2897
Anal. Chem. 1992, 64, 2897-2903
Influence of Tyrosine on the Dual Electrode Electrochemical Detection of Copper(I1)-Peptide Complexes Hweiyan Tsai and Stephen G. Weber' Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
The bluret reactlon makes peptldeselectrochemlcally actlve. W l s t h e baslsofanelectrochemkalmethodfor thedetectlon of peptldesfollowlng thelr llquld chromatographlcseparatlon. Thbpaper discusses the Influence of tyrodne, an electroacthm amlno add, on the detectlon of Cu( I I)-peptlde (bluret) complexes contalnlng It. The dual electrode detector has an upstream anode and a downstream cathode. Tyroslna contalnlngpeptidesyield anodk signals that are approximately the sum of the tyrodne signal and the signal from the bluret complex of a nonelectroactlvemodel peptlde, phenylalanylglycylglyclne (FGG). The cathodlc signal Is depressed In comparhn to FOG. Thls Is traced to the presence of an Intramolecular reaction between Cu( I I I ) and a reaction product rewltlng from the oxldatlon of the tyroslnyl reddue. The rate constant for the correspondlngintenndecularreactlon Is significant ( 106-107 M-' s-l), but In practical analytical sltuatlons, the concentratlons of the reactants wlll be small, 80 the reaction wlll not be a major factor. Sensltlvttles for several bloacthm peptldes are reported. The dependence of the signals on the posltlon of tyroslne In a trlpeptlde Is also studied.
INTRODUCTION Electrochemical detection in liquid chromatography has provided a powerful tool for the determination of peptides in biological mixtures. Peptides containing electroactive amino acids, such as tyrosine, tryptophan, and cysteine can be detected directly through electrochemical method8.l Peptides containing one or more primarylsecondary amines can be detected through electrochemical or fluorometric detection after derivatization with o-phthalaldehyde or 2,3naphthalenedicarbaldehyde.' Of course, not all important peptides contain easily detected amino acids. Recently, we have introduced a method for the electrochemicaldetection of peptides that are not electroactive, such as bradykinin, followingtheir separation by HPLC2-' The detection system is based on the biuret r e a ~ t i o n . ~ The > ~formation of Cu(I1)peptide complexes makes the peptides electrochemically active.'^^ The advantage of the biuret reaction is that it involves the amide backbone of the peptide, and thus it requires nothing in the way of particular functional groups in the peptide, but neither is it as unselective as to react with all amines. As the detection of the complex is by ita reversible
* T o whom correspondence should be addressed.
(1) Dou, L.; Mazzeo, J.; KNU,I. S. Biochromatography 1990,5, 74. (2) Tsai, H.;Weber, S. G. J. Chromatog. 1991,542, 345. (3) Tsai, H.; Weber, S. G. J. Chromatog. 1990,515,451. (4) Warner, A. M.; Weber, S. G. Anal. Chem. 1989,61,2664. (5) Schlabach, T. Anal. Biochem. 1984,139,309. (6) Gomd, A.; Bardawill, C.; Daid, M. J. Biol. Chem. 1949,177,751. (7) Margerum, D. W.; Wong, L. F.; Bossu, F. P.; Chellappa, K. L.; Czamecki, J. J.; Kirksey, S. T., Jr.; Neubecker, T.A. In Bioinorganic Chemistry II; Raymond, K. N., Ed.; ACS Symposium Series No. 162; American Chemical Society: Washington, D.C., 1977; p 281. (8) Metal ions in Biological Systems; Sigel, H., Ed.; Marcel Dekker, Inc.: New York and Basel, 1981; Vol. 12.
