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Electrochemical, chemical and spectrophotometric investigation of the copper(II)-bicinchoninic acid reagent used for protein measurements
Analytica Chimica Acta, 221 (1989) 223-238 Elsevier Science Publishers B.X., Amsterdam -
223 Printed in The Netherlands
ELECTROCHEMICAL, CHEMICAL AND SPECTROPHOTOMETRIC INVESTIGATION OF THE COPPER(BICINCHONINIC ACID REAGENT USED FOR PROTEIN MEASUREMENTS
ROBERT D. BRAUN*, KAREN J. WIECHELMAN and AUGUST A. GALL0 Department of Chemistry, University of Southwestern Louisiana, Lafayette, LA 70504-4370 (U.S.A.) (Received 15th September 1988)
SUMMARY The use of a reagent containing copper (II), bicinchoninic acid (BCA ) and tartrate buffered at pH 11.25 was studied voltammetrically, coulometrically, spectrophotometrically and chemically. The reagent exhibits three cathodic waves at rotating platinum disk and rotating glassy carbon electrodes. The two more-positive cathodic waves correspond to electrochemical reduction to copper (I) -bisbicinchoninate, Cu (BCA ), 3- . The third cathodic wave is caused by reduction to metallic copper. A reaction mechanism is proposed that shows the major chemical species in the solution and the electrochemical reaction products. Voltammetric and chemical studies indicate that the reagent should be used with care for protein assays because it is subject to multiple chemical interferences.
Bicinchoninic acid (BCA) (Fig. 1) is a well known reagent [ 11 that is used for the determination of Cu+, with which it forms a purple complex. Recently, BCA has been used in conjunction with the biuret reaction as a reagent for the determination of protein [ 21. In each of the assays, Cu2+ in a solution containing BCA is chemically reduced by the assayed substance to form a purple Cu+-BCA complex that subsequently is measured spectrophotometrically.
9
-0-C
BCA
WA’-
Fig. 1. Structures of bicinchoninic acid (BCA) and its anion (BCA*- ).
0003-2610/89/$03.50
0 1989 Elsevier Science Publishers B.V.
224
Owing to the potential importance of this reagent for protein determinations, a study was undertaken to define the chemical reaction that occurs between the protein, copper and BCA. The electrochemical techniques used included voltammetry at a rotating platinum disk electrode (r.p.d.e.) and at a rotating glassy carbon electrode (r.g.c.e. ) and controlled-potential coulometry. The results of voltammetric measurements of potential interferences were compared with chemical measurements obtained on the same species by using the Cu2+-BCA reagent. EXPERIMENTAL
Chemicals The sodium salt of anhydrous bicinchoninic acid was obtained from Pierce and ACS-grade copper(I1) sulfate pentahydrate from Curtin Matheson Scientific. All other reagents were of analytical-reagent grade and were used without purification. Deionized water was used to prepare the solutions. The standard BCA reagent either was used as purchased from Pierce or was prepared from analytical-reagent grade chemicals as described elsewhere [ 21. The reagent consisted of 1% Na,BCA, 2% Na,CO,*H,O, 0.16% disodium tartrate, 0.4% NaOH and 0.95% NaHC03. If necessary, the pH of the solution was adjusted to 11.25 with either 50% NaOH or solid NaHCO,. In some of the studies, one or more of the components of the reagent were omitted in order to study the reaction mechanism. Except as described elsewhere, the solutions used for the electrochemical studies were prepared by using a 2-ml microburet (Gilmont) to add 0.100 ml of 0.163 M copper (II) sulfate solution to 5.00 ml of the BCA reagent or to the reagent from which one or more of the components had been omitted. The solution containing Cu”+ and the BCA reagent is green. Several compounds were chemically tested with the reagent and were examined electrochemically. The tested chemicals were hydrogen peroxide, DLtryptophan, benzaldehyde, benzyl alcohol, L-ascorbic acid, hydroquinone, oaminophenol, L-tyrosine, 1-naphthol, pyrocatechol, 4-aminobenzoic acid, DL/3-3,4-dihydroxyphenylalanine, L-cysteine hydrochloride, phenol, resorcinol, aniline, biuret and glycylglycine. Apparatus Linear scan voltammetry was performed using an r.p.d.e. and an r.g.c.e. The electrodes were rotated at 30 Hz using a Sargent synchronous rotator. Prior to each scan the r.p.d.e. was cleaned by dipping it successively in nitric acid and deionized water. The electrode was subsequently polished with a laboratory tissue while rotating. In all electrochemical experiments, potential control and current measurements were made with a Model 174a Polarographic Analyzer (E, G & G Princeton Applied Research) coupled to an Omnigraphic 2000
X-Y recorder (Houston Instrument) for polarography and voltammetry, or to an Omniscribe strip-chart recorder (Houston Instrument) for controlled-potential coulometry. Controlled-potential coulometry was done at a platinum gauze electrode. Prior to each electrochemical study, the sample solution was deaerated with high-purity nitrogen that had been passed through a gas train consisting of two wash towers containing acidic ammonium metavanadate and amalgamated zinc, and a final wash tower containing deionized water. A three-compartment, water-jacketed cell was used for the electrochemical studies. The outer reference electrode and auxiliary electrode compartments were connected near the bottom of each compartment to the central sample compartment by glass tubes containing medium-porosity glass frits. The temperature of the cell was maintained at 25.0 % 0.1 ‘C throughout the studies by pumping water from a waterbath through the water-jacket. During coulometric studies the sample was stirred by bubbling nitrogen through the solution. Spectrophotometric studies were done with a Lambda 3B ultraviolet/visible spectrophotometer ( Perkin-Elmer ). Except where stated otherwise, chemical studies were performed using commercially available BCA reagent. The tested samples were heated in the presence of the reagent at 30’ C for 30 min. RESULTS AND DISCUSSION
Studies were not done at mercury electrodes owing to the chemical reduction of the copper (II)-BCA reagent to the copper (I) complex by mercury.
Voltammetry In order to understand the voltammetric waves obtained in solutions of the Cu’+-BCA reagent, voltammograms at the r.p.d.e. and r.g.c.e. were recorded of the carbonate/hydrogencarbonate buffered solution alone and with the other components of the reagent added individually. The pH 11.25 buffer solution, potassium hydrogen tartrate at the concentration in the reagent used by Smith et al. [2], and BCA (0.10 g per 10 ml) were electroinactive throughout the potential range of the study. The voltammograms at the r.p.d.e. all contained a single peak at about -0.28 V (vs. the saturated calomel electrode) corresponding to the reduction of platinum oxide [ 31 on the electrode surface. A voltammogram at the r.p.d.e. of a lo-ml portion of the buffer solution to which 12 mg of potassium hydrogen tartrate and 0.200 ml of 0.163 M CuS04*5Hz0 had been added is shown in Fig. 2. The concentration of Cu2+ in the solution is identical with that used in the Cu2+-BCA reagent. The Cu2+tartrate-buffer solution is light blue. The voltammogram of the solution contains three cathodic waves (I,, II, and III,) associated with Cu2+ reduction in addition to the peak at - 0.3 V. Similar results were obtained at the r.g.c.e. Figure 3A is a voltammogram at the r.p.d.e. of a lo-ml portion of the pH
226
B
40 20 0
III, I’,
1, A
--:.-1:” 0 0
-0.4 Potential
-0.8
-1.2
Potential
-0.4 (V)
-0.8
(V)
Fig. 2. Voltammogram at the r.p.d.e. of a lo-ml portion of the buffer solution to which 0.200 ml of 0.163 M CuSO., and 12 mg of potassium hydrogen tartrate had been added. Fig. 3. Voltammograms at the r.p.d.e. of a lo-ml portion of the buffer solution to which (A) 0.10 g of BCA and 0.200 ml of 0.163 M CuSOl and (B ) solution A to which 10 mg of potassium hydrogen tartrate had been added. 11.25 carbonate/hydrogencarbonate buffer solution to which 0.10 g of BCA and 0.200 ml of 0.163 M CuS04 had been added. The voltammogram contains three cathodic waves (I,, II, and III,) corresponding to reduction of copper (II) species. Wave III, was often accompanied by a current maximum. Similar results were obtained by using the r.g.c.e. The cathodic waves are similar to those shown in Fig. 2 for solutions which contained no BCA but did contain tartrate. Wave II, in Fig. 2, however, occurs at a significantly more positive potential than the corresponding wave in Fig. 3A. Although the voltammograms of the two solutions are similar, the appearances of the two solutions differed. The solution containing tartrate and no BCA was light blue whereas the solution containing BCA and no tartrate was green. The green color indicates the formation of a Cu2+-BCA complex. The blue color could be caused by a Cu2+tartrate complex [ 41, by the Cu2+ -water complex [5] or by a mixture of the two. Addition of 0.010 g of potassium hydrogen tartrate to the buffered Cu2+BCA solution caused the solution to become lighter green. A voltammogram of the resulting solution is shown in Fig. 3B. The addition of tartrate in excess of that used in the BCA reagent has no effect on the potential of wave I, or III,, but does shift wave II, to a more positive potential. The potential shifts to a value intermediate between those observed for wave II, in solutions containing tartrate but no BCA (Fig. 2) and those observed in solutions containing BCA but no tartrate (Fig. 3A). The shift in potential and the color change indicate that tartrate also forms a complex with Cu2+. Because the potential of wave II, in the presence of both tartrate and BCA is intermediate between that of the corresponding wave in the Cu2+- tartrate solution and that in the Cu2+BCA solution, it is likely that BCA and tartrate are simultaneously complexed to cu2+.
