Voltammetric Studies on Chromotrope 2B-Protein Interaction and Its Analytical Application

Journal of the Chinese Chemical Society, 2006, 53, 1141-1147 1141 Voltammetric Studies on Chromotrope 2B-Protein Interaction and Its Analytical Application Wei Sun* ( ), Kui Jiao ( ), Jun-Ying Han ( ) and Gui-Yun Xu ( ) College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, P. R. China In this paper the interaction of chromotrope 2B (Ch2B) with proteins was studied by the electrochemical method. Ch2B is an azo dye and shows irreversible electrochemical responses on the mercury electrode in a ph 3.0 Britton-Robinson (B-R) buffer solution. After the addition of human serum albumin (HSA) into the Ch2B solution, an interaction took place, and a supramolecular complex was formed in the mixed solution. The electrochemical parameters of the Ch2B-HSA interaction system were calculated and compared. The results showed that in the absence and presence of HSA in Ch2B solution, the electrochemical parameters such as the formal potential E 0, the electrode reaction standard rate constant k s, etc. showed no significant changes, which indicated that an electro-inactive supramolecular biocomplex was formed. The free concentration of Ch2B in reaction solution was decreased, and this resulted in the decrease of the peak current. The binding constant and the binding ratio were calculated as 7.85 10 9 and 1:2, respectively, and the interaction mechanism was discussed. Based on the decrease of the peak current, this new electrochemical method was proposed for the determination of HSA in the concentration range of 2.0~25.0 mg/l with the linear regression equation as Ip" (na) = 50.56C (mg/l) 6.72 ( = 0.995). This method was further used to determine other different kinds of proteins, such as bovine serum albumin (BSA), oval albumin, etc.. The new method was successfully applied to detect the content of albumin in healthy human serum samples with the results in good agreement with the traditional Coomassie Brilliant Blue G-250 spectrophotometric method. Keywords: Chromotrope 2B; Serum albumin; Voltammetry; Interaction; Protein assay. INTRODUCTION The quantitative analysis of protein is very important in clinical tests and biological techniques. Many methods have been proposed to determine the content of proteins, such as fluorometric methods, 1,2 spectrophotometric methods, 3,4 chemiluminescent methods 5,6 and light scattering techniques. 7,8 Among them, the commonly accepted methods are spectrophotometry such as the Lowry method, 9 Bradford method 10 and Bromophenol Blue method, 11 which are mostly based on the interaction of dyes with proteins. However, these methods are often limited by their sensitivity, selectivity or stability. Compared with other analytical methods, the electrochemical method is seldom used in protein analysis. The electrochemical method is an effective technique in studying organic compounds and their interaction with biomolecules. The interactions of many substances such as metal complexes, drugs, and dyes with DNA have been studied by different electrochemical methods. 12-16 Because the electrochemical reaction occurs on the interface of electrode and liquid, it is especially suitable for a small amount of sample with a low detection limit and a wide dynamic range. There are few reports on the investigation of the interaction of protein with small molecules. Our group has reported the interaction of some small molecules 17-19 such as alizarin red S, beryllon III, etc. with protein by different electrochemical methods. Li et al. have studied the interaction of some electro-active small molecules such as 9,10- anthraquinone and tetraphenylporphyrin tetrasulfonate (TPPS) with different kinds of proteins such as hemoglobin, albumin and antibodies. 20-22 * Corresponding author. E-mail: sunwei_1975@public.qd.sd.cn