oxidation and reduction at a series dual electrode amperometric detector, there is the possibility that the presence of electroactive functional groups in the peptide, such as those occurring on cysteine, tryptophan, and tyrosine, will interfere with the quantitative analysisof the peptides by amperometry. This paper concerns tyrosine-containing peptides. There are several possibilities for the reaction of tyrosinecontaining Cu(I1)-peptide complexes (Cu(I1)-(Y-peptide)) in the detection system. These heterogeneouselectrochemical reactions may occur at the anode: Cu(I1)-(Y-peptide) Cu(I1)-(Y-peptide) Cu(I1)-(Y-peptide)
-
-
Cu(II1)-(Y-peptide)
Y,,-Cu(II)-peptide
Y,,-Cu(III)-peptide
+ e-
(1)
+ ne-
(2)
+ ne-
(3)
In these reactions Y-peptide is a tyrosine-containing peptide and Yo, is some oxidized form of tyrosine. In practice the anodic potentials used in these determinations (0.7-0.9 V vs AglAgCl, 3 M NaC1) is in the diffusionlimited regime for tyrosineeJOand for the biuret complex of simple tripeptide~.',~We fully anticipate that reaction 3 will predominate. If it does not, then the current at the anode will be less than the s u m of the currents from the biuret and tyrosine oxidations. It is kn0wnll-~4that tyrosine can undergo chemicalreactions following electron transfer. These may be followed by further homogeneous intramolecular electron-transfer reactions, thus it is necessary to consider the following intramolecular reactions: Y,,-Cu(III)-peptide Y,,-Cu(III)-peptide
-
-
Yoxr-Cu(III)-peptide
(4)
Yo,,o,-Cu(II)-peptide
(5)
In reaction 4, the transformation of Yo, to yo,^ allows the intramolecularelectron transfer shown in reaction 5 to occur. If reaction 4 is fast, this sequence of events will occur near the anode, and the Cu(I1) product of reaction 5 will have the chance to be oxidized again. If reaction 4 is slow enough, it will not influence the measurement. If reaction 4 occurs on a time scale that is intermediate (0.001-0.1a), then the result will be a decrease in the concentration of Cu(II1) in the solution downstream from the anode. At the relatively long time scales of the mass transport process from the anode to the cathode in the dual electrode ~~
(9) Spatola, A. F.; Benovitz, D. E. J. Chromatogr. 1985,327, 166. (10) Harriman, A. J. Phys. Chem. 1987,91,6102. (11) Reynaud, J. A,; Malfoy, B.; Bere, A. J. Electroanal. Chem. Interfacial Electrochem. 1980, 116, 595. (12) Malfoy, B.; Reynaud, J. A. J. Electroanal. Chem. Znterjacial Electrochem. 1980,114, 213. (13) Vermillion, F. J., Jr.; Pearl, I. A. J. Electrochem. SOC. 1964,111, 1392. (14) Deasy, C. L. J. Org. Chem. 1974,39, 1429.
0003-2700/92/0384-2897%03.00/0 @ I992 American Chemical Society
2898
ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992
detection, tyrosine oxidations are chemically irreversible; cyclic staircase voltammetry on a similar time scale shows no reverse wave. Thus, the only carrier of signal for the cathode is Cu(II1). As a consequence, the occurrence of reactions 4 and 5 in a particular time scale is deleterious to the cathodic detection of the biuret complex. In addition to these reactions, a host of intermolecular reactions can be anticipated to occur between copper centers and the various intermediates of tyrosine as well as with tyrosine itself. The objective of this work is to understand the influence of tyrosine on the electrochemistry of the biuret complexes of peptides containing it in sufficient detail to be able to predict the sensitivity of a determination. Unfortunately, because of the lack of molecular structure information provided by simple amperometry and voltammetry, we do not anticipateunderstanding the mechanism of the oxidation in molecular detail, but rather a t the level indicated in eqs 1-5. In particular we wish to know the extent of the influence of tyrosine, both within the molecule and as a separate entity, on the electrochemicsty of the biuret complex. EXPERIMENTAL SECTION Instrumentation. A Waters pump, Model 510,was used to pump the postcolum reagents and a Waters pump, Model 6000, was used to pump the mobile phase. These two pumps were operated by a Waters 680 automated gradient controller in the isocratic mode at a flow rate of 1.0 mL/min. The mixing ratio was 50 % mobile phase and 50% postcolumn reagent. A reversedphase column, Waters Delta Pak c18 (2 X 150 mm), was placed between the injector and the mixing Y in the system. A Biotage PBD column (4.6 X 250 mm) was used for chromatography with a basic mobile phase. For the flow injection analysis (FIA) experiments 2.8-mL knotted Teflon tubing (0.08-cm i.d.) was placed between the injector and the detector in place of the column. The detector was a BAS dual glassy-carbon electrode with a Model LC-4B potentiostat to control the potential (vs Ag/AgCl, 3 M NaCl) and measure the anodic current. The cathode current-to-voltage converter was laboratory made. A DT2802 interface board, DT777 screw terminal panel (Data Translation, Marlboro, MA), and DTK Keen 2530 computer and EZchrom version 4.5 (Scientific Software Inc., San Ramon, CA) were used for data acquisition and integration of the area of the peaks in the chromatograms. Cyclic staircase voltammetry was performed with Model CS-1090electroanalysis system (Cypress Systems, Inc., Lawrence, KS). Anodic currents are negative. Reagents. The following reagents were used without further purification: sodium bicarbonate and sodium hydroxide pellets (Mallinckrodt,Paris, Kentucky),acetonitrile (HPLCgrade, EM Science, Gibbstown, NJ), trifluoracetic acid (TFA) (Aldrich, Milwaukee, WI), cupric sulfate, sodium phosphate monobasic (Fisher, Fair Lawn, NJ). Potassium sodium tartrate (Aldrich) was recrystallized from water before use. Water was pawed through an activated carbon bed and doubly deionized by a Millipore Milli-Q system. G l y G l m and GlyTyrGly were manufactured by Research Plus, Bayonne, NJ. Other peptides were manufactured by Sigma, St. Louis, MO. One letter symbols for the amino acids (A = Ala, D = Asp, F = Phe, G = Gly, H = His, I =ne, K = L p , L =Leu, P =Pro, R = Arg, V = Val, and Y = Tyr) will be used later. Because the biuret reaction requires an alkaline solution and our unpublished experimental results showed leas electrode fouling when biuret reagents were in a buffer containing 0.1 M NaOH/0.2 M NaHCOs, pH 9.8,we used this bicarbonate buffer for all the experimentsreported. The mobile phase for reversedphase HPLC contained0.1 % (v/v)TFAin 10% (v/v)acetonitrile water,except where mentioned in the text. Postcolumn reagents (biuret reagents) contained 0.1 mM cupric sulfate, 0.3 mM potassium sodium tartrate, 0.1 M sodium hydroxide, and 0.2 M sodium bicarbonate. The pH was 9.80-9.85. Biuret reagents were used as the mobile phase in combination with a base-stable PBD column and in the flow injection analysis (FIA) system, except where mentioned in the text.