227
In order to determine the effect that tartrate has on the voltammetric waves, a solution was prepared that contained 0.048 g of BCA and 0.100 ml of 0.163 M CuS04 in 5.00 ml of the pH 11.25 buffer. A voltammogram at the r.p.d.e. of the solution was recorded. Subsequently, six stepwise additions of solid potassium hydrogen tartrate to the solution were made and a voltammogram was recorded after each addition. Sufficient potassium hydrogen tartrate was eventually added to make the final tartrate to Cu2+ mole ratio in the solution about 8. The mole ratio of BCA to Cu2+ in all of the solutions was 7.5. Some of the results are shown in Fig. 4. The addition of tartrate to the solution resulted in a decrease in the height (current) of waves II, and III,. Although the height of wave II, decreased as tartrate was added, the wave never completely disappeared. The height of wave I, could not be accurately measured owing to overlap of the wave with the platinum oxide peak. The decreased heights of waves II, and III, with increased tartrate concentration are indicative of the formation of one or more Cu2+tartrate complexes that are either not electroactive, or less electroactive (smaller diffusion coefficient) than either Cu2+ or the Cu2+-BCA complex that provides the solution with its green color. It is likely that tartrate competes with BCA2- for complexation sites around the Cu2+. Voltammetric wave heights can be assumed to be directly proportional (with a zero intercept in the absence of a more easily reduced or oxidized species) to the concentrations of the corresponding electroactive species. If a chemical reaction between an electroactive species and an added reactant (tartrate in this case) proceeds stoichiometrically to completion or near completion, plots of the wave height as a function of the amount of reactant added should contain at least two linear regions. The mole ratio of the reactants at the extrapolated intersection of the two regions should correspond to the ratio of the reactants in the balanced chemical equation. The method is analogous to spectrophotometric methods [ 61 for determining the mole ratio of ligand to metal in a complex.
0
I
I 0
I Potential
I - 0.4
I
I -0.8
I.
W
Fig. 4. Voltammograms at the r.p.d.e. of (A) the buffer solution containing C&O4 and BCA, and solution A to which (B) 1.1, (C) 6.3 and (D) 14.9 mg of potassium hydrogen tartrate had been added.
228
The wave heights decreased non-linearly as a function of added tartrate for waves II, and III,. The data indicate that tartrate reacts incompletely to form one or more electroinactive reaction products, i.e., tartrate competes with BCA for complexation sites around the Cu2+. An overall equilibrium such as that shown in Eqn. 1 is established. CU(BCA),~-~”
+nT2-
=CU(BCA),_,T,~-~“+~BCA~-
(1)
BCA2- in Eqn. 1 and elsewhere in the paper represents the anion of BCA, and T2- represents tartrate. In Eqn. 1, m can have an integral value between 1 and n. The experimental evidence presented so far is inadequate for determining either n or m. If the reaction occurs between uncomplexed Cu2+ and tartrate, the equilibrium can be represented as shown in Eqn. 2, where p is an integer. Cu2+ +pT2- =CUT,‘-~~
(2)
In Eqns, 1 and 2 the water molecules that are complexed to Cu2+ have been omitted for simplicity. Up to six molecules of water [ 71 can be attached to each copper ion. Of the six molecules, four are relatively strongly bound to the Cu2+. In the presence of BCA”- or T2-, each ion of the bidentate complexing agent probably replaces two water molecules around the Cu2+. In order to study the effect of tartrate on Cu2+ reduction in the absence of BCA, a solution was prepared by adding 0.100 ml of 0.163 M CuSO, to 5.00 ml of the pH 11.25 buffer solution. As in the previous study, voltammograms were recorded of this solution and of the solution after addition of various amounts of solid potassium hydrogen tartrate. Initially the solution was light blue (the copper (II)-water complex) and the CuS04 did not dissolve completely. In basic solutions copper (II) sulfate is sparingly soluble and a complexing agent is required in order to increase the solubility. Voltammograms (Fig. 5) of the CuSO, solution contained waves I, and III, at both the r.p.d.e. and r.g.c.e. As tartrate was added, CuS04 dissolved. A total of 4.0 mg of potassium hydrogen tartrate was needed to dissolve all of the CuSO,, which corresponds to a mole ratio of tartrate to Cu2+ in the solution of 1.3. Because copper (II) was dissolving during the study, a plot of wave height as a function of amount of tartrate added was meaningless. In order to study the effect of BCA on the voltammetric waves, a solution was prepared by adding 9.4 mg of potassium hydrogen tartrate and 0.100 ml of 0.163 M CuS04 solution to 5.00 ml of pH 11.25 buffer. Voltammograms were recorded successively of that solution and the solution to which 16 stepwise additions of solid BCA were made. The mole ratios of BCA to Cu2+ and of BCA to tartrate in the final solution were 7.4 and 2.4, respectively. The initial solution, which contained no BCA, was blue. The voltammogram of the solution contained two cathodic voltammetric waves (I, and III,) as shown in Fig. 6A. Apparently the presence of excess tartrate (about twice the
I
0. 0
T
I
I
I
I
,
C
6
5 k 0’
Potential Potential
W)
(V)
Fig. 5. Superimposedvoltammograms at the r.p.d.e. of (A) the buffer solution and (B) a saturated solution of CuSO, in the buffer. Fig. 6. Voltammograms of (A) a solution containing 5.00 ml of buffer, 0.100 ml of 0.163 M CuSO, and 9.4 mg of potassium hydrogen tartrate to which (B) 14.6 and (C) 45.8 mg of disodium BCA had been added.
amount normally used in the reagent) prevents the reduction corresponding to wave II, (in the absence of BCA). It is interesting that at lower concentrations of tartrate, wave II, is observed in the absence of BCA (Fig. 2). The decreasing height of wave II, with the addition of tartrate was also observed in the complexation studies described earlier. As was previously observed, a current maximum was often associated with wave III,. As BCA was added to the solution, wave III, generally decreased in height, as shown in Fig. 6B and C, while wave II, appeared and increased in height. The proximity of the platinum oxide peak to wave I, made it impossible to make accurate measurements of its height. Plots of the wave heights (Fig. 7) for waves II, and III, as a function of the total molar amount of BCA added to the solution contained three linear portions. At the extrapolated intersections of the linear portions in each curve, the mole ratios of BCA to Cu2+ were 0.87 and 2.10 for wave II, and 1.06 and 2.37 for wave III,. Several conclusions can be drawn from these studies. Because wave II, increases in height as BCA is added, it is likely that BCA is replacing tartrate in the Cu2+ complex (reverse of Eqn. 1 ), and wave II, is caused by a BCA complex with Cu2+. Because two intersections occur in the plots of wave height as a function of amount of BCA added at mole ratios of BCA to Cu2+ of 1 and 2, it is likely that two tartrates in the complex are replaced stepwise. Since wave II, does not appear until the second BCA starts to add to the complex, it is probable that the wave is caused by reduction of CU(BCA),~-. Although a mixed complex forms as shown in Eqn. 3, the bis complex with BCA is electroactive
2
6 BCA (10e5mol)
10
Fig. 7. Plots of voltammetric wave heights as a function of moles of BCA added: (A) wave II,; (B) wave III,.