1142 J. Chin. Chem. Soc., Vol. 53, No. 5, 2006 Sun et al. In this present work, a new voltammetric method for protein assay with chromotrope 2B (Ch2B) as bioprobe was developed. Ch2B has been used as the spectrophotometric reagent for protein assay. 23 It is an azo dye with the molecular structure shown in Fig. 1, and it can take place a redox reaction on the mercury working electrode. Electrochemical studies on the interaction of Ch2B with protein at the mercury electrode were carried out according to the changes of electrochemical responses of the reaction solution. The electrochemical parameters of the Ch2B-HSA reaction system were calculated. The conditions for the interaction and the electrochemical determination were optimized. Under the optimal conditions, the proposed electrochemical method was further applied to the determination of the albumin content in human serum samples and the results were compared with the traditional Coomassie Brilliant Blue G-250 spectrophotometric method. EXPERIMENTAL SECTION Apparatus Cyclic voltammetric experiments were operated on a DS Model 2004 electrochemical analyzer (Shandong Dongsheng Electronic Instruments, China) with a DS-991 static mercury drop electrode (Shandong Dongsheng Electronic Instruments, China) as the working electrode, a saturated calomel electrode (SCE) as the reference electrode and a platinum wire as the auxiliary electrode. The second-order derivative linear sweep voltammetric determination was carried out using a Model JP-303 polarographic analyzer (Chengdu Apparatus Factory, China) with a traditional three-electrode system composed of a dropping mercury electrode (DME) working electrode, a SCE reference electrode and a platinum wire auxiliary electrode. A Cary Model 50 probe spectrophotometer (Varian, Australia) were used to record the UV-Visible absorption spectra. The ph values of the buffer solution were measured with a phs-25 acidimeter (Shanghai Leici Instrument Factory, Fig. 1. The molecular structure of chromotrope 2B. China). All the experiments were carried out at 25 C 1 C. Reagents Different kinds of proteins such as human serum albumin (HSA, 99%, Shanghai Biomedical Products Research Institute), bovine serum albumin (BSA, 99%, Sigma), oval albumin (OVA, Sigma), lipase (Sigma), and bovine hemoglobin (BHb, Tianjin Chuanye Biotechnology Company) were used as commercial products. The 1.0 g/l stock solution of different proteins were prepared by directly dissolving them in doubly distilled water from an all-quartz still and were stored at 4 C. The working solutions were obtained by diluting the stock solution with water before use. A 1.0 10-3 mol/l Ch2B solution (Shanghai Xinzhong Chemical Reagent Factory) was used as stock solution and diluted to the working concentration when used. A 0.2 mol/l Britton-Robinson (B-R) buffer solution was used to control the ph of the tested solutions. The Coomassie Brilliant Blue G-250 (CBB G-250, Shanghai Chemical Reagent Company) solution for spectrophotometric determination of protein was prepared according to the common procedure. Human serum samples were kindly provided by the Hospital of Qingdao University of Science and Technology. All other reagents used were of analytical reagent grade and doubly distilled water was used throughout. Procedure Into a 10 ml volumetric flask were successively added with 3.0 ml of 1.0 10-4 mol/l Ch2B, 1.0 ml of 0.2 mol/l B-R (ph 3.0) buffer solution and an appropriate amount of standard HSA solution or human serum sample solution. The mixture was diluted to the scale of 10 ml with water and shaken to mix homogeneously. After reacting at 25 C for 10 min, the voltammetric curve was recorded in the potential range from0vto-0.80 V (vs. SCE). Under the same conditions, a solution without the addition of HSA was used as the blank response. The voltammetric peak current (Ip) was obtained and the difference of the peak currents ( Ip=Ip 0 Ip) was used to show the changes of electrochemical responses of the reaction system. For an analytical comparison, the CBB G-250 spectrophometric method was used to determine the albumin concentration in the same samples with the traditional procedure and the absorbance value at 595 nm was recorded.