Table I. E X ~ I I IRemesaion D~~ Reaulta.
anode no. of injections
FGG
10 10 13 10 12 10
YGG
cathode
slope ( 8 ) [+I 3.5 (0.07) [0.998] 3.8 (0.08)[0.9951 3.6 (0.07)[0.997] 10.8 (0.1)[0.9991 10.8 (0.1)[0.999] 10.3 (0.3)[0.995]
Slow ( 8 ) 191 0.72 (0.01)[0.9981 0.62 (0.01)C0.9991 0.78 (0.01)[OM91 0.13 (0.002)[0.9991 0.12 (0.001)[0.9991 0.10 (o.CNl4) 10.9941
0 The slope i s the sensitivity (nC/pmol), and s is the standard deviation of the slope. For each peptide, the three seta of data were taken on different days.
,
5.00
1
-
0.00
-5.00
-
-15.00
-
-20 00
L
1 800
680
560
Potential
440
320
200
ImV)
Cycilc staircase voltammogram of & In biuret reagent at a glassycarbon electrode. The scan rate was 200 mV/s. & was 0.5 mM in 1.97 mM CuSO, (tartrate/Cu*+molar ratio = 3), 0.1 M NaOH, and 0.2 M NaHC03,pH 9.8, biuret reagent. Flgure 1.
Sensitivitieshave been determined by regressing the response on the concentration in the range 0-20 pM; the 9valuea of the regressions are all greater than 0.99. Some example reaults in the regression are shown in Table I. The pooled standard deviation for all calibration curves is 0.4 nC/pmol for the anode and 0.02 nC/pmol for the cathode. Major contributors to the random error were variations in the electrode surfaces, temperature fluctuations,and electrode fouling from polymerizationof Y.
RESULTS AND DISCUSSION Before the influence of the tyrosine on the electrochemical detection of peptides was determined, cyclic staircase voltammetry a t a glassy-carbon electrode was used to determine whether or not there was an influence of tyrosine on the electrochemistry of Cu(I1)-peptide complexes. Figure 1 is the voltammogram of A3 in biuret reagent. It shows a quasireversible wave (AI3 = 150 mV) at 0.70 V. Figure 2 represents the voltammogram of tyrosine in the bicarbonate buffer and in biuret reagent a t different scan rates. The voltammograms of tyrosine show an irreversible wave a t 0.460.60 V. When Cu2+is present with tyrosine, the oxidation peak shifta to a more positive potential and the peak current is smaller, but the effect of Cu2+is small. The effect of Cu2+ on the shift of oxidation potentials and current decrease is scan rate dependent: the faster the scan rate the smaller the effect. Figure 3 reveals the voltammogram of the mixture of tyrosine and ABin biuret reagent. It shows two oxidation waves a t 0.53 and 0.70 V, which correspond to the oxidation of tyrosine and the Cu(II)-A3 complex, respectively. The height of the reduction wave of Cu(III)-As is decreased. Figure
-
ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992 6.00
2
0 n
a
2
-2
2
Y
C L
5
0
2U8
r
1
-6.00
2
I
II c D
-4
L
2
-6
-12.00
-18.00
-8
ado
6d0
460
260
Potential (mV)
-24.00 800
680
560
440
320
200
Potential imV)
Fburr 3. Cycllc staircase voltammogram of the mlxtue of tyro8ina and & In biwet reagent at a giassy-carbon electrode. The scan rate was 200 mV/s. Bothtyroslne and & were 0.5 mM in the biuret reagent descrlbed In Figure 1.