at the potential associated with wave II,. For the sake of simplicity, the unattached ligands in Eqn. 3 are not shown. CuTz2- =CUT(BCA)~blue
=CU(BCA),~-
(3)
green (wave II,)
The linear variations in the heights of waves II, and III, are indicative of chemical reactions that proceed nearly to completion. Consequently, BCA2is more strongly complexed to Cu2+ than is tartrate. This conclusion is supported by the non-linear variations in wave heights observed when tartrate was added to Cu2+ solutions containing BCA. In order to study the effect of BCA on the voltammetric waves recorded in the absence of tartrate, a solution was prepared by adding 0.100 ml of 0.163 M CuS04 to 5.00 ml of pH 11.25 buffer. Voltammograms were recorded of this solution and the solution after each of 16 stepwise additions of solid BCA. As previously described, the solution initially was blue, and the CuS04 did not dissolve completely. The voltammogram of the initial solution was similar to that shown in Fig. 5. As BCA was added, the color of the solution changed from blue to blue-green and eventually to green. Copper (II) sulfate gradually dissolved with the addition of BCA, and dissolved completely after the seventh addition (0.0132 g total). This corresponds to a mole ratio of BCA to Cu2+ in the solution of 2.1. The CU(BCA),~- complex is highly soluble at pH 11.25. As described earlier, in a similar experiment in which tartrate rather than BCA was added, the mole ratio of tartrate to Cu2+ resulting in complete dissolution was 1.3. As BCA was
231
added, wave III, decreased in height and wave II, appeared. Because the total dissolved concentration of copper changed during the study, plots of wave height as a function of added BCA were of little use. Controlled-potential coulometry Controlled-potential coulometric studies were done on a platinum gauze working electrode. In each case a voltammogram was recorded prior to and after each electrolysis. Prior to studying CL?++solutions in the presence of T2or BCA2-, voltammetric and coulometric studies were made of CuSO, solutions in the pH 11.25 carbonate/hydrogencarbonate buffer. Solutions were prepared by adding 0.050-ml portions of 0.163 M CuS04 to 5.00-ml portions of the buffer solution, or by adding O.lOO-ml portions to 10.00 ml of buffer solution. Even at a concentration of 1.6 mM, the CuSO* did not dissolve completely in the buffer. The solution was pale blue. The low concentration of dissolved Cu2+ is responsible for the small cathodic wave heights (I, and III,) observed in Fig. 5. Two controlled-potential electrolyses were performed at -0.2 V prior to wave I, in order to determine the effect of the platinum oxide peak on the results. The calculated number (n) of moles of electrons for each mole of Cu2+ in the solution was 0.1 + 0.1. The uncertainties listed in the measurements are standard deviations. Voltammograms recorded after the electrolyses were identical with those recorded prior to the electrolyses, i.e., no apparent electrochemical reaction occurred. Controlled-potential coulometry also was performed at -0.4 V (four trials) and at -0.5 V (one trial) on the plateau of wave I,, but prior to wave III,. The calculated value of n was 1.05 ? 0.09 (five trials). During the electrolyses the CuSO, dissolved completely and the solutions changed from blue to colorless. A yellow deposit formed on the electrode during each reduction. Voltammograms recorded after the electrolyses were identical with the voltammogram of the buffer solution, i.e., both cathodic waves disappeared. Apparently Cu2+ is reduced to either Cu20 or to Cu,COa during the electrochemical reaction. Both Cu20 and Cu2C03 are insoluble and are yellow. Cu,O is usually prepared [ 81 by chemical reduction of an alkaline solution of Cu2+ with hydrazine. The deposit rapidly dissolved in HCl (aq. ) without evolution of gas bubbles (no apparent CO, emission). Consequently, it is likely that the deposit is Cu,O. Controlled-potential coulometry was also performed on the rising portion of wave III, at -0.8 V, and near the top of wave III, at -0.9 V. The calculated value of n for the electrolyses was 2.0 5 0.1 (two trials). During each electrolysis CuS04 dissolved completely; the solution changed from blue to colorless and a copper plate formed on the electrode. Voltammograms recorded after the electrolyses were identical with that of the buffer solution. Consequently, wave III, can be attributed to the two-electron reduction of Cu2+ to metallic Cu.
232
A set of five electrolyses of the Cu ‘+-BCA-tartrate-buffer reagent were performed on the plateau of wave I, at -0.5 V. Both commercial reagent and laboratory-prepared solutions were used. The calculated number of moles of electrons used for each mole of Cu2+ in the solution was 1.020.2. From the results, it is clear that copper (II) in the solution is reduced to copper (I). During each electrolysis the solution changed from green to purple, indicating the formation of the copper (I)-BCA complex. Unlike the reductions in the absence of BCA and tartrate, no CuzO deposit formed on the electrode. The voltammogram recorded after each electrolysis contained no cathodic wave, even though the applied potential was not sufficiently negative to cause direct reduction of the species responsible for waves II, and III,. Either all three waves correspond to reductions to different products of the same chemical species, or the species responsible for the three waves are in chemical equilibrium with each other, thereby causing all three to disappear when one is completely removed from the solution. In light of the results in Cu2+-buffer solutions, the latter possibility is more likely. Copper did not plate on the electrode during the five reductions. Electrolyses at - 0.8 V on the plateau of wave II, but before III, yielded an average n of 1.04 + 0.08 (three trials). During the electrolyses, the solution changed from green to purple as during the studies at -0.5 V. The voltammogram recorded after each electrolysis had no cathodic wave, i.e., all three cathodic waves disappeared. No copper plate formed during the reduction. Wave II, therefore also corresponds to a one-electron reduction to a copper (I) species. Controlled-potential electrolyses were also done on the plateau of wave III, at -0.95 and - 1.00 V. The calculated n values for the electrolyses were 1.11 and 1.29, respectively. In both cases the solution changed from green to purple and some copper plate formed on the electrode. During the electrolysis at - 1.00 V, electrogravimetry was used to determine that 14% of the copper initially present in the solution plated on to the electrode. Even less plated on to the electrode during the study at -0.95 V. Values of n that are greater than 1 but less than 2 support the conclusion that at these potentials Cu2+ is simultaneously reduced to a copper (I) species and to metallic copper. The change in the color of the solution to purple is indicative of a one-electron reduction and the formation of the copper plate is indicative of a two-electron reduction. From the coulometric studies it is possible to conclude that waves I, and II, correspond to one-electron reductions of copper (II) species to a copper (I) species. Wave III, corresponds to reduction of copper (II) to metallic copper. Reduction to metallic copper also explains the maximum associated with wave III,, which is caused by copper plating on the electrode surface. Polarographic maxima in Cu2+ solutions have been associated with reduction to copper (0) by other workers [ 91. The calculated n after reduction on wave III, is less than 2 because the one-electron reductions corresponding to waves I, and II, are
233
simultaneously occurring to yield a product that is not electrochemically reducible to metallic copper. Spectrophotometry The copper (I) complex with BCA is purple. The purple complex forms upon chemical or electrochemical reduction of copper (II) in the presence of BCA. Tartrate is not required in order to obtain a purple solution. Complexation between Cu+ and BCA was studied spectrophotometrically using the mole-ratio method [ 61. Because copper (I) salts are sparingly soluble at pH 11.25, it was necessary to do the studies by coulometrically generating Cu+ from a CuS04 solution containing a known amount of BCA. Solutions were prepared by adding 0.992 ml of 0.163 M CuS04 and 0.039 g of Na,BCA to 10.0 ml of the pH 11.25 buffer. The mole ratio of Cu2+ to BCA in the initial solutions was 1.5. The working electrode was controlled at a potential of - 0.3 V on the plateau of cathodic wave I,. The electrochemical reaction occurring at that potential was earlier shown to correspond to reduction of Cu2+ to Cu+. Prior to starting the electrolysis, a O.lO-ml portion of the sample solution in the cell was withdrawn and diluted to 10.00 ml in a volumetric flask. The visible spectrum of the diluted solution (Fig. 8A) was recorded. A recorded current was allowed to flow through the cell until approximately one tenth of the Cu2+ had been reduced. The current flow was then stopped and another 0.10 ml portion of the cell solution was removed and diluted to 10.00 ml. The absorbance of the solution was measured at 558 nm on the spectrophotometric peak corresponding to the purple Cu+-BCA complex. The process was repeated until all of the CL?+ in the solution had been electrochemically reduced to Cu+. A typical spectrum obtained after reduction of Cu2+ to Cu+ is shown in Fig. 8B.
0.
A I
400
I
I
I
1
560 Wavelength (nm)
1
I
I
720
I 0
I
I
I
I
0.8 mol Cu*lmo~ BCA
04
I
I
II
1.2
Fig. 8. Visible spectra of (A) a solution containing the Cu 2+-BCA complex and (B) a solution containing the electrochemically formed Cu+-BCA complex. The maximum in curve B occurs at 558 nm. Fig. 9. Plot of absorbance at 558 nm as a function of the mole ratio of electrogenerated Cu+ to BCA in a buffered solution.