Voltammetric Studies on Chromotrope 2B and Protein J. Chin. Chem. Soc., Vol. 53, No. 5, 2006 1143 RESULTS AND DISCUSSION UV-Vis absorption spectra Fig. 2 shows the UV-Vis absorption spectra of Ch2B in the absence and presence of HSA. In ph 3.0 B-R buffer solution and in the scan range of 300~700 nm, Ch2B had a maximum absorption peak at 515 nm (curve 1) and HSA had no absorption (curve 4). When HSA was mixed with Ch2B, the absorbance of Ch2B at 515 nm decreased without the movement of maximum absorption wavelength (curve 2, 3). The more protein was added, the greater the absorbance decreased, which indicated that a binding reaction between Ch2B and HSA had taken place, and a new complex was formed under the selected experimental condition. The results were in close accordance with the literature results given in a reference. 23 Based on the decrease of absorbance value at 515 nm, the binding constant, K f, of Ch2B-HSA complex can be obtained according to the following equation: 24,25 A 0 /(A-A 0 )= G /( H-G - G )+ G /( H-G - G )(K f [HSA]) where A 0 and A are the absorbance value of the free guest and the apparent one, G and H-G are the absorption coefficients of the guest and complex, respectively. By changing the concentration of HSA in CH2B solution, the absorbance value was obtained and the relationship of A 0 /(A-A 0 ) with 1/[HSA] was plotted. The linear regression equation was A 0 /(A-A 0 )=3.0 10-9 /[HSA] + 6.8 (n = 8, = 0.991). The ratio of the intercept to the slope gives the value of the Fig. 2. UV-Vis absorption spectra of Ch2B-HSA reaction system. 1. ph 3.0 B-R + 3.0 10-5 mol/l Ch2B; 2. 1 + 20.0 mg/l HSA; 3. 1 +100.0 mg/l HSA; 4. ph 3.0 B-R + 100.0 mg/l HSA. binding constant K f as 2.27 10 9 L/mol. Irreversible electrochemical process of Ch2B Ch2B is an electroactive substance with an azo group ( N=N ) in its molecular structure (shown in Fig. 1), so it can be easily reduced on the mercury electrode. It has been widely used as a chelating agent in polarographic adsorptive waves for the determination of different metals. The cyclic voltammogram of Ch2B was recorded under the selected conditions, and the result showed that the reductive reaction of Ch2B on the mercury electrode was an irreversible process (curve 1 of Fig. 3). It had two reductive peaks at -0.26 V (P 1 ) and -0.40 V (P 2 )(vs. SCE), respectively, which corresponded to the two-step one-electron reduction of the azo group on the Hg electrode, and no oxidative peaks appeared in the same potential range of 0 ~ -0.8 V. The multi-sweep cyclic voltammograms indicated that with the increase of the scanning cycle, the reductive peak currents decreased greatly, which was the characteristic of the strong adsorption behavior of Ch2B on the Hg electrode. The relation of the peak potential against the ph of the buffer solution was investigated. With the increase of buffer ph, the peak potential moved negatively, and the linear relation was obtained in the ph range of 1.5~6.0, which indicated that there were hydrogen ions participating in the electrode reaction. In organic polarography, the half-wave potential of an azo compound with the functional group as N=N often appears at -0.40 V (vs. SCE). 26 So the second reduction peak at -0.40 V (P 2 ) was chosen for investigation in all subsequent experiments. At the same time, it was not easily disturbed by the electrochemical reduction of oxygen dissolved in solution that often appeared around -0.20 V(vs. SCE). Cyclic voltammogram of HSA and Ch2B Fig. 3 shows the cyclic voltammograms of Ch2B in the absence and presence of HSA. When HSA was added into the Ch2B solution, the current of the two reductive peaks of Ch2B decreased greatly without the shift of peak potentials, and no new reductive peaks appeared in the same potential scan range. The results also showed that there were interactions between HSA and Ch2B. In order to investigate the reaction mechanism of the Ch2B-HSA interaction system, the variation of electrochemical parameters of Ch2B in the absence and presence of HSA were calculated and compared. Because of the

