Polarographic Behavior of Co (II)− BSA or− HSA Complex in the

Anal. Chem. 2003, 75, 6346-6350

Polarographic Behavior of Co(II)-BSA or -HSA Complex in the Presence of a Guanidine Modifier Dengbai Luo,* Jingui Lan, Chun Zhou, and Chenxia Luo

College of Chemistry and Life Science, South-Central University for Nationalities, Wuhan 430074, P. R. China

An extremely sensitive adsorptive wave of BSA at about -1.73 V (vs SCE) has been obtained in the solution containing 8 × 10-7 mol/L CoCl2, 0.2 mol/L guanidine hydrochloride, and 0.2 mol/L NaOH by using singlesweep polarography. The interaction of BSA with guanidine in an alkaline solution results in its Co(II) complex with a positive excess charge. Thus, the complex is strongly adsorbed by the DME as a result of static electrical attraction. The adsorption efficiently accumulates the electrochemical active complex of Co(II)-BSA onto the DME. In the following potential scan, the complex produces a sensitive adsorptive reduction peak, which can be used to determine low-level BSA. In the absence of guanidine, the complex of Co(II)-BSA with a negative excess charge is repulsed by the DME. The reduction current is very small. The peak current depends on both BSA and Co(II) ion concentrations. HSA is similar to BSA in polarographic behavior. At the optimal conditions, the peak height is linearly proportional to the BSA or HSA concentration in the range of 0.005-20 mg/L (correlation coefficient 0.999). The detection limit for BSA or HSA is 0.002 mg/L. Lysozyme, common amino acids, and metal ions have no interference with the protein determination. The new method could be useful in protein studies.

nation of BSA in a range of 0.16-0.65 mg/L. The linear range is too narrow to practice. Chronopotentiometric stripping analysis (CPSA) was shown to be valuable for the determination of some peptides and metallothionein (MT) measuring the signal of catalytic hydrogen evolution in the presence or absence of cobalt ion. The highest sensitivity was obtained with MT in pH 8.0 borate buffer; a subnanomolar concentration was detectable.9-11 Because of the importance of proteins in life science, rapid, sensitive, and simple methods for their determination are still required. Guanidine is a denaturant of proteins, and is very often used in the studies of molecule structure and function of proteins.12,13 The association of guanidine with protein molecule causes a change of its space configuration, so some reaction can take place. On the other hand, the association may change the charge state of the protein molecule. Little use has been made of this property. This paper reports for the first time that the complex of Co(II) with BSA or HSA modified with protonated guanidine in a NaOH solution produces an extremely sensitive polarographic wave which corresponds to the reduction of Co(II) in the complex adsorbed on the dropping mercury electrode. The polarographic wave is capable of determining BSA or HSA concentration from 0.005 to 20 mg/L. Furthermore, chemical modification of proteins is useful not only in structure and function studies of proteins, but also in electroanalytical chemistry of proteins.

The catalytic hydrogen wave of proteins containing SH groups in ammonia-buffered cobalt ion solution was first reported by Brdicka.1 Since then, this Brdicka wave has received much attention, and has been extensively applied in biochemical, clinical, pharmaceutical2 and environmental3 analysis. The original method was improved by modern techniques. By using pulse technique,4 quantification of 0.1-1.0 mg/L BSA could be made. Over 1000 papers5-7 focus on the studies of its reduction mechanism. The common explanation is that the double catalytic hydrogen wave is due to the catalytic evolution of hydrogen of the active Co(0)BSA complex.5 Recently, a so-called parallel catalytic hydrogen wave of BSA appeared in the literature.8 It allowed the determi-