234
The procedure was done three times in solutions containing BCA but no tartrate and once in a solution containing both BCA and 16.1 mg of tartrate. In each case a plot was prepared of absorbance as a function of the ratio of the number of moles of electrogenerated Cu+ to the number of moles of BCA in the solution. The plots contained two linear portions that, on extrapolation, intersected at a mole ratio of BCA to Cu+ of 2.1 +O.l (four trials). A typical plot is shown in Fig. 9. The results obtained in the absence of tartrate were essentially identical with those obtained in solutions that contained tartrate. From the results it is apparent that the complex contains two BCA’- moieties for each Cu+, as suggested by Smith et al. [ 21. Consequently, the formula of the complex is Cu (BCA ) 23-. Normally, mole-ratio plots are expected to have a horizontal (constant absorbance) portion after the intersection of the two linear regions, corresponding to a constant concentration of the monitored complex. In Fig. 9, the absorbance decreases after the intersection. This decrease could be associated with the formation of either a mono/BCA complex or a dinuclear complex. Evidence does not exist that would make it possible to determine whether either of these complexes actually exists. Chemical and electrochemical reactions The probable chemical equilibria that occur in the tested solutions containing copper (II) are summarized in the following equations: T Cu(H,O),
2+
*T 2-
e
Cu(HzO),T
blue
-
CU(H,O),T,
blue
CU(H>O)~~+
+
2BCA2-
_ --
(4)
blue
CU(H,O),(BCA),~-
blue
(5)
green
2-
CutH,O),T>
2-
2-
c
Cu(H,O),T
(BCA)
e
CU(H,O),WA),~-
(6)
blue
Equations 4 and 5 show the equilibria between the copper (II)-water complex and the tartrate and BCA complexes. Each tartrate or BCA ion replaces two water molecules around the Cu 2+ . The stoichiometries of these equilibria were established during the voltammetric studies obtained as the concentration of the complexing agent was varied. It is likely that the four water mole-
235
cules that are nearer to the Cu2+ are replaced, leaving the two water molecules at the greater distance from the Cu2+. Reduction of the complex between copper (II) and a single tartrate is probably responsible for cathodic wave II, in the absence of BCA. The existence of a copper (II)-monotartrate complex is supported by the studies described earlier in which the mole ratio of tartrate to Cu2+ at which all of the CuS04 dissolved was 1.3. The corresponding copper (II )-monoBCA complex is not as likely to exist since the mole ratio of BCA to Cu2+ at which the CuS04 dissolved was 2.1. Equation 6 shows the equilibria between the predominant species in the solution containing both BCA and tartrate. The presence of the mixed monotartrate-monoBCA complex was the conclusion arrived at Eqn. 3, where the water molecules were not shown for the sake of simplicity. The unattached complexing agent molecules and ions are not shown in Eqn. 6, in order to simplify the equations. All of the electrochemical reactions are irreversible, as indicated by the absence of anodic waves after the coulometric studies and during triangular-wave voltammetric studies. The electrochemical reactions associated with cathodic wave I, are summarized in Eqns. 7 and 8. ~CU(H,O),~+ +OH-+2e--+Cu,O(s)+H,O++llH,O blue
CU(H,O),~+ +2BCA2-+e-+Cu(BCA)23-+6H20 blue
(7)
yellow
(8)
purple
The reaction shown in Eqn. 7 occurs in the absence of BCA at pH 11.25, while the reaction shown in Eqn. 8 occurs in the presence of BCA. The reaction shown in Eqn. 8 is responsible for the color change to purple during protein assays. Coulometric data were used to define the reductions from copper (II) to copper (I). Wave I, is present in the absence of tartrate or BCA, and therefore can be assigned to reduction of the copper (II)-water complex. In the presence of BCA, the green color of the copper (II)-BCA complex masks the light blue color of the copper (II)-water complex so that the initial solution appears green rather than blue. Owing to the equilibria in Eqn. 6, as CU(H,O),~+ is electrochemically removed from the solution the concentrations of all other copper(I1) species also decrease and eventually disappear. The existence of CU(BCA),~- in Eqn. 8 was demonstrated spectrophotometrically after controlled-potential coulometry on wave I, (Fig. 9). Wave II, has been shown to be associated with a one-electron reduction.
236
Wave II, is observed in Cu2+ solutions that contain BCA, and mixtures of tartrate and BCA, but it has not been observed in solutions containing neither tartrate nor BCA. In the presence of BCA wave II, can be assigned to the oneelectron reduction of a Cu ‘+-BCA complex to a Cu+-BCA complex. In the studies in which BCA was added to a solution containing Cu2+ and tartrate, wave II, appeared only after sufficient BCA had been added to start forming the binary complex from the monoBCA complex. Consequently, the reduction is that shown in Eqn. 9. CU(H,O),(BCA),~-+~--*C~(BCA),~-+~H,O green
(9)
purple
In the absence of BCA but in the presence of tartrate, a wave that is anodic of the wave corresponding to the reduction in Eqn. 9 but cathodic of wave I, is observed (Fig. 2, wave II,). If sufficient tartrate is added, the wave disappears. It is likely that the wave is caused by the one-electron reduction of the copper (II )-monotartrate complex shown in Eqn. 4. As sufficient tartrate is added to cause the electroinactive Cu ( H20)2T22- to form, the wave disappears. Because BCA has been shown to be a stronger complexing agent than tartrate for Cu2+, it is unlikely that the copper (II)-monotartrate complex exists in the presence of BCA. That conclusion is supported by the absence of the voltammetric wave in solutions containing BCA. Consequently, the complex is not shown in Eqn. 6. Cathodic wave III, is observed in CuSO, solutions in the absence of tartrate and BCA. As either tartrate or BCA is added to the solution, the wave height has been shown to decrease. Electrolytic data have shown that the wave corresponds to a two-electron reduction to metallic copper. The wave can be assigned to the reduction shown in Eqn. 10. Cu(H20),2++2e-+Cu(s)
+6H2O
The wave height decreases as tartrate chemical equilibria shown in Eqn. 6.
(IO) or BCA is added owing to shifts in the
Comparison of electrochemical and chemical results From the previous description of the electrochemical reaction, it is apparent that any chemical substance that is sufficiently reactive to reduce copper (II) chemically to copper (I) should be capable of being assayed using the Cu2+BCA-tartrate reagent. Essentially, this means that any chemical species that has an anodic wave that at least partially overlaps with wave I, should be capable of being assayed with the reagent. In order to determine the potential that is sufficient to cause the color of the reagent to change from green to purple, the potential of the working electrode
231
in the reagent was successively stepped from +0.40 V toward more negative potentials in 0.05-V steps. If a color change had not occurred after 30 s (at 25 ‘C ), it was assumed that the potential was not sufficiently negative to cause the reaction to occur. The potential at which the solution first changed from green to purple was -0.15 V. In the comparisons described in this section, a positive voltammetric test consequently means that the tested compound has an anodic voltammetric wave that exhibits a current that is greater than that of the buffer solution at -0.15 V. Solutions containing about 1OmM of each tested solution were used for the voltammetric tests. Chemical studies were performed as described by Smith et al. [ 21 in a bath heated at 30” C for 30 min. If the solution containing the tested compound caused the solution to turn purple within the 30-min period, the chemical test was positive. The compounds tested and the results obtained using the chemical and voltammetric methods are shown in Table 1. In most cases the two methods agree. In those cases in which disagreement occurs, the voltammetric response was negative while the chemical response was positive. The difference in response can be attributed to the longer reaction time and higher temperature used for the chemical test, and to the higher sensitivity of the chemical test. From the results it is possible to conclude that use of the reagent for protein assays as suggested by Smith et al. [2] is subject to many interferences. The method should be applied with caution only under carefully controlled conditions in the absence of even mild reducing agents. Voltammetry of those compounds in Table 1 revealed that essentially any compound which can be more easily oxidized than the buffer-solvent system, i.e., which has an anodic wave, yields a positive chemical test. This is in agreement with the results of Wiechelman et al. [lo]. TABLE 1 Summary of the results of the chemical tests with the Cu*+-BCA-tartrate reagent and the voltammetric tests Compound
DL-Try-ptophar+ 4-Acetamidophenol Benzaldehyde’ Benzyl alcohol L-Ascorbic acid Hydroquinone o-Aminophenol L-Tyrosineb 1-Naphtholc
Result’
Compound
Voltammetric
Chemical
+ -
+ + +
+ + + -
+ + + + +
Pyrocatechol I-Aminobenzoic acid D@3,4-Dihydroxyphenylalanine L-Cysteine Phenolb Resorcinol Anilineb Biuret Glycylglycine
Result” Voltammetric
Chemical
+ + -
+ + + + + + -
+
“A+ sign indicates the compound responded positively and a - sign negatively to the test. ‘The chemical reaction occurred slowly. ‘The low solubility of the compound accounts for the negative voltammetric test.
238 REFERENCES 1 2 3 4 5 6 7 8 9 10
V.N. Tikhonov and I.S. Must&in, Zh. Anal. Khim., 20 (1965) 390. P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F. H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke, B.J. Olson and D.C. Klenk, Anal. Biochem., 150 (1985) 76. R.N. Adams, Electrochemistry at Solid Electrodes, Dekker, New York, 1969, pp. 191-205. J.J. Lingane, Electroanalytical Chemistry, 2nd edn., Interscience, New York, 1966, pp. 206, 380. F.A. Cotton and G. Wilkinson, Advanced Inorganic Chemistry, Interscience, New York, 1962, pp, 756,757. R.D. Braun, Introduction to Instrumental Analysis, McGraw-Hill, New York, 1987, pp. 289291. J.N. Butler, Ionic Equilibrium, Addison-Wesley, Reading, MA, 1964, pp. 5-7. F.A. Cotton, G. Wilkinson and P.L. Gaus, Basic Inorganic Chemistry, 2nd edn., Wiley, New York, 1987, p. 508. F.W. Hawkridge, T.W. Holt and H.H. Bauer, Anal. Chim. Acta, 58 (1972) 203. K.J. Wiechelman, R.D. Braun and J.D. Fitzpatrick, Anal. Biochem., 175 (1988) 231.