1144 J. Chin. Chem. Soc., Vol. 53, No. 5, 2006 Sun et al. Fig. 3. Cyclic voltammograms of Ch2B-HSA binding reaction system. 1. 3.0 10-5 mol/l Ch2B in ph 3.0 B-R; 2. 1 + 3.0 mg/l HSA, scan rate: 120.0 mv/s. strong adsorption behavior and the irreversible electrode process of the reductive reaction of Ch2B on the mercury electrode, Laviron s equation 27 was used to calculate the electrochemical parameters. Ep=E 0 + RT/( nf){ln [(RTk s )/( nf)] ln } Table 1. The electrochemical parameters of Ch2B in the absence and presence of HSA Parameters Ch2B Ch2B-HSA E 0 (V) án k s (s -1 ) -0.384 0.870 1.514-0.384 0.865 1.480 where is the electron transfer coefficient, k s the standard rate constant of the surface reaction, the scanning rate and E 0 the formal potential. According to the above equation, if E 0 is known, Ep is in linear with ln and the n value can be calculated from the slope and k s from the intercept. The E 0 value can be deduced from the intercept of Ep vs. plot on the ordinate by extrapolating the line to = 0. Based on this method, the electrochemical parameters were calculated for the Ch2B- HSA reaction system. All the results are listed and compared in Table 1. Obviously the values of E 0, n and k s of Ch2B in the absence and presence of HSA had no significant changes, so the interaction of Ch2B with protein formed an electroinactive complex. Stoichiometry of HSA-Ch2B supramolecular complex With references to the method proposed by Li, 28 the binding number and the equilibrium constant of Ch2B- HSA complex can be calculated based on the changes of reductive peak current. It was assumed that Ch2B and HSA produced the single complex of HSA-mCh2B. Has + mch2b HSA-mCh2B (1) The equilibrium constant ( s ) was expressed as follows: [HSA -mch2b] s = (2) [HSA][Ch2B] m And the following equation can be deduced step by step: I max =kc HSA (3) I = k[hsa-mch2b] (4) [HSA] + [HSA-mCh2B] = C HSA (5)

Voltammetric Studies on Chromotrope 2B and Protein J. Chin. Chem. Soc., Vol. 53, No. 5, 2006 1145 or Therefore: I max - I = k(c HSA -[HSA-mCh2B]) = k[hsa] (6) Introducing equations (2), (4) and (6) gave: 1/ I=1/ I max + (1/ s I max )(1/[Ch2B] m ) (7) log[ I/( I max - I)] = log s + mlog[ch2b] (8) where I was the difference of peak current in the presence and absence of HSA, I max corresponded to the obtained value when the concentration of Ch2B was extremely higher than that of HSA. C HSA, [HSA], [HSA-mCh2B] were corresponding to the total, free and bound concentration of protein in the solution, respectively. From the equation (8) the relation of log[ I/( I max - I)] versus log[ch2b] was calculated and plotted as a line with the linear regression equation as log[ I/( I max - I)] = 9.895 + 1.984 log[ch2b]. From the intercept and the slope, m=2and s =7.85 10 9 were deduced. The binding constant was close to the value obtained by absorption spectrophotometry. Thus a stable 1:2 complex of HSA-2Ch2B was formed in the selected conditions. Optimal reaction conditions In order to improve the sensitivity of the detection, all the analytical data were obtained with a JP 303 polarographic analyzer and the second-order derivative linear sweep voltammetric method. The ph of the buffer solution greatly influences the binding reaction, and the optimal reaction ph was selected in the range of 1.5~6.0. The results showed that at ph 3.0 the difference of the peak currents reached its maximum, so ph 3.0 B-R buffer solution was used throughout in this experiment. Different buffers such as B-R, HOAc-NaOAc, NH 3 -NH 4 Cl were tested, and in B-R buffer solution the response was the maximum. So the B-R buffer of ph 3.0 is recommended in this paper. After Ch2B and HSA were mixed, the difference of peak currents reached the maximum after reaction for about 5 min and remained unchanged for at least 2 hours. So this new method gave enough time for daily measurements. The effect of the reaction temperature on the interaction was tested at 15 C, 25 C, 30 C and 37 C, respectively. The results showed that there was little influence on the binding reaction with the increase of the reaction temperature and 25 C was used throughout. Different adding orders of the reagents of Ch2B, HSA and B-R buffer were tested, and the best result was that Ch2B, B-R buffer and proteins were added in sequence. This result indicated that the electronic coupling model made Ch2B bind to HSA. The scan rate and the standing time of the mercury drop of the instrument for the assay were studied. The peak current increased with the increase of the potential scan