-20 1000 -30
aoo
60 0
400
io
i
200
P o t e n t i a l (mV)
10
0 -10
-20
i
-20
4 ii
,
(C)
-304,
,
I
I
400 200 0 P o t e n t i a l (mV) Flguro 4. Cyclic staircase voltammograms of YQQ at a glamy-carbon electrode. Scan rate was 200 mV/s. (a) (-) Is 0.5 mM YQQ In the bicarbonate buffer, and (b) (- -) Is 0.5 mM YQQ In the same bluret reagent as used In Figure 1.
-30
800
600
-
4a is the voltammogram of YGG in bicarbonate buffer at scan rate of 200 mV/s which shows an oxidation wave at 0.52 V, which is less positive than tyrosine. This is consistent with the literature.16J6 Figure 4b is the voltammogram of YGG in biuret reagent. It apparently shows the oxidation waves of the Cu(I1)-YGG complex. There is no observable reduction wave of Cu(1II)-YGG. Figure 5 represents the voltammograms of Y, YGG, GYG, and GGY at scan rates of 20 and 2000 mV/s. The redox potentials of these Y-containing
compounds are similar. The shapes and relative magnitudes of the voltammograms of these Y-containing peptides are scan rate dependent; this reveals that the rates of the various steps17Jein the electrochemical oxidation of tyrosine depend on the position of the tyrosine in these peptides. The cyclic staircase voltammetry indicates that the anodic peaks of tyrosine and of the Cu(1I)-tripeptide complexes are additive; Cu(I1)-YGG has the same anodic appearance as the mixture of tyrosine and the Cu(II)-& complex. But the cathodic peak of the Cu(I1)-tripeptide complex decreases when tyrosine is present with the complexes. This influence is larger when tyrosine is in the complex (intramolecular influence) than when tyrosine is just present in the solution with the complex (intermolecularinfluence). In order to get a more quantitative determination of the intra- and intermolecular influence of tyrosine and to gather data useful for predicting sensitivities,solutions of YGG, tyrosine, and FGG made up in biuret reagent were injected into the biuret reagent flow stream for flow injection analysis. Anodic and cathodic (generator and collector) currents were measured.
(15)M o w , S.A.;Van Loon, G. R.Life Sci. 1985, 37, 1795. (16)Bennett, G.W.;Brazell, M. P.; Marsden, C. A. Life Sci. 1981,29, 1001.
(17)Hawley, M.D.;Tatawawadi, S. V.; Piekkareki, S.; Adame, R. N. J. Am. Chem. SOC.1967,89,447. (18)Young, T.E.;Babbitt, B. W. J. Org. Chem. 1983,48,662.
-40 d”
,
I
1000
I
aoo
I
I
400
I
200
P o t e n 4 3 (mv) Figure 2. Cyclic stakcase voltammogram of tyrosine at a glassycarbon electrode. Tyroslne was 0.5 mM in 0.1 M NaOH and 0.2 M NaHCOs bicarbonate buffer sohtlon (- - -)and biuret reagent containing 2.47 mM CuSO+(-). Scan rates were (a,top) 5 mV/s (b, mlddle) 200 mV/s, and (c, bottom) 2000 mV/s.
2900
ANALYTICAL CHEMISTRY, V a . 64, NO. 23, DECEMBER 1, 1992
-24
-
:'. -36 -40
(?
\.J
/
l
I
l
l
600
800
l
400
/
1
I
200
0
Potential ( m v 1 Figure 5. Cyclic staircase voltammograms of Y and Y-containing peptides at a glassyearbon electrode. Scan rates were (a)20 mV/s and (b) 2000 mV/s. Y (- -), YGG (-), GYG (- -), and GGY (-) were 0.5 mM In 0.1 M NaOHl0.2 M NaHC03 buffer. The electrode was polished with 0.05-pm AI after every scan.
-
-
".I 3 0
a \
3 3
u C
r u r(
>
.r( .r( u
c m ffl I
.5 E
(
I
I
.a
.6 .7 V vs. Ag/AgCl, 3 M NaC1)
t
Flguro 6. Hydrodynamic voltammograms for Y (0),YGG ( O ) , GYG (O), and GGY (A). Flow Injection analysis conditions: flow rate was 1.0 mL/min of 0.1 M NaOH and 0.2 M NaHC03 buffer. The Injected volumes were 20 pL. The sensitivities plotted are from regresslng the response on concentration in the range from 2 to 20 wM.