EXPERIMENTAL SECTION Apparatus. A JP-2 oscillopolarograph and a JP-3 polarograph (Chengdu Instrumental Factory) were used for measurements of the single-sweep polarographic wave and its derivative wave. The three-electrode system used consisted of a dropping mercury electrode (DME), a platinum counter electrode, and a saturated calomel reference electrode (SCE). The parameters for model JP-2 were as follows: scan rate 250 mV/s, potential scan range 0.5 V, drop time 7 s, and rest time 5 s. For model JP-3 they were adjustable. Initial scan potential was -1.40 V for JP-2, normally -1.20 V for JP-3; the electrolytic cell a a 10-mL beaker. An EG&G PAR (Princeton Applied Research) model 174A polarographic

* Corresponding author. E-mail: [email protected]. (1) Brdicka, R. Collect. Czech. Chem. Commun. 1933, 5, 112-128. (2) Brezina, M.; Zuman, P. Polarography in Medicine, Biochemistry and Pharmacology; Interscience Publisher: New York, 1958. (3) Geret, F.; Rainglet, F.; Cosson, R. P. Mar. Environ. Res. 1998, 46, 545. (4) Palecek, E.; Pechan, Z. Anal. Biochem. 1971, 42, 59-71. (5) Gao, X. Polarographic Catalytic Wave; Science Publisher: Beijing, China, 1991; Chapter 7. (6) Kolthoff, I. M.; Kihara, S. Anal. Chem. 1977, 49, 2108-2109. (7) Raspor, B. J. Electroanal. Chem. 2001, 503, 159-162.

(8) Guo, W.; Liu, L.; Lin, H.; Song, J. Sci. China, Ser. B: Chem. 2001, 31, 519-524. (9) Tomschik, M.; Havran, L.; Fojta, M.; Palecek, E. Electroanalysis 1998, 10, 403-409. (10) Tomschik, M.; Havran, L.; Palecek, E.; Heyrovsky, M. Electroanalysis 2000, 12, 274-279. (11) Kizek, R.; Trnkova, L.; Palecek, E. Anal. Chem. 2001, 73, 4801-4807. (12) Toyooka, T.; Imai, K. Anal. Chem. 1985, 57, 1931-1937. (13) Hori, K.; Matsubara, K.; Miyazawa, K. Biochim. Biophys. Acta 2000, 1474, 226-236.

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analyzer with a model 303 static mercury drop electrode (SMDE), a platinum counter electrode, and a Ag/AgCl reference electrode was used for cyclic voltammetric experiments. A BioPhotometer (Eppendorf Company) was used to measure spectroscopically the protein concentrations. A model J2705 biological anatomy lens (Wuhan Education Instrument Factory) with an amplified factor of 30 was used for detection of the gas product of the electrode reaction. A model LD-6-4 centrifugal (Dalin Medical Instrumental Factory; rotating rate: 0-4000 rpm) was used for centrifugation of urine samples. All experiments were carried out at room temperature. Reagents. Bovine serum albumin (BSA) was purchased from B. M. Company. Human serum albumin (HSA) was purchased from Sigma Company. The stock solutions of BSA and HSA were prepared with water and stored at 4 °C. The protein concentrations were accurately measured spectroscopically using the 280 values as follows: BSA, 6.6; HSA, 5.3.14 Lysozyme (Lyso) was purchased from Shanghai Promega. Guanidine hydrochloride (CH5N3HCl) was GR grade. Other chemicals used were AR grade. All reagents were used as received. Twice distilled water was used for the experiments. Procedure. Sample solution was added to a 10-mL beaker and mixed with 1 mL of 2 mol/L guanidine hydrochloride, 1 mL of 2 mol/L NaOH, 0.8 mL of 0.01 mmol/L CoCl2, and 1 mL of 5% Na2SO3. The mixture was diluted to 10 mL with water. The derivative cathodic single-sweep polarogram was recorded. The peak at -1.73 V (vs SCE) was measured. RESULTS AND DISCUSSION Single-Sweep Polarographic Wave of the Complex of Co(II) with BSA Modified with Guanidine. Co(II) ion in alkaline solution easily forms a precipitate. But when the concentration is low, the solution is clear. Figure 1A is the polarogram for this solution. There is no measurable reduction wave. Addition of BSA to the solution produces a very small wave Po at about -1.78 V (Figure 1B). Once more, with addition of guanidine hydrochloride into the same solution, a sharp peak P appears at around -1.73 V (Figure 1C). Peak P is about 100 times peak Po. The peak shape shows it is a typical adsorptive wave. The pH value of this solution is slightly lower than that in Figure 1B. In the normal single-sweep polarography, peak P partly overlaps with the background discharge, but it is well separated in the derivative mode. To find out the relative solution components to peak P, the polarographic behavior of guanidine hydrochloride and its mixture with Co(II) and with BSA in NaOH solution was studied. The results show that peak P is attributed to the interaction of Co(II), BSA, and guanidine. In fact, guanidine transforms wave Po into peak P. The detail is shown in Figure 4. It is interesting that the lower BSA concentration, the greater the signal enhancement multiple. This new electrochemical behavior is useful. The peak P current depends on both BSA and Co(II) concentrations and can be used for the determination of low-level BSA. The behavior of HSA is similar to that of BSA (Figure 1D), but the peak current is larger, and the potential a little negative, probably because of the structure difference between BSA and HSA. It is well-known that cysteine as well as BSA produces a Brdicka catalytic hydrogen wave. In (14) Perez-Ruiz, T.; Martinez-Lozano, C.; Tomas, V.; Fenoll, J. Analyst 2000, 125, 507-510.