rate within 200~1000 mv/s and the mercury drop standing time from 5 to 15 s. When the dropping mercury time was more than 15 s, the mercury drop would fall down automatically from the electrode. So the scan rate and the standing time were selected as 900 mv/s and 15 s, respectively. Interference of coexisting substances The influences of interference substances such as metal ions, amino acids and carbohydrates on the determination were tested by adding them in the binding reaction solution. Table 2 showed that the commonly observed Table 2. Effect of interference substances on the determination of 10.0 mg/l HSA Coexisting substance Concentration (mg/l) Relative error/% Coexisting substance Concentration ( mol/l) Relative error/% L-Tryptophan 5.0-0.41 Mg 2+ 20.0-0.076 L-Valine 5.0-0.26 Ca 2+ 20.0 0.54 L-Serine 5.0-0.32 Cu 2+ 20.0 3.97 L-Lysine 5.0-0.33 Zn 2+ 20.0 2.78 L-Leucine 5.0-0.17 Mn 2+ 20.0-0.19 L-Arginine 5.0-0.29 Fe 3+ 20.0 0.39 Urea 0.1 mol/l 1.69 Co 2+ 20.0 3.97 Glucose 4.0 g/l -2.68 K + 20.0 0.77

1146 J. Chin. Chem. Soc., Vol. 53, No. 5, 2006 Sun et al. Table 3. The working curves for the determination of different proteins Protein Linear range (mg/l) Linear regression equation Detection limit (3, mg/l) Correlation coefficient ( ) HSA 2.0~25.0 Ip" (na) = 50.56C (mg/l) - 6.72 1.84 0.995 BSA 4.0~40.0 Ip" (na) = 64.92C (mg/l) - 223.25 3.46 0.995 BHb 3.0~40.0 Ip" (na) = 58.67C (mg/l) + 30.50 1.59 0.995 OVA 1.0~50.0 Ip" (na) = 95.08C (mg/l) - 330.66 0.91 0.988 Lipase 1.0~100.0 Ip" (na) = 19.51C (mg/l) + 172.19 0.88 0.997 Table 4. The results for the determination of HSA in human serum samples Sample This method (g/l) RSD (%, n=5) Recovery % CBB G-250 method (g/l) RSD (%, n=5) 1 101.2 2.56 94.7 105.2 3.84 2 96.7 3.03 100.3 97.7 1.19 3 77.5 2.31 98.9 75.7 4.30 metal ions and amino acids in blood samples could be allowed with higher concentrations. Different concentrations of NaCl in the range of 2.0 10-3 ~8.0 10-2 mol/l were added in the binding solution to investigate the interference and the results proved to have significant influences on the interaction. The peak current decreased with the increase of the concentration of NaCl, which indicated that the interaction of Ch2B with HSA was mainly caused by electrostatic attraction. The electrostatic shielding effect of the charges of higher concentrations of NaCl on binding reaction with the increase of Na + concentration was unbeneficial to the formation of HSA-Ch2B complex. Calibration curves of different proteins According to the standard procedure, a linear relationship of the peak current against the concentration of different kinds of proteins were established, and all the analytical parameters are listed in Table 3. The results showed that different proteins had different responses, and the sensitivities were enough for routine sample determination. Samples determination The proposed method was further applied to the determination of the albumin content in healthy human serum samples, and the analytical results are listed in Table 4. By comparing the results with the commonly used Coomassie Brilliant Blue G-250 (CBB G-250) spectrophotometric method, it was clear that the proposed electrochemical detection method was reliable, practical and reproducible. CONCLUSION The reaction models between small molecules with protein can be divided as the electrostatic binding and the hydrophobic interaction. In a ph 3.0 buffer solution, the amino acid residues such as lysine, arginine, etc. on the molecular chains of HSA (isoelectric point pi 4.7) are positively charged, while the free species of Ch2B is negatively charged. So it is possible that Ch2B interacts with HSA by electrostatic attraction and other weak responses such as ionic, van der Waals and hydrogen bonding to form an electro-inactive supramolecular complex, which could not be reduced on the Hg electrode surface. In the presence of HSA, the equilibrium concentration of free Ch2B in solution decreases, which results in the decrease of the peak current and can be further used for electrochemical protein assay. ACKNOWLEDGEMENTS Financial supported from the National Natural Science Foundation of China (No. 20405008, 20375020), the Natural Science Foundation of Qingdao City (No. 04-2-JZ- 114) and Doctoral Foundation of QUST (No. 0022125) are acknowledged.