Table 11. Inter- and Intramolecular Influence of Tyrosine on the Sensitivity of Cu(II)-PeDtide Complexes Sensitivity (nC/pmol) peptide anode cathode Cu(I1)-FGG complex 3.6 0.75 ~~~~~~
Y
Cu(I1)-YGG complex (Y&FGG)mixture (Y+FGG)calc
5.6 10.5 10.3 9.2
0.03 0.12 0.56 0.78
Figure'6 shows the hydrodynamic voltammograms of Y, YGG, GYG, and GGY. At a potential of 0.8 V, which is well positive of the anodic peaks in cyclic staircase voltammetry, the diffusion-controlled range is nearly obtained for all peptides, except GGY. Higher potentials bring more drift, thus a potential of 0.8 V was used. The poor correspondence between the cyclic and hydrodynamic diffusion-controlled regimes is not understood. Table I1 shows the inter- and intramolecular influence of tyrosine on the sensitivity of Cu(I1)-peptide complexes. If there were no homogeneous chemical reactions in the detector, then the anodic and cathodic sensitivities of YGG or the mixture of tyrosine and FGG should be approximately equal, and these should equal the sum of the sensitivities from tyrosine and FGG individually, (Y + FGG),d,. The anodic
sensitivities of both the tyrosine-containing injections (YGG and Y + FGG) are in fact, comparable to,though slightly higher than, the calculated values. The cathodic sensitivity of the Cu(I1)-YGG complex shows that the intramolecular influence of tyrosine is to decrease the cathodic sensitivity to 15 % of the calculated one, which indicates that 86% of the Cu3+ complex is reduced in solution before it reaches the downstream cathode. The cathodic sensitivity of the mixture of Y and FGG shows that the intermolecular influence of tyrosine in this concentration range is to decrease the cathodic sensitivity to 72 % of the calculated one. The decrement in cathodic sensitivities from the intramolecular effect is severe and worth considering in more detail. The natural first step in understanding a sequence of electrochemical steps is to determine the number of the electronstransferred. Two complexeswhose electrochemistry is reversible and well studied' are the pentaglycine and hexaglycine complexes of Cu2+ which demonstrate oneelectron transfers. A t an anodic potential of 0.8 V the sensitivity for the Cu(II)-Ge complex is 2.24 nC/pmol; that for Cu(II)-Gs is 2.79 nC/pmol. Y (Table 11)is detected with a sensitivity of 5.6 nC/pmol. The hydrodynamic voltammogram of Y is not ideally shaped, and even the well-behaved oligoglycine complexes have sensitivities that are in no better agreement than 20%,so the number of electrons transferred cannot be determined unambiguously. The most likelyvalue is 2-3. The pathways and products of the anodic oxidation of tyrosine depend on the concentration of tyrosine and the time of the electrolysis. At high surface concentrations, the one-electron oxidation of tyrosine proceeds from step I to step IIB (Figure7)11J2J7-19 and the dimerization of the tyrosine free radical forms polymeric films on the electrode surface causing electrode fouling. We observed that the sensitivity decreased after several injections of 40 p M tyrosine. At low surface concentrations, the oxidation can proceed from step I via step IIA through step V depending on the time scale. The number of electrons transferred, in the oxidation of Y in the current study is 2-3. Thus, the oxidation of Y creates speciesI1and I11(Figure7) SpeciesI1and IV, which is formed from 111, are good reducing agents. IV must be considered as the intramolecular cyclization proceeds rapidly at pH 10. To become species I1or IV, severalchemical steps are required. In fact the cyclic staircase voltammetry in Figure 2 is consistent with ECE behavior.24 The ratios of peak current to square root of sweep rate for Y in buffer in Figure 2 are 5 mV/s, 1.29; 200 mV/s, 0.44; and 2000 mV/s, 0.38. The conclusion is that species 11,or IV, is only formed some time after the initial oxidation. The redox potential of the Cu3+/2+-tripeptide complex is more positive than the orthohydroquinones I1 and IV. The Cu3+complex is formed directly upon the anodic oxidation of, for example, the Cu(I1)-YGG complex, and the tyrosine moiety undergoes an initial oxidation, followed by a chemical reaction which yields a reductant capable of reducing the Cu3+to Cu2+. Thus, the reason why the cathodic sensitivity is decreased to such a degree has to do with an unfortunate coincidence between the reaction time of the genericreaction shown in eq 4 and the anode-cathode mass transport time. ~
(19)Young, T.E.;Griswold, J. R.; Hulbert, M. H. J. Org. Chem. 1974, 39,1980. (20) Rivaa, G. A.; Solis, V. M. Anal. Chem. 1991,63, 2762. (21) Karpinski, Z. J. Electrocatal., Mater. Symp. Electrochem. Sect. Pol. Chem. SOC.,1987, 168. (22) Marcus, R. A. Electrochim. Acta 1968,13, 995. (23) Marcus, R. A. J. Chem. Phys. 1966,43,679. (24) Bard, A. J.; Faulkner, L.R.Electrochemical Methode: htndamentals and Applications; John Wiley & Sons, Inc.: New York, 1980; p 462.