Figure 1. Derivative single-sweep polarograms for (A) 8 × 10-7 mol/L CoCl2 + 0.2 mol/L NaOH and containing (B) 7.4 × 10-8 mol/L BSA, (C) 7.4 × 10-8 mol/L BSA + 0.2 mol/L guanidine hydrochloride, (D) 2.9 × 10-8 mol/L HSA + 0.2 mol/L guanidine hydrochloride, and (E) 1 × 10-5 mol/L cysteine + 0.2 mol/L guanidine hydrochloride. Rest time, 5 s; scan rate, 250 mV/s.

our case, cysteine has no relative response (Figure 1E). Cystine is the same as cysteine. Addition of guanidine hydrochloride to the Brdicka solution, BSA does not give any response. DC polarography was also used to study the peak P. No detectable wave was observed. Peak P is an adsorptive wave, and a continuing dropping mercury electrode cannot be used as its working electrode. Obviously, the peak reported here is distinct from the Brdicka wave. Verification of Complex Reduction Wave. A simple technique to prove a catalytic hydrogen wave is examining whether there are hydrogen bubbles on the electrode surface while the electrode reaction is going. This method was used to investigate the electrode reaction mechanism of peak P. The mixture solution of BSA, Co(II), guanidine hydrochloride, and NaOH was electrolyzed at the peak potential with the SMDE. The ocular lens of the biological anatomy lens was directed to the SMDE. It was clearly seen that there was no gas bubble on the surface of the SMDE, even for electrolysis of 30 min. A similar procedure was used to study the Brdicka catalytic hydrogen wave. Gas bubbles on the SMDE surface were easily seen. The favorite medium for peak P is a strong alkali solution. As mentioned later, when the solution pH value gets lower than 12, the peak completely disappears. In a strong alkali solution, catalytic hydrogen evolution cannot take place because of the lack of a proton donor. The signal of a catalytic hydrogen wave increases with buffer concentration. Figure 5B shows peak P gradually decreases as guanidine hydrochloride concentration is increased higher than 0.26 mol/ L. It disagrees with the behavior of a catalytic hydrogen wave. These results show peak P is not a catalytic hydrogen wave. Figure 2 shows the cyclic voltammograms of relative components in guanidine hydrochloride and NaOH solution. There were no reduction and oxidation peaks for BSA (Figure 2B). CoCl2 produced a reduction wave P1 at about -1.34 V (Figure 2C). The Analytical Chemistry, Vol. 75, No. 22, November 15, 2003