Voltammetric Studies on Chromotrope 2B and Protein J. Chin. Chem. Soc., Vol. 53, No. 5, 2006 1147 Received December 1, 2005. REFERENCES 1. Ma, C. Q.; Li, K. A.; Tong, S.Y. Anal. Chim. Acta 1996, 333, 83. 2. Ma, C. Q.; Li, K. A.; Tong, S. Y. Anal. Chim. Acta 1997, 338, 255. 3. Wei, Q.; Wu, D.; Du, B.; Zhang, H.; Ou, Q.Y. Chin. J. Chem. 2004, 22, 714. 4. Gao, H. W.; Zhao, J. F. J. Chin. Chem. Soc. 2003, 50, 329. 5. Tsukagoshi, K.; Okumura,Y.; Akasaka, H.; Nakajima, R.; Hara, T. Anal. Sci. 1996, 12, 525. 6. Hara, T.; Torigama, M.; Tsukagoshi, K. Bull. Chem. Soc. Jpn. 1998, 61, 2996. 7. Chen, L. H.; Zhao, F. L.; Li, K. A. Chin. J. Chem. 2002, 20, 368. 8. Fan, L.; Liu, S. P.; Luo, H. Q. Chin. J. Chem. 2003, 21, 56. 9. Lowry, O. H.; Rosebrough, N. J.; Farr, L. A.; Randall, R. J. J. Biol. Chem. 1951, 193, 265. 10. Bradford, M. M. Anal. Biochem. 1976, 72, 248. 11. Flores, R. L. Clin. Chem. 1965, 11, 478. 12. Carter, M. T.; Bard, A. J. J. Am. Chem. Soc. 1987, 109, 7528. 13. Radi, A. Anal. Chim. Acta 1999, 386, 63. 14. Sun, W.; Shang, Z. M.; Li, Q. J.; Jiao, K. J. Chin. Chem. Soc. 2005, 52, 1269. 15. Ye, B. X.; Wang, C. H.; Jing, A. H. J. Chin. Chem. Soc. 2003, 50, 457. 16. Wang, S. F.; Peng, T. Z.; Yang, C. F. Biophys. Chem. 2003, 104, 239. 17. Sun, W.; Jiao, K. Talanta 2002, 56, 1073. 18. Sun, W.; Jiao, K.; Wang, X. L.; Lu, L. D. Electroanal. 2005, 17, 162. 19. Sun, W.; Jiao, K.; Wang, X. L.; Lu, L.D. J. Electroanal Chem. 2005, 578, 37. 20. Zhang, H. M.; Zhu, Z. W.; Li, N. Q. Fresenius J. Anal. Chem. 1999, 363, 408. 21. Zhu, Z. W.; Li, N. Q. Mi krochim. Acta 1999, 130, 301. 22. Zeng, Y. N.; Liu, J. Y.; Li, Y. Z. Electrochem.Commun. 2002, 4, 679. 23. Hu, Q. H.; Liu, S. P.; Luo, H. Q. J. Southwest China Norm. Univ. 2001, 26, 594. 24. Ibrahim, M. S. Anal. Chim. Acta 2001, 443, 63. 25. Ibrahim, M. S.; Shehatta, I. S.; Al-Nayeli, A. A. J. Pharm. Biomed. Anal. 2002, 28, 217. 26. Wang, J. Analytical Electrochemistry (2nd editon); Wiley- VCH: New York, 2001; p 64. 27. Laviron, E. J. Electroanal. Chem. 1979, 101, 19. 28. Li, N. Q.; Min, J. Chin. J. Anal. Chem. 1989, 17, 346.