ANALYTICAL CHEMISTRY,VOL. 64,NO. 23,DECEMBER 1, 1992 2901 Table 111. Influence of the Position of Tyrorine in Peptides on the Sensitivity of Cu(I1)-Peptide Complexer sensitivity (nC/pmol) with Cua+ no Cu2+ peptide anode anode cathode FGG 3.6 0.75 Y 3.8 5.6 0.03 YGG 3.1 10.5 0.12 GYG 1.7 9.1 0.06 GGY 1.4 6.1 0.06
OH'
c _
-e
Step I1 A
-
H .O
more e
"
w
Hmo HO
0o '
(11)
O'
I
Step V
Flgum7. Reactkn scheme proposed forthe electrochemicaloxidation of tyrosi~.ll-12.17-19
Let us turn now to the less severe intermolecular effect. Because the redox potential of Cu3+/2+-tripeptidecomplexes is slightly higher than that of tyrosine, there is a thermodynamic driving force for electron transfer between Cu3+and tyrosine. As discwed above, there are several steps following the initial oxidation of Y in which reducing agents other than Y are formed (Figure 7). Thus, it is a complex mixture in which some Cu3+is reduced. Consequently, we only can do a semiquantitativedeterminationof the reaction rate constant by assuming that the intermolecular reaction is
Y or Y,
+ Cu(II1)-FGG
-
Yo, or Yox,ox+ Cu(I1)-FGG
(6)
then the rate equation can be written as -d[Cu(III)FGGl/dt = k2[Cu(III)-FGG] [Yl
(7)
where kz has the units of a second-order reaction rate constant and t is the reaction time. We can only approximate the concentration of the reactive species, as its actual identity is not known. If we let A = [Cu(III)-FGGI and B = [Yl, A = A0 and B = Bo at t = 0, and A = A0 - x and B = Bo - r at t > 0, where x is the extent of the reaction, then we can rewrite eq 7 as dx/dt = k2(Ao- %)(Bo- x )
(8)
If A0 # BO,then the integrated rate equation is If A0 = Bo, then the integrated rate equation is l/(Ao - X ) = k2t + l/Ao
(10)
A data set consisting of cathodic peak area for injections of Y and FGG over the range from 2 to 20 pM can be converted to the number of moles of Cu3+reduced because the cathodic sensitivity for the Cu(I1)-FGG is known. That number of moles in the peak volume (estimated to be 0.75 mL) leads to concentrations which correspond to A0 - x for data taken
from injection of mixtures of FGG and Y or to A0 for data taken in the absence of Y. All cathodic peak areas are corrected for the contribution that Y makes to the cathodic signal. Regression of l/(Ao - z)vs l/Ao, should yield a slope of 1 and intercept of kzt. The actual slope is 0.9 (a0.02) and the intercept is 1.2 (f0.4)X 106 M-I. The value for tis difficult to evaluate accurately, but we can use the average velocity from the surface of the cell out into the solution a distance correspondingto the diffusion layer thickness at the end of one electrode and the center-to-center distance between the electrodes to estimate it. The ratio of the sensitivity (in Coulombs per mole) to Faraday's constant for a one-electron transfer is the cell's coulometricefficiency,which from theory26 leads directly to the diffusion layer thickness at the end of the anode. This diffusion layer thickness is estimated to be 10 pm. We take the flow to be laminar, so a mathematical velocity from the electrode out to the end of the diffusion layer can be calculated to be 2.7 cm/s. The center-to-center distance of the electrodes is 0.38 cm, so the reaction time ( t ) is about 0.14 8. Therefore the second-order reaction rate constant is on the order of 8 X 106 M-I 8-l. One may expect from this that 25 p M Y will decrease the cathodic response of a 1 pM Cu(I1)-tripeptide complex to 50%. Although the cathodic sensitivity of Y is much smaller than that of Cu(11)-tripeptide complexes (Table 11), the contribution of Y to the cathodic current at such large ratios of concentration is not insignificant. Also, high concentrations of Y cause electrode fouling and nonreproducible responses. Thus, experimentaleffortato c o n f i i this rate constant over a wider range of concentrations are plagued by uncertainty. As a practical matter, samples with high concentrationsof Y which would foul the electrode surface would be diluted anyway. Therefore,practically speaking, the intermolecularinfluence of Y on the cathodic response is negligible. Table I11 shows the influence of the position of tyrosine on the sensitivity of Cu(I1)-peptide complexes. When the flow stream is only 0.1 M NaOH and 0.2 M NaHC03 buffer, the anodic sensitivities of tyrosine and YGG are about the same and are twice those of GYG and GGY. This is probably related to the primary amine on tyrosine and YGG, the presence of which permits the intramolecular cyclization which forms dopachrome, which then further oxidizes. Increased sensitivity of the a-amine-containingmolecules is also seen in cyclic staircase voltammetry at a scan rate of 2 V/s (Figure 5). An anodic reaction in cyclic staircase voltammetryat 2 V/s occurs on about the same time scale as the exposure time of the compounds to the anode in FIA. As described above, the exposure time is about 0.1 s at the electrode surface. The anodic sensitivity of tyrosine in biuret reagents is higher than that of tyrosine in bicarbonate buffer. This is different than the result from the cyclic staircase voltammetry (Figure 2). We do not understand the effect of Cu2+on the oxidation of tyrosine, and the literature is contradictory. Desay" showed that Cu2+catalyzes the oxidation of tyrosine to form dopachrome. As expeded, the oxidationof COOH-terminal tyrosine (26) Weber,