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Figure 2. Cyclic voltammograms for (A) 0.1 mol/L guanidine hydrochloride + 0.1 mol/L NaOH and containing (B) 5.9 × 10-8 mol/L BSA, (C) 2 × 10-6 mol/L CoCl2, and (D) 5.9 × 10-8 mol/L BSA + 2 × 10-6 mol/L CoCl2. Rest time, 20 s; scan rate, 100 mV/s.

potential of oxidation wave of Co(0) was fairly positive and overlapped with the oxidation current of Hg in a strong basic medium. Figure 2D showed that when CoCl2 and BSA coexisted, a new sharp cathodic peak P appeared at around -1.74 V, and the reduction wave P1 was smaller than that in Figure 2C. These results indicate that peak P corresponds to the reduction of the Co(II)-BSA complex formed in the alkaline guanidine solution. Co(II) ion in the complex was reduced to Co(0). The sharp peak again shows the strong adsorption of the complex at SMDE. The anodic process was like that for CoCl2. Function of Guanidine in Formation of Peak P. Guanidine could react with Co(II) ion to form a blue complex in NaOH solution. When BSA was added into this blue solution, the color decayed gradually. At last the solution became colorless. The ligand exchange reaction took place. The Co(II)-guanidine complex was converted to the Co(II)-BSA complex. Therefore, the substance that produces peak P is the Co(II)-BSA complex. There is no ternary complex of Co(II) with BSA and guanidine. The function of guanidine in forming peak P is not as a ligand but as a modifier of the BSA molecule. In protein studies, guanidine as a denaturant of protein competes with the peptide chain for a hydrogen bond, destroys the hydrophobic association inside the molecule, and has the molecule extend and expose the hydrophobic groups. It may increase the ability of adsorption of BSA and Co(II)-BSA complex at the DME. As a result, the polarographic current is possibly magnified in the presence of guanidine hydrochloride. Urea is often used for the same purpose in protein studies. The substitution of urea for guanidine does not produce the peak P. Clearly, the interaction of guanidine with a BSA molecule possesses special character. Guanidine is an organic strong alkali; its pKa is 13.6.15 In NaOH solution, guanidine exists in two species: guanidine and protonated guanidine with a positive charge. The BSA molecule (15) Zhang, X. Principle of Chemical Analysis; Science Publisher: Beijing, China, 1991; p 3.

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Figure 3. Plots of (a) ip vs v and (b) ip vs v1/2. 8 × 10-7 mol/L CoCl2 + 0.2 mol/L NaOH solution containing (A) 7.4 × 10-8 mol/L BSA + 0.2 mol/L guanidine hydrochloride and (B) 7.4 × 10-7 mol/L BSA. Rest time, 10 s; initial scan potential, -1.45 V.

associated with protonated guanidine possesses a positive excess charge, which originally is negatively charged in the NaOH solution. The coordination of Co(II) with SH groups of protein forms a product RS2Co 7 without changing the charge of the protein molecule. Therefore, the Co(II) complex with BSA modified with protonated guanidine carries a positive excess charge. It is strongly adsorbed by the DME negatively charged as a result of static electrical attraction and accumulated at the DME surface. In the electrode reaction, the complex at the DME surface is reduced and produces a remarkable adsorptive current. In the absence of guanidine hydrochloride, Co(II)-BSA complex in basic medium has a negative excess charge and is strongly repulsed by the DME and hardly adsorbed; thus, there is only a small reduction current. Compared with guanidine, although urea can also associate with BSA molecule, it cannot induce strong adsorption of the Co(II)-BSA complex at the DME. Therefore, urea cannot cause peak P. Cysteine and cystine cannot bond with guanidine. It is reasonable that they have no relative response. Figure 3 shows the relationship between peak current and potential scan rate from 200 to 900 mV/s for the Co(II)-BSA complex in the presence (A) and absence (B) of guanidine hydrochloride. The peak current is linearly proportional to the scan rate v (correlation coefficient 0.9996) and deviates from linear correlation to v1/2 toward the current axis in the presence of guanidine (Figure 3A), which is the characteristic property of a surface reaction. In the absence of guanidine (Figure 3B), the peak current, measurable only at higher concentration, is linearly proportional to v1/2 (correlation coefficient 0.9993). Obviously, the Co(II)-BSA complex in NaOH solution with guanidine hydrochloride produces an adsorptive reduction current. In contrast, the Co(II)-BSA complex in NaOH solution without guanidine hydrochloride does not show any adsorption at the DME. Such a characteristic of guanidine has never been reported.