223 Printed in The Netherlands
ELECTROCHEMICAL, CHEMICAL AND SPECTROPHOTOMETRIC INVESTIGATION OF THE COPPER(BICINCHONINIC ACID REAGENT USED FOR PROTEIN MEASUREMENTS
ROBERT D. BRAUN*, KAREN J. WIECHELMAN and AUGUST A. GALL0 Department of Chemistry, University of Southwestern Louisiana, Lafayette, LA 70504-4370 (U.S.A.) (Received 15th September 1988)
SUMMARY The use of a reagent containing copper (II), bicinchoninic acid (BCA ) and tartrate buffered at pH 11.25 was studied voltammetrically, coulometrically, spectrophotometrically and chemically. The reagent exhibits three cathodic waves at rotating platinum disk and rotating glassy carbon electrodes. The two more-positive cathodic waves correspond to electrochemical reduction to copper (I) -bisbicinchoninate, Cu (BCA ), 3- . The third cathodic wave is caused by reduction to metallic copper. A reaction mechanism is proposed that shows the major chemical species in the solution and the electrochemical reaction products. Voltammetric and chemical studies indicate that the reagent should be used with care for protein assays because it is subject to multiple chemical interferences.
Bicinchoninic acid (BCA) (Fig. 1) is a well known reagent [ 11 that is used for the determination of Cu+, with which it forms a purple complex. Recently, BCA has been used in conjunction with the biuret reaction as a reagent for the determination of protein [ 21. In each of the assays, Cu2+ in a solution containing BCA is chemically reduced by the assayed substance to form a purple Cu+-BCA complex that subsequently is measured spectrophotometrically.
9
-0-C
BCA
WA’-
Fig. 1. Structures of bicinchoninic acid (BCA) and its anion (BCA*- ).
0003-2610/89/$03.50
0 1989 Elsevier Science Publishers B.V.
224
Owing to the potential importance of this reagent for protein determinations, a study was undertaken to define the chemical reaction that occurs between the protein, copper and BCA. The electrochemical techniques used included voltammetry at a rotating platinum disk electrode (r.p.d.e.) and at a rotating glassy carbon electrode (r.g.c.e. ) and controlled-potential coulometry. The results of voltammetric measurements of potential interferences were compared with chemical measurements obtained on the same species by using the Cu2+-BCA reagent. EXPERIMENTAL
Chemicals The sodium salt of anhydrous bicinchoninic acid was obtained from Pierce and ACS-grade copper(I1) sulfate pentahydrate from Curtin Matheson Scientific. All other reagents were of analytical-reagent grade and were used without purification. Deionized water was used to prepare the solutions. The standard BCA reagent either was used as purchased from Pierce or was prepared from analytical-reagent grade chemicals as described elsewhere [ 21. The reagent consisted of 1% Na,BCA, 2% Na,CO,*H,O, 0.16% disodium tartrate, 0.4% NaOH and 0.95% NaHC03. If necessary, the pH of the solution was adjusted to 11.25 with either 50% NaOH or solid NaHCO,. In some of the studies, one or more of the components of the reagent were omitted in order to study the reaction mechanism. Except as described elsewhere, the solutions used for the electrochemical studies were prepared by using a 2-ml microburet (Gilmont) to add 0.100 ml of 0.163 M copper (II) sulfate solution to 5.00 ml of the BCA reagent or to the reagent from which one or more of the components had been omitted. The solution containing Cu”+ and the BCA reagent is green. Several compounds were chemically tested with the reagent and were examined electrochemically. The tested chemicals were hydrogen peroxide, DLtryptophan, benzaldehyde, benzyl alcohol, L-ascorbic acid, hydroquinone, oaminophenol, L-tyrosine, 1-naphthol, pyrocatechol, 4-aminobenzoic acid, DL/3-3,4-dihydroxyphenylalanine, L-cysteine hydrochloride, phenol, resorcinol, aniline, biuret and glycylglycine. Apparatus Linear scan voltammetry was performed using an r.p.d.e. and an r.g.c.e. The electrodes were rotated at 30 Hz using a Sargent synchronous rotator. Prior to each scan the r.p.d.e. was cleaned by dipping it successively in nitric acid and deionized water. The electrode was subsequently polished with a laboratory tissue while rotating. In all electrochemical experiments, potential control and current measurements were made with a Model 174a Polarographic Analyzer (E, G & G Princeton Applied Research) coupled to an Omnigraphic 2000
X-Y recorder (Houston Instrument) for polarography and voltammetry, or to an Omniscribe strip-chart recorder (Houston Instrument) for controlled-potential coulometry. Controlled-potential coulometry was done at a platinum gauze electrode. Prior to each electrochemical study, the sample solution was deaerated with high-purity nitrogen that had been passed through a gas train consisting of two wash towers containing acidic ammonium metavanadate and amalgamated zinc, and a final wash tower containing deionized water. A three-compartment, water-jacketed cell was used for the electrochemical studies. The outer reference electrode and auxiliary electrode compartments were connected near the bottom of each compartment to the central sample compartment by glass tubes containing medium-porosity glass frits. The temperature of the cell was maintained at 25.0 % 0.1 ‘C throughout the studies by pumping water from a waterbath through the water-jacket. During coulometric studies the sample was stirred by bubbling nitrogen through the solution. Spectrophotometric studies were done with a Lambda 3B ultraviolet/visible spectrophotometer ( Perkin-Elmer ). Except where stated otherwise, chemical studies were performed using commercially available BCA reagent. The tested samples were heated in the presence of the reagent at 30’ C for 30 min. RESULTS AND DISCUSSION
Studies were not done at mercury electrodes owing to the chemical reduction of the copper (II)-BCA reagent to the copper (I) complex by mercury.
Voltammetry In order to understand the voltammetric waves obtained in solutions of the Cu’+-BCA reagent, voltammograms at the r.p.d.e. and r.g.c.e. were recorded of the carbonate/hydrogencarbonate buffered solution alone and with the other components of the reagent added individually. The pH 11.25 buffer solution, potassium hydrogen tartrate at the concentration in the reagent used by Smith et al. [2], and BCA (0.10 g per 10 ml) were electroinactive throughout the potential range of the study. The voltammograms at the r.p.d.e. all contained a single peak at about -0.28 V (vs. the saturated calomel electrode) corresponding to the reduction of platinum oxide [ 31 on the electrode surface. A voltammogram at the r.p.d.e. of a lo-ml portion of the buffer solution to which 12 mg of potassium hydrogen tartrate and 0.200 ml of 0.163 M CuS04*5Hz0 had been added is shown in Fig. 2. The concentration of Cu2+ in the solution is identical with that used in the Cu2+-BCA reagent. The Cu2+tartrate-buffer solution is light blue. The voltammogram of the solution contains three cathodic waves (I,, II, and III,) associated with Cu2+ reduction in addition to the peak at - 0.3 V. Similar results were obtained at the r.g.c.e. Figure 3A is a voltammogram at the r.p.d.e. of a lo-ml portion of the pH
226
B
40 20 0
III, I’,
1, A
--:.-1:” 0 0
-0.4 Potential
-0.8
-1.2
Potential
-0.4 (V)
-0.8
(V)
Fig. 2. Voltammogram at the r.p.d.e. of a lo-ml portion of the buffer solution to which 0.200 ml of 0.163 M CuSO., and 12 mg of potassium hydrogen tartrate had been added. Fig. 3. Voltammograms at the r.p.d.e. of a lo-ml portion of the buffer solution to which (A) 0.10 g of BCA and 0.200 ml of 0.163 M CuSOl and (B ) solution A to which 10 mg of potassium hydrogen tartrate had been added. 11.25 carbonate/hydrogencarbonate buffer solution to which 0.10 g of BCA and 0.200 ml of 0.163 M CuS04 had been added. The voltammogram contains three cathodic waves (I,, II, and III,) corresponding to reduction of copper (II) species. Wave III, was often accompanied by a current maximum. Similar results were obtained by using the r.g.c.e. The cathodic waves are similar to those shown in Fig. 2 for solutions which contained no BCA but did contain tartrate. Wave II, in Fig. 2, however, occurs at a significantly more positive potential than the corresponding wave in Fig. 3A. Although the voltammograms of the two solutions are similar, the appearances of the two solutions differed. The solution containing tartrate and no BCA was light blue whereas the solution containing BCA and no tartrate was green. The green color indicates the formation of a Cu2+-BCA complex. The blue color could be caused by a Cu2+tartrate complex [ 41, by the Cu2+ -water complex [5] or by a mixture of the two. Addition of 0.010 g of potassium hydrogen tartrate to the buffered Cu2+BCA solution caused the solution to become lighter green. A voltammogram of the resulting solution is shown in Fig. 3B. The addition of tartrate in excess of that used in the BCA reagent has no effect on the potential of wave I, or III,, but does shift wave II, to a more positive potential. The potential shifts to a value intermediate between those observed for wave II, in solutions containing tartrate but no BCA (Fig. 2) and those observed in solutions containing BCA but no tartrate (Fig. 3A). The shift in potential and the color change indicate that tartrate also forms a complex with Cu2+. Because the potential of wave II, in the presence of both tartrate and BCA is intermediate between that of the corresponding wave in the Cu2+- tartrate solution and that in the Cu2+BCA solution, it is likely that BCA and tartrate are simultaneously complexed to cu2+.