S. G.;Purdy, W. C. Anal. Chim. Acta 1978, 100, 631.
2902
ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992
Table VI. Sensitivity (nC/pmol) of Bioactive Peptides Containing Tyrosine (Precolumn Reaction Followed by Chromatography on PBD) peptides anode cathode GGFL 2.0 0.47 YGGFL 1.6 0.36 RKDVY 6.2 0.08
Table IV. Sensitivity (nC/pmol) of Tripeptide9 from the CISColumn Separation Following Postcolumn Reaction peptide anode cathode Cu(I1)-FGG complex 3.8 0.62 6.4 0.05 Y Cu(I1)-YGG complex 12.9 0.23 Table V. Sensitivity (FIA) (nC/pmol) of Bioactive Peptides Containing Tyrosine peptides anode cathode Des-Tyr'-enkephalin GGFL 2.7 0.60 9.1 0.33 enkephalin YGGFL thymopoietin I1 fragment 32-36 R K D W 7.7 0.13 angiotensin I1 DRVYIHPF 8.2 0.20 andotensin I11 R W I H P F 7.9 0.13
2
I
1-I
4.00
0
8
4
(2
.
min
-8.00
-12 00
Flgure 8. Chromatogram of enkephalin peptides. The mobile phaae was 50 mM NaH2W4 and 15% (v/v) acetonitrile-water. The postcolumn reagent was 0.1 mM CuS04,0.3 mM tartrate, 0.1 M NaOH, and 0.2 M NaHCO3. The mobile phase: postcolumn reagent ratio was 40360, and the flow rate was 2.0 mL/min. A 2Gpl ailquot of an 8 pM mixturewas Injected. Peaks are Des-Tyrl-Leu-enkephalin (1) and Lew enkephalin (2). The top trace Is the response of the anode, and the bottom one Is the response of the cathode.
i
i / p
L 700
600
500
400
4 300
200
Potential ( v V )
Flgure 8. Cyclic staircase voltammogram of the Cu(II)-& complex at a glassy-carbon electrode. The scan rate was 200 mV/s. Oe was 0.5 mM in 3.0 mM CuS04,(tartrate/Cu*+ ratio = 3), 0.1 M NaOH, and 0.2 M MaHCO3. pH 9.8, buffer solution.