Figure 4. Derivative single-sweep polarograms for 3.7 × 10-7 mol/L BSA in 1 × 10-6 mol/L CoCl2 + 0.1 mol/L NaOH and guanidine hydrochloride of different concentrations: 0 (a), 0.001, (b), 0.004, (c), 0.008, (d), and 0.012 mol/L (e). Other conditions as in Figure 1.

Figure 4 clearly shows the effect of guanidine on the polarographic behavior of the Co(II)-BSA complex, in which the concentration of BSA is higher and of guanidine is lower. Figure 4a is the polarogram for BSA in CoCl2 + NaOH solution without guanidine. At this time, the wave P0 at about -1.78 V is defined. The half wave potential of Co(II) in 1 mol/L NaOH is -1.44 V (vs SCE).16 Thus, P0 corresponds to the reduction of the Co(II)BSA complex; Co(II) is reduced to Co(0). The additions of guanidine hydrochloride from 0.001 to 0.012 mol/L (Figure 4be) into the above solution do not produce a new peak, but cause positive shifts of the peak potential, peak current increments, and the peak shape changes. For example, in 0.012 mol/L guanidine solution, the potential shift is +60 mV, peak current increment is 5.4 times, and the peak turns to a typical adsorptive wave. Guanidine does not act as the second ligand of cobalt ion; otherwise, the potential shift should be negative, at least not positive. Guanidine associates with the BSA molecule. The increment of guanidine concentration results in an increment of the amount of positive charge of BSA molecule and the adsorption of the complex at DME and the reduction current, as well. The static electrical attraction makes the electrode reaction more easily carried out, and so the reduction potential shifts to positive. The polarographic catalytic wave strongly depends on the pH value of the buffer solution. In Na2HPO4-Na3PO4 (pH > 12), peak P with a positive potential shift can be observed, but the peak current is smaller than that in NaOH solution. In lower pH buffer solution, the peak completely disappears, although there is protonated guanidine in the solution. Analytical Application. Figure 5 shows the dependence of the BSA peak current upon the CoCl2 (A), guanidine hydrochlo(16) Guo, H. Instrumental Analysis; High Education Publisher: Beijing, China, 1964; p 87.

Figure 5. Effect of CoCl2 (A), guanidine hydrochloride (B), NaOH (C), and Na2SO3 (D) concentrations on peak height of 7.4 × 10-8 mol/L BSA. Solution conditions: A, 0.2 mol/L guanidine hydrochloride + 0.2 mol/L NaOH; B, 8 × 10-7 mol/L CoCl2 + 0.2 mol/L NaOH; C, 8 × 10-7 mol/L CoCl2 + 0.2 mol/L guanidine hydrochloride; D, 8 × 10-7 mol/L CoCl2 + 0.2 mol/L guanidine hydrochloride + 0.2 mol/L NaOH. Other conditions as in Figure 1.

