration of BSA was kept constant. The results are presented in Fig. 7. Both RLS and spectrophotometric methods gave the same results. The maximum binding number of ARS to BSA was found to be 8. It is obvious that RLS is more sensitive which shows a clearer inflexion. 3.9. Sample determination Under the appropriate condition, six samples were assayed and the results were compared with Coomassie Brilliant Blue (CBB) method using HSA as the standard (see Table 3). It can be clearly shown that the determination of the proteins is reliable, practical and sensitive. 3.10. The discussion of reaction mechanism and the reason of the enhancement of RLS Under the experimental conditions (pH = 3.60), the proteins such as BSA, HSA, and chymotrypsin are macromolecules with positive charges (their isoelectric point pI values are listed in Table 2), while ARS is negatively charged. From Fig. 2, it can be seen that RLS peak of ARS–BSA is close to the absorption peak. This spectral character showed clearly the enhanced resonance RLS mechanism. H2 R− anion interacted with positive charged protein molecules which resulted in the assembling of ARS on the protein. When the dye assembles on the protein, not only does the molecular size increase, but also is the efficient cross-section for light enhanced greatly. Both electrostatic force and the induced chromophore aggregation on the
Fig. 5. Effects of surfactant on the RLS intensity of ARS (···) and mixture of ARS and BSA at pH 3.60 (—). ARS 2.0 × 10−4 mol l−1 ; BSA: 5.0 g ml−1 .
H. Zhong et al. / Talanta 62 (2004) 37–42
41
Fig. 6. RLS response for various proteins. BSA ( ); HSA (夹); ␥-G (䉱); hemoglobin (䊉); protamin sulfate (䉬); lysozyme (䊏); egg albumin ( ); chymotrypsin (䉲). ARS 2.0 × 10−4 mol l−1 , pH 3.60.
Table 2 Standard regression equation of proteinsa Proteins
Regression equation
BSA HSA Protamine sulfate ␥-G Hemoglobin Chymotrypsin Egg albumin
I I I I I I I I I
Lysozyme a b
= 5.14 + 2.49c = 5.73 + 2.51c = 3.62 + 0.60c = 4.98 + 3.31c = 4.27 + 4.31c = 4.08 + 0.59c = 6.64 + 1.83c = 14.49 + 0.63c = 1.43 + 0.65c × 10−4
Correlation coefficient
Linear range (g ml−1 )
Determination limitb (ng ml−1 )
pI
0.9995 0.9994 0.9987 0.9992 0.9994 0.9985 0.9996 0.9993 0.9971
0.2–24.9 0.2–15.5 0.3–17.1 0.2–28.0 0.3–18.4 0.3–18.0 0.7–6.8 6.8–34.8 0.5–9.3
9.59 9.51 39.8 7.21 5.54 40.5 13.0 37.9 36.72
4.8–4.9 4.7 10–12 5.8–6.6 6.9 1.0 4.6–4.7
mol l−1 ;
Concentration of ARS 2.0 pH 3.60. Calculated from three times the S.D. of 11 blank measurements.
Fig. 7. The determination of stoichiometry by two methods. RLS method (䊊); spectrophotometric method (×). BSA: 5.0 mg ml−1 .
11.0–11.2
42
H. Zhong et al. / Talanta 62 (2004) 37–42
Table 3 Analytical results for protein in human seruma Sample
This method (mg ml−1 )
R.S.D.
CBB method (mg ml−1 )
1 2 3 4 5 6
90.1 83.2 76.8 88.1 81.6 92.7
1.6 1.3 2.2 3.4 2.8 4.7
89.7 83.6 76.7 87.6 81.9 93.8
parable with CBB method. Under the same conditions, the stoichiometry was determined by both RLS and spectrophotometric methods. The maximum binding number of ARS on BSA is 8.
References
R.S.D.: relative standard deviation. a Each result was the average of seven measurements.
[1] [2] [3] [4] [5]
biological macromolecule lead to the significant enhancement of RLS.
[6] [7]
4. Conclusion A new method for the determination of proteins was developed based on the fact that the weak RLS intensity of ARS is greatly enhanced by the addition of proteins. The method is sensitive, simple and repeatable. It is applied to determination of human serum and the results were com-
[8] [9] [10] [11] [12] [13] [14]
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