227
In order to determine the effect that tartrate has on the voltammetric waves, a solution was prepared that contained 0.048 g of BCA and 0.100 ml of 0.163 M CuS04 in 5.00 ml of the pH 11.25 buffer. A voltammogram at the r.p.d.e. of the solution was recorded. Subsequently, six stepwise additions of solid potassium hydrogen tartrate to the solution were made and a voltammogram was recorded after each addition. Sufficient potassium hydrogen tartrate was eventually added to make the final tartrate to Cu2+ mole ratio in the solution about 8. The mole ratio of BCA to Cu2+ in all of the solutions was 7.5. Some of the results are shown in Fig. 4. The addition of tartrate to the solution resulted in a decrease in the height (current) of waves II, and III,. Although the height of wave II, decreased as tartrate was added, the wave never completely disappeared. The height of wave I, could not be accurately measured owing to overlap of the wave with the platinum oxide peak. The decreased heights of waves II, and III, with increased tartrate concentration are indicative of the formation of one or more Cu2+tartrate complexes that are either not electroactive, or less electroactive (smaller diffusion coefficient) than either Cu2+ or the Cu2+-BCA complex that provides the solution with its green color. It is likely that tartrate competes with BCA2- for complexation sites around the Cu2+. Voltammetric wave heights can be assumed to be directly proportional (with a zero intercept in the absence of a more easily reduced or oxidized species) to the concentrations of the corresponding electroactive species. If a chemical reaction between an electroactive species and an added reactant (tartrate in this case) proceeds stoichiometrically to completion or near completion, plots of the wave height as a function of the amount of reactant added should contain at least two linear regions. The mole ratio of the reactants at the extrapolated intersection of the two regions should correspond to the ratio of the reactants in the balanced chemical equation. The method is analogous to spectrophotometric methods [ 61 for determining the mole ratio of ligand to metal in a complex.
0
I
I 0
I Potential
I - 0.4
I
I -0.8
I.
W
Fig. 4. Voltammograms at the r.p.d.e. of (A) the buffer solution containing C&O4 and BCA, and solution A to which (B) 1.1, (C) 6.3 and (D) 14.9 mg of potassium hydrogen tartrate had been added.
228
The wave heights decreased non-linearly as a function of added tartrate for waves II, and III,. The data indicate that tartrate reacts incompletely to form one or more electroinactive reaction products, i.e., tartrate competes with BCA for complexation sites around the Cu2+. An overall equilibrium such as that shown in Eqn. 1 is established. CU(BCA),~-~”
+nT2-
=CU(BCA),_,T,~-~“+~BCA~-
(1)
BCA2- in Eqn. 1 and elsewhere in the paper represents the anion of BCA, and T2- represents tartrate. In Eqn. 1, m can have an integral value between 1 and n. The experimental evidence presented so far is inadequate for determining either n or m. If the reaction occurs between uncomplexed Cu2+ and tartrate, the equilibrium can be represented as shown in Eqn. 2, where p is an integer. Cu2+ +pT2- =CUT,‘-~~
(2)
In Eqns, 1 and 2 the water molecules that are complexed to Cu2+ have been omitted for simplicity. Up to six molecules of water [ 71 can be attached to each copper ion. Of the six molecules, four are relatively strongly bound to the Cu2+. In the presence of BCA”- or T2-, each ion of the bidentate complexing agent probably replaces two water molecules around the Cu2+. In order to study the effect of tartrate on Cu2+ reduction in the absence of BCA, a solution was prepared by adding 0.100 ml of 0.163 M CuSO, to 5.00 ml of the pH 11.25 buffer solution. As in the previous study, voltammograms were recorded of this solution and of the solution after addition of various amounts of solid potassium hydrogen tartrate. Initially the solution was light blue (the copper (II)-water complex) and the CuS04 did not dissolve completely. In basic solutions copper (II) sulfate is sparingly soluble and a complexing agent is required in order to increase the solubility. Voltammograms (Fig. 5) of the CuSO, solution contained waves I, and III, at both the r.p.d.e. and r.g.c.e. As tartrate was added, CuS04 dissolved. A total of 4.0 mg of potassium hydrogen tartrate was needed to dissolve all of the CuSO,, which corresponds to a mole ratio of tartrate to Cu2+ in the solution of 1.3. Because copper (II) was dissolving during the study, a plot of wave height as a function of amount of tartrate added was meaningless. In order to study the effect of BCA on the voltammetric waves, a solution was prepared by adding 9.4 mg of potassium hydrogen tartrate and 0.100 ml of 0.163 M CuS04 solution to 5.00 ml of pH 11.25 buffer. Voltammograms were recorded successively of that solution and the solution to which 16 stepwise additions of solid BCA were made. The mole ratios of BCA to Cu2+ and of BCA to tartrate in the final solution were 7.4 and 2.4, respectively. The initial solution, which contained no BCA, was blue. The voltammogram of the solution contained two cathodic voltammetric waves (I, and III,) as shown in Fig. 6A. Apparently the presence of excess tartrate (about twice the
I
0. 0
T
I
I
I
I
,
C
6
5 k 0’
Potential Potential
W)
(V)
Fig. 5. Superimposedvoltammograms at the r.p.d.e. of (A) the buffer solution and (B) a saturated solution of CuSO, in the buffer. Fig. 6. Voltammograms of (A) a solution containing 5.00 ml of buffer, 0.100 ml of 0.163 M CuSO, and 9.4 mg of potassium hydrogen tartrate to which (B) 14.6 and (C) 45.8 mg of disodium BCA had been added.
amount normally used in the reagent) prevents the reduction corresponding to wave II, (in the absence of BCA). It is interesting that at lower concentrations of tartrate, wave II, is observed in the absence of BCA (Fig. 2). The decreasing height of wave II, with the addition of tartrate was also observed in the complexation studies described earlier. As was previously observed, a current maximum was often associated with wave III,. As BCA was added to the solution, wave III, generally decreased in height, as shown in Fig. 6B and C, while wave II, appeared and increased in height. The proximity of the platinum oxide peak to wave I, made it impossible to make accurate measurements of its height. Plots of the wave heights (Fig. 7) for waves II, and III, as a function of the total molar amount of BCA added to the solution contained three linear portions. At the extrapolated intersections of the linear portions in each curve, the mole ratios of BCA to Cu2+ were 0.87 and 2.10 for wave II, and 1.06 and 2.37 for wave III,. Several conclusions can be drawn from these studies. Because wave II, increases in height as BCA is added, it is likely that BCA is replacing tartrate in the Cu2+ complex (reverse of Eqn. 1 ), and wave II, is caused by a BCA complex with Cu2+. Because two intersections occur in the plots of wave height as a function of amount of BCA added at mole ratios of BCA to Cu2+ of 1 and 2, it is likely that two tartrates in the complex are replaced stepwise. Since wave II, does not appear until the second BCA starts to add to the complex, it is probable that the wave is caused by reduction of CU(BCA),~-. Although a mixed complex forms as shown in Eqn. 3, the bis complex with BCA is electroactive
2
6 BCA (10e5mol)
10
Fig. 7. Plots of voltammetric wave heights as a function of moles of BCA added: (A) wave II,; (B) wave III,.