ride (B), NaOH (C), and Na2SO3 (D) concentrations. When the CoCl2 concentration is lower than 1 × 10-7 mol/L, the relationship of the peak current and Co(II) concentration is linear. If it is higher than 4 × 10-7 mol/L, the peak current of the BSA is stable. A guanidine hydrochloride concentration from 0.12 to 0.26 mol/L has almost no influence on the peak current. The peak decreases slowly with higher concentration. A NaOH concentration higher than 0.1 mol/L is preferred to obtain a large and stable peak. CoCl2, guanidine hydrochloride, and NaOH concentrations of 8 × 10-7, 0.2, and 0.2 mol/L, respectively, were selected for the determination of BSA or HSA. Na2SO3 almost has no effect upon the BSA signal from 0 to 2%, but it increases the stability of the peak due to the removal of dissolved oxygen; 0.5% Na2SO3 was chosen for the work. The temperature of the solution also has influence on the peak stability. BSA is more stable at lower temperature in alkaline solution. The peak height was stable for more than 2 h at ∼20 °C for a 5 mg/L BSA solution in the selected condition. At the optimal conditions, the derivative peak height is linearly proportional to the BSA concentration in the range of 0.005-20 mg/L (correlation coefficient 0.999). The detection limit of BSA is 0.002 mg/L. HSA has the same concentration linear range and detection limit. Figure 6 is the polarograms for BSA in concentrations below 0.1 mg/L. The peaks are well-defined, and the corresponding calibration plot is excellent, with a correlation coefficient 0.997. Common amino acids and metal ions were examined for their possible interference with the determination of BSA. At an appropriate level, none of them had influence on the determination. Lysozyme had no response and did not interfere with the Analytical Chemistry, Vol. 75, No. 22, November 15, 2003

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Figure 6. Derivative single-sweep polarograms of BSA for increasing concentrations: 0 (a), 0.02 (b), 0.04 (c), 0.06 (d), 0.08 (e), and 0.10 (f) mg/L. Solution containing 8 × 10-7 mol/L CoCl2, 0.2 mol/L guanidine hydrochloride, 0.2 mol/L NaOH, and 0.5% Na2SO3. Other conditions as in Figure 1.

BSA determination. The spatial structure is not suitable for the peak, although it contains four disulfide bonds. A healthy human urine sample was centrifuged at 3000 rpm for 10 min, and 0.1 mL of the upper clear solution was used for the determination of protein content. The quantification was based on standard addition. The average value of the determination results calculated by HSA standard was 30.6 mg/L (n ) 5), RSD 1.44%. The HSA added to the urine sample before centrifugation was found back with 96.7-105.6% efficiency, as compared to a standard prepared in the supporting electrolyte. CONCLUSION The polarographic behavior of the Co-BSA complex is varied. In NH3-NH4Cl solution, the complex produces a catalytic hydro-

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gen wave, which has been studied and practiced extensively for about 70 years. In guanidine hydrochloride and NaOH solution, we have found it produces a very sensitive new peak. Guanidine plays a special role in the formation of the peak. As a denaturant, guanidine can associate with the protein molecule, and as an organic strong alkali, guanidine in solution is dissociated partly into protonated guanidine. The BSA molecule bonded with protonated guanidine possesses a positive excess charge. The Co(II) complex with BSA modified with protonated guanidine is strongly adsorbed and efficiently accumulated at DME as a result of static electrical attraction and produces a highly sensitive adsorptive reduction current. The new peak is different from a Brdicka wave1 or peak H.11 For a BSA or HSA assay, the new technique can provide not only low-level determination but also a very broad linear relation concentration range. This simple and rapid method should be useful in biochemistry and clinical and environment analysis. The level of protein quantification will be much lower by combining the principle with adsorptive stripping voltammetry and chronopotentiometric stripping analysis.11 Such use of guanidine may open up opportunities for studies of other proteins without SH and S-S groups. ACKNOWLEDGMENT D.L. acknowledges the financial support from the Natural Science Foundation of Hubei Province, China (99J063), and the Natural Science Foundation of South-Central University for Nationalities, Wuhan, China. Received for review February 11, 2003. Accepted August 19, 2003. AC0300643