peptides which have no free amine gave no indication of the formation of dopachrome or dopaquinone. Karpinski21 showed that Cu2+interacted with two sites on dopaquinone molecules and inhibited the ring closure reaction as well as the oxidation of dopa; i.e. Cu2+inhibited the formation of dopachrome. Table IV shows the sensitivity of the Cu(I1)-tripeptide complexes in the liquid chromatography-electrochemical detection system. The results agree with the FIA results quite well. The slight increase in the anodic sensitivity is probably due to the higher diffusion coefficient of the solute in acetonitrile. The responses from FIA discussed above, because there is no separation, could be from one or more coeluting substances. When chromatographywas used, there was only one peak. This is a good indication that the peptides were pure. The goal of this work is to predict approximately the sensitivity of bioactive peptides containing tyrosine in the detection system based on the formation of Cu(I1)-peptide complexes. The tripeptide data can be used to make semiquantitative predictions of the sensitivity for bioactive peptides. The anodic sensitivity of the Cu(I1)-peptide complexes containing tyrosine is higher than the electrochemically inactive Cu(I1)-peptide complexes. On the basis of data in Tables 11-IV, the anodic sensitivity will be about 8 (f2) nC/pmol and the cathodic sensitivity will be about 0.1-0.2 nC/pmol. Both sensitivities are higher than average when Y is on the amine terminal and lower than average
when it is on the carboxyl terminal. The detection limit (twice rms noise) for the anodic detection of these peptides is 40100 fmol for a 20-pL injection on the basis of pp baseline noise measurement taken for 30 s and divided by 5 to approximate the standard deviation. Table V shows the FIA sensitivity of bioactive peptides containing tyrosine. The anodic sensitivities are very consistant with our prediction, about 8 nC/pmol. The anodic and cathodic sensitivities of enkephalin, which has Y at its amine terminal, are higher than those for thymopoietin I1 fragment 32-36, withy at the carboxylterminal. The cathodic sensitivities of the bioactive Cu(I1)-peptide complexes are larger than those of the Cu(I1)-tripeptide complexes. This fortunate result may be because the redox potential of C U ~ + / ~ + is less positive for larger peptides.' Figure 8 is the voltammogram of the c u ( n ) 4 6 complex. The voltammogram show that the Cu(II)-G6 complex has a more reversible electrochemical reaction (AI3 = 90mV) and the oxidation potential (Epa= 0.51 V) is less positive than the Cu(II)-As complex. According to Marcus' the reaction between Cu3+ and the products of the oxidation of tyrosine will be slower when the difference in the redox potential is lower. The resultant lower energy Cu3+ formed by oxidizing Cu2+ complexes of larger bioactive peptides is more stable toward intramolecular electron transfer which destroys the complexes. Therefore, the cathodic sensitivity of the tyrosinecontainingbioactive Cu(II)-peptide complexes is higher than Cu(I1)-tripeptide complexes. Chromatography has been done with the precolumn reaction and the PBD columns and with a CUcolumn followed by the postcolumn reaction. Table VI shows the sensitivities obtained from the precolumn reaction. The polarity of the Cu(II)-peptide complexes is different than that of the peptides themselves. Thew Cu(II)-peptide complexesare only slightly retained on the PBD column. There is no separation of the various peptides under the conditions used (mobile phase: 0.1 mM CuSOa, 0.3 mM tartrate, 0.1 M NaOH, and 0.2 M NaHC03). More work on establishing conditions for the
ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, W02
separation of Y-containing peptides by the PBD column is needed. Sensitivities obtained from the precolumn reaction are slightly lower than those obtained by FIA. This could be due to the reactivity of the packing material2 or the air oxidation of Y in alkaline solution. The separation of Y-containing peptides has been done2'3-28 with reversed-phase CIScolumns, so it is straightforward to follow the conditions of separation used in the literature and then introduce the biuret reagent after the separation. Figure 9 shows the reversed-phase separation and detection of des-Tyrl-Leu-enkephalin(GGFL)and Leuenkephalin. Leu-enkephalin, which has Y at the amine terminal, has a higher anodic sensitivity, but a lower cathodic sensitivity, than des-Tyrl-Leu-enkephalinwhich is not electroactive. The following summarizesthe influence of tyrosine on the electrochemical detection of Cu(I1)-peptide complexes. (1) Tyrosine increases the anodic sensitivity of the Cu(11)-peptide complexes by a factor of 2-4. The sensitivity is about 8 (f2) nC/pmol. The anodic detection limit is about 40-100 fmol for the bioactive peptides wed. (26) Mousa, S.; Couri, D. J. Chromatogr. 1983,267, 191. (27) Meek, J. L. J. Chromatogr. 1983,266,401. (28) Sauter, A.; Frick, W. J. Chromatogr. 1984, 297, 215.
2909
(2) Tyrosine decreases the cathodic eensitivity. The tripeptide8 may be the worst case. An analytically significant sensitivity is obtained from larger peptides. (3)The intermolecularinfluence of tyrosine on the cathodic sensitivity is smaller than the intramolecular influence. The intermolecular influence is negligible at concentrations in the trace range. (4) The position of tyrosine in the peptides is important. When tyrosine is at the amine terminal position, the anodic sensitivity is higher and cathodic Sensitivity is about twice that obtained when tyrosine is at other positions in the peptides.
ACKNOWLEDGMENT We gratefully acknowledge support from the National Institutes of Health under Grant GM44842.
RECEIVED for review June
29, 1992. Accepted September 4, 1992. Registry No. FGG, 23576-42-3;YGG, 21778-69-8;Y, 60-184;GYG,6099-08-7;GGY, 17343-07-6;GGFL960254-83-3;YGGFL, 58822-25-6;RKDW, 99109-47-4;DRWIHPF, 447491-3; R W IHPF, 13602-53-4; CUSOI, 7758-98-7.