at the potential associated with wave II,. For the sake of simplicity, the unattached ligands in Eqn. 3 are not shown. CuTz2- =CUT(BCA)~blue
=CU(BCA),~-
(3)
green (wave II,)
The linear variations in the heights of waves II, and III, are indicative of chemical reactions that proceed nearly to completion. Consequently, BCA2is more strongly complexed to Cu2+ than is tartrate. This conclusion is supported by the non-linear variations in wave heights observed when tartrate was added to Cu2+ solutions containing BCA. In order to study the effect of BCA on the voltammetric waves recorded in the absence of tartrate, a solution was prepared by adding 0.100 ml of 0.163 M CuS04 to 5.00 ml of pH 11.25 buffer. Voltammograms were recorded of this solution and the solution after each of 16 stepwise additions of solid BCA. As previously described, the solution initially was blue, and the CuS04 did not dissolve completely. The voltammogram of the initial solution was similar to that shown in Fig. 5. As BCA was added, the color of the solution changed from blue to blue-green and eventually to green. Copper (II) sulfate gradually dissolved with the addition of BCA, and dissolved completely after the seventh addition (0.0132 g total). This corresponds to a mole ratio of BCA to Cu2+ in the solution of 2.1. The CU(BCA),~- complex is highly soluble at pH 11.25. As described earlier, in a similar experiment in which tartrate rather than BCA was added, the mole ratio of tartrate to Cu2+ resulting in complete dissolution was 1.3. As BCA was
231
added, wave III, decreased in height and wave II, appeared. Because the total dissolved concentration of copper changed during the study, plots of wave height as a function of added BCA were of little use. Controlled-potential coulometry Controlled-potential coulometric studies were done on a platinum gauze working electrode. In each case a voltammogram was recorded prior to and after each electrolysis. Prior to studying CL?++solutions in the presence of T2or BCA2-, voltammetric and coulometric studies were made of CuSO, solutions in the pH 11.25 carbonate/hydrogencarbonate buffer. Solutions were prepared by adding 0.050-ml portions of 0.163 M CuS04 to 5.00-ml portions of the buffer solution, or by adding O.lOO-ml portions to 10.00 ml of buffer solution. Even at a concentration of 1.6 mM, the CuSO* did not dissolve completely in the buffer. The solution was pale blue. The low concentration of dissolved Cu2+ is responsible for the small cathodic wave heights (I, and III,) observed in Fig. 5. Two controlled-potential electrolyses were performed at -0.2 V prior to wave I, in order to determine the effect of the platinum oxide peak on the results. The calculated number (n) of moles of electrons for each mole of Cu2+ in the solution was 0.1 + 0.1. The uncertainties listed in the measurements are standard deviations. Voltammograms recorded after the electrolyses were identical with those recorded prior to the electrolyses, i.e., no apparent electrochemical reaction occurred. Controlled-potential coulometry also was performed at -0.4 V (four trials) and at -0.5 V (one trial) on the plateau of wave I,, but prior to wave III,. The calculated value of n was 1.05 ? 0.09 (five trials). During the electrolyses the CuSO, dissolved completely and the solutions changed from blue to colorless. A yellow deposit formed on the electrode during each reduction. Voltammograms recorded after the electrolyses were identical with the voltammogram of the buffer solution, i.e., both cathodic waves disappeared. Apparently Cu2+ is reduced to either Cu20 or to Cu,COa during the electrochemical reaction. Both Cu20 and Cu2C03 are insoluble and are yellow. Cu,O is usually prepared [ 81 by chemical reduction of an alkaline solution of Cu2+ with hydrazine. The deposit rapidly dissolved in HCl (aq. ) without evolution of gas bubbles (no apparent CO, emission). Consequently, it is likely that the deposit is Cu,O. Controlled-potential coulometry was also performed on the rising portion of wave III, at -0.8 V, and near the top of wave III, at -0.9 V. The calculated value of n for the electrolyses was 2.0 5 0.1 (two trials). During each electrolysis CuS04 dissolved completely; the solution changed from blue to colorless and a copper plate formed on the electrode. Voltammograms recorded after the electrolyses were identical with that of the buffer solution. Consequently, wave III, can be attributed to the two-electron reduction of Cu2+ to metallic Cu.
232
A set of five electrolyses of the Cu ‘+-BCA-tartrate-buffer reagent were performed on the plateau of wave I, at -0.5 V. Both commercial reagent and laboratory-prepared solutions were used. The calculated number of moles of electrons used for each mole of Cu2+ in the solution was 1.020.2. From the results, it is clear that copper (II) in the solution is reduced to copper (I). During each electrolysis the solution changed from green to purple, indicating the formation of the copper (I)-BCA complex. Unlike the reductions in the absence of BCA and tartrate, no CuzO deposit formed on the electrode. The voltammogram recorded after each electrolysis contained no cathodic wave, even though the applied potential was not sufficiently negative to cause direct reduction of the species responsible for waves II, and III,. Either all three waves correspond to reductions to different products of the same chemical species, or the species responsible for the three waves are in chemical equilibrium with each other, thereby causing all three to disappear when one is completely removed from the solution. In light of the results in Cu2+-buffer solutions, the latter possibility is more likely. Copper did not plate on the electrode during the five reductions. Electrolyses at - 0.8 V on the plateau of wave II, but before III, yielded an average n of 1.04 + 0.08 (three trials). During the electrolyses, the solution changed from green to purple as during the studies at -0.5 V. The voltammogram recorded after each electrolysis had no cathodic wave, i.e., all three cathodic waves disappeared. No copper plate formed during the reduction. Wave II, therefore also corresponds to a one-electron reduction to a copper (I) species. Controlled-potential electrolyses were also done on the plateau of wave III, at -0.95 and - 1.00 V. The calculated n values for the electrolyses were 1.11 and 1.29, respectively. In both cases the solution changed from green to purple and some copper plate formed on the electrode. During the electrolysis at - 1.00 V, electrogravimetry was used to determine that 14% of the copper initially present in the solution plated on to the electrode. Even less plated on to the electrode during the study at -0.95 V. Values of n that are greater than 1 but less than 2 support the conclusion that at these potentials Cu2+ is simultaneously reduced to a copper (I) species and to metallic copper. The change in the color of the solution to purple is indicative of a one-electron reduction and the formation of the copper plate is indicative of a two-electron reduction. From the coulometric studies it is possible to conclude that waves I, and II, correspond to one-electron reductions of copper (II) species to a copper (I) species. Wave III, corresponds to reduction of copper (II) to metallic copper. Reduction to metallic copper also explains the maximum associated with wave III,, which is caused by copper plating on the electrode surface. Polarographic maxima in Cu2+ solutions have been associated with reduction to copper (0) by other workers [ 91. The calculated n after reduction on wave III, is less than 2 because the one-electron reductions corresponding to waves I, and II, are
233
simultaneously occurring to yield a product that is not electrochemically reducible to metallic copper. Spectrophotometry The copper (I) complex with BCA is purple. The purple complex forms upon chemical or electrochemical reduction of copper (II) in the presence of BCA. Tartrate is not required in order to obtain a purple solution. Complexation between Cu+ and BCA was studied spectrophotometrically using the mole-ratio method [ 61. Because copper (I) salts are sparingly soluble at pH 11.25, it was necessary to do the studies by coulometrically generating Cu+ from a CuS04 solution containing a known amount of BCA. Solutions were prepared by adding 0.992 ml of 0.163 M CuS04 and 0.039 g of Na,BCA to 10.0 ml of the pH 11.25 buffer. The mole ratio of Cu2+ to BCA in the initial solutions was 1.5. The working electrode was controlled at a potential of - 0.3 V on the plateau of cathodic wave I,. The electrochemical reaction occurring at that potential was earlier shown to correspond to reduction of Cu2+ to Cu+. Prior to starting the electrolysis, a O.lO-ml portion of the sample solution in the cell was withdrawn and diluted to 10.00 ml in a volumetric flask. The visible spectrum of the diluted solution (Fig. 8A) was recorded. A recorded current was allowed to flow through the cell until approximately one tenth of the Cu2+ had been reduced. The current flow was then stopped and another 0.10 ml portion of the cell solution was removed and diluted to 10.00 ml. The absorbance of the solution was measured at 558 nm on the spectrophotometric peak corresponding to the purple Cu+-BCA complex. The process was repeated until all of the CL?+ in the solution had been electrochemically reduced to Cu+. A typical spectrum obtained after reduction of Cu2+ to Cu+ is shown in Fig. 8B.
0.
A I
400
I
I
I
1
560 Wavelength (nm)
1
I
I
720
I 0
I
I
I
I
0.8 mol Cu*lmo~ BCA
04
I
I
II
1.2
Fig. 8. Visible spectra of (A) a solution containing the Cu 2+-BCA complex and (B) a solution containing the electrochemically formed Cu+-BCA complex. The maximum in curve B occurs at 558 nm. Fig. 9. Plot of absorbance at 558 nm as a function of the mole ratio of electrogenerated Cu+ to BCA in a buffered solution.
234
The procedure was done three times in solutions containing BCA but no tartrate and once in a solution containing both BCA and 16.1 mg of tartrate. In each case a plot was prepared of absorbance as a function of the ratio of the number of moles of electrogenerated Cu+ to the number of moles of BCA in the solution. The plots contained two linear portions that, on extrapolation, intersected at a mole ratio of BCA to Cu+ of 2.1 +O.l (four trials). A typical plot is shown in Fig. 9. The results obtained in the absence of tartrate were essentially identical with those obtained in solutions that contained tartrate. From the results it is apparent that the complex contains two BCA’- moieties for each Cu+, as suggested by Smith et al. [ 21. Consequently, the formula of the complex is Cu (BCA ) 23-. Normally, mole-ratio plots are expected to have a horizontal (constant absorbance) portion after the intersection of the two linear regions, corresponding to a constant concentration of the monitored complex. In Fig. 9, the absorbance decreases after the intersection. This decrease could be associated with the formation of either a mono/BCA complex or a dinuclear complex. Evidence does not exist that would make it possible to determine whether either of these complexes actually exists. Chemical and electrochemical reactions The probable chemical equilibria that occur in the tested solutions containing copper (II) are summarized in the following equations: T Cu(H,O),
2+
*T 2-
e
Cu(HzO),T
blue
-
CU(H,O),T,
blue
CU(H>O)~~+
+
2BCA2-
_ --
(4)
blue
CU(H,O),(BCA),~-
blue
(5)
green
2-
CutH,O),T>
2-
2-
c
Cu(H,O),T
(BCA)
e
CU(H,O),WA),~-
(6)
blue
Equations 4 and 5 show the equilibria between the copper (II)-water complex and the tartrate and BCA complexes. Each tartrate or BCA ion replaces two water molecules around the Cu 2+ . The stoichiometries of these equilibria were established during the voltammetric studies obtained as the concentration of the complexing agent was varied. It is likely that the four water mole-
235
cules that are nearer to the Cu2+ are replaced, leaving the two water molecules at the greater distance from the Cu2+. Reduction of the complex between copper (II) and a single tartrate is probably responsible for cathodic wave II, in the absence of BCA. The existence of a copper (II)-monotartrate complex is supported by the studies described earlier in which the mole ratio of tartrate to Cu2+ at which all of the CuS04 dissolved was 1.3. The corresponding copper (II )-monoBCA complex is not as likely to exist since the mole ratio of BCA to Cu2+ at which the CuS04 dissolved was 2.1. Equation 6 shows the equilibria between the predominant species in the solution containing both BCA and tartrate. The presence of the mixed monotartrate-monoBCA complex was the conclusion arrived at Eqn. 3, where the water molecules were not shown for the sake of simplicity. The unattached complexing agent molecules and ions are not shown in Eqn. 6, in order to simplify the equations. All of the electrochemical reactions are irreversible, as indicated by the absence of anodic waves after the coulometric studies and during triangular-wave voltammetric studies. The electrochemical reactions associated with cathodic wave I, are summarized in Eqns. 7 and 8. ~CU(H,O),~+ +OH-+2e--+Cu,O(s)+H,O++llH,O blue
CU(H,O),~+ +2BCA2-+e-+Cu(BCA)23-+6H20 blue
(7)
yellow
(8)
purple
The reaction shown in Eqn. 7 occurs in the absence of BCA at pH 11.25, while the reaction shown in Eqn. 8 occurs in the presence of BCA. The reaction shown in Eqn. 8 is responsible for the color change to purple during protein assays. Coulometric data were used to define the reductions from copper (II) to copper (I). Wave I, is present in the absence of tartrate or BCA, and therefore can be assigned to reduction of the copper (II)-water complex. In the presence of BCA, the green color of the copper (II)-BCA complex masks the light blue color of the copper (II)-water complex so that the initial solution appears green rather than blue. Owing to the equilibria in Eqn. 6, as CU(H,O),~+ is electrochemically removed from the solution the concentrations of all other copper(I1) species also decrease and eventually disappear. The existence of CU(BCA),~- in Eqn. 8 was demonstrated spectrophotometrically after controlled-potential coulometry on wave I, (Fig. 9). Wave II, has been shown to be associated with a one-electron reduction.
236
Wave II, is observed in Cu2+ solutions that contain BCA, and mixtures of tartrate and BCA, but it has not been observed in solutions containing neither tartrate nor BCA. In the presence of BCA wave II, can be assigned to the oneelectron reduction of a Cu ‘+-BCA complex to a Cu+-BCA complex. In the studies in which BCA was added to a solution containing Cu2+ and tartrate, wave II, appeared only after sufficient BCA had been added to start forming the binary complex from the monoBCA complex. Consequently, the reduction is that shown in Eqn. 9. CU(H,O),(BCA),~-+~--*C~(BCA),~-+~H,O green
(9)
purple
In the absence of BCA but in the presence of tartrate, a wave that is anodic of the wave corresponding to the reduction in Eqn. 9 but cathodic of wave I, is observed (Fig. 2, wave II,). If sufficient tartrate is added, the wave disappears. It is likely that the wave is caused by the one-electron reduction of the copper (II )-monotartrate complex shown in Eqn. 4. As sufficient tartrate is added to cause the electroinactive Cu ( H20)2T22- to form, the wave disappears. Because BCA has been shown to be a stronger complexing agent than tartrate for Cu2+, it is unlikely that the copper (II)-monotartrate complex exists in the presence of BCA. That conclusion is supported by the absence of the voltammetric wave in solutions containing BCA. Consequently, the complex is not shown in Eqn. 6. Cathodic wave III, is observed in CuSO, solutions in the absence of tartrate and BCA. As either tartrate or BCA is added to the solution, the wave height has been shown to decrease. Electrolytic data have shown that the wave corresponds to a two-electron reduction to metallic copper. The wave can be assigned to the reduction shown in Eqn. 10. Cu(H20),2++2e-+Cu(s)
+6H2O
The wave height decreases as tartrate chemical equilibria shown in Eqn. 6.
(IO) or BCA is added owing to shifts in the
Comparison of electrochemical and chemical results From the previous description of the electrochemical reaction, it is apparent that any chemical substance that is sufficiently reactive to reduce copper (II) chemically to copper (I) should be capable of being assayed using the Cu2+BCA-tartrate reagent. Essentially, this means that any chemical species that has an anodic wave that at least partially overlaps with wave I, should be capable of being assayed with the reagent. In order to determine the potential that is sufficient to cause the color of the reagent to change from green to purple, the potential of the working electrode
231
in the reagent was successively stepped from +0.40 V toward more negative potentials in 0.05-V steps. If a color change had not occurred after 30 s (at 25 ‘C ), it was assumed that the potential was not sufficiently negative to cause the reaction to occur. The potential at which the solution first changed from green to purple was -0.15 V. In the comparisons described in this section, a positive voltammetric test consequently means that the tested compound has an anodic voltammetric wave that exhibits a current that is greater than that of the buffer solution at -0.15 V. Solutions containing about 1OmM of each tested solution were used for the voltammetric tests. Chemical studies were performed as described by Smith et al. [ 21 in a bath heated at 30” C for 30 min. If the solution containing the tested compound caused the solution to turn purple within the 30-min period, the chemical test was positive. The compounds tested and the results obtained using the chemical and voltammetric methods are shown in Table 1. In most cases the two methods agree. In those cases in which disagreement occurs, the voltammetric response was negative while the chemical response was positive. The difference in response can be attributed to the longer reaction time and higher temperature used for the chemical test, and to the higher sensitivity of the chemical test. From the results it is possible to conclude that use of the reagent for protein assays as suggested by Smith et al. [2] is subject to many interferences. The method should be applied with caution only under carefully controlled conditions in the absence of even mild reducing agents. Voltammetry of those compounds in Table 1 revealed that essentially any compound which can be more easily oxidized than the buffer-solvent system, i.e., which has an anodic wave, yields a positive chemical test. This is in agreement with the results of Wiechelman et al. [lo]. TABLE 1 Summary of the results of the chemical tests with the Cu*+-BCA-tartrate reagent and the voltammetric tests Compound
DL-Try-ptophar+ 4-Acetamidophenol Benzaldehyde’ Benzyl alcohol L-Ascorbic acid Hydroquinone o-Aminophenol L-Tyrosineb 1-Naphtholc
Result’
Compound
Voltammetric
Chemical
+ -
+ + +
+ + + -
+ + + + +
Pyrocatechol I-Aminobenzoic acid D@3,4-Dihydroxyphenylalanine L-Cysteine Phenolb Resorcinol Anilineb Biuret Glycylglycine
Result” Voltammetric
Chemical
+ + -
+ + + + + + -
+
“A+ sign indicates the compound responded positively and a - sign negatively to the test. ‘The chemical reaction occurred slowly. ‘The low solubility of the compound accounts for the negative voltammetric test.
238 REFERENCES 1 2 3 4 5 6 7 8 9 10
V.N. Tikhonov and I.S. Must&in, Zh. Anal. Khim., 20 (1965) 390. P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F. H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke, B.J. Olson and D.C. Klenk, Anal. Biochem., 150 (1985) 76. R.N. Adams, Electrochemistry at Solid Electrodes, Dekker, New York, 1969, pp. 191-205. J.J. Lingane, Electroanalytical Chemistry, 2nd edn., Interscience, New York, 1966, pp. 206, 380. F.A. Cotton and G. Wilkinson, Advanced Inorganic Chemistry, Interscience, New York, 1962, pp, 756,757. R.D. Braun, Introduction to Instrumental Analysis, McGraw-Hill, New York, 1987, pp. 289291. J.N. Butler, Ionic Equilibrium, Addison-Wesley, Reading, MA, 1964, pp. 5-7. F.A. Cotton, G. Wilkinson and P.L. Gaus, Basic Inorganic Chemistry, 2nd edn., Wiley, New York, 1987, p. 508. F.W. Hawkridge, T.W. Holt and H.H. Bauer, Anal. Chim. Acta, 58 (1972) 203. K.J. Wiechelman, R.D. Braun and J.D. Fitzpatrick, Anal. Biochem., 175 (1988) 231.