658
Anal. Chem. 1984, 56, 658-663
Investigation of 6-Methylpterin Electrochemistry by Dual-Electrode Liquid Chromatography/Electrochemistry Craig E. Lunte a n d Peter T. Kissinger* Department of Chemistry, Purdue University, W e s t Lafayette, Indiana 47907
An lnvestlgatlon of the eiectrochemlstry of 6-methylpterln is reported. The dual-electrode amperometrlc detector is used In the serles configuration to determine coupled electrochemlcai reactions in a complicated reactlon scheme. Products generated at the upstream electrode are detected and voltammetrlcally characterized at the downstream eiectrode. The parallel configuration of the dual-electrode detector is used to identify the products of enzymatlc, electrochemical, and chemical oxidation of 6-methyl-5,6,7,8-tetrahydropterin. The parallel conflguratlon Is also used to study the rate of tautomerlzation of quinonoid knethyldlhydropterln. Through these investigations, appllcatlons of the dual-electrode amperometrlc detector to the study of electrochemlcal mechanlsrns are demonstrated.
In recent years, liquid chromatography/electrochemistry (LCEC) has been shown to be a powerful tool for the study of biological systems (1). LCEC has been employed to determine endogenous compounds of biological importance, to study the metabolism of xenobiotics, and to determine the activity of several enzymes. In this report, we describe the application of LCEC to the fundamental study of redox reactions. In particular, the electrochemistry of 6-methylpterin has been investigated. A reduced pterin is required for the enzymatic hydroxylation of phenylalanine, tyrosine, and tryptophan (2,3). In these enzymatic hydroxylation reactions, the tetrahydropterin cofactor is oxidized to a dihydropterin in conjunction with the hydroxylation of the amino acid (AA) (reaction 1). The dihydropterin is then reduced by NADH and the enzyme, dihydropteridine reductase, to regenerate the tetrahydropterin (reaction 2).
AA
monooxygenase
+ tetrahydropterin + O2 hydroxylated AA + dihydropterin + H 2 0 (1)
dihydropterin
+ NADH + H+
dihydropteridine reductase
tetrahydropterin
+ NAD+ (2)
The natural cofactor has been shown to be 5,6,7,8-tetrahydrobiopterin (4). However, several 6-alkyl-substituted pterins can serve as the cofactor (5). 6-Methyl-5,6,7,8-tetrahydropterin (MPH4) shows the highest activity of any of the pseudocofactors. In the absence of dihydropteridine reductase or NADH, the initial dihydropterin tautomerizes to a 7,8-dihydropterin. It has been assumed that the initial oxidation product of tetrahydropterins, and therefore the intermediate of the enzymatic hydroxylations, is a quinonoid dihydropterin (6, 7). Several possible structures exist for the quinonoid dihydropterin and, as yet, there is no conclusive evidence as to which, if any, is correct (8,9). In fact, other structures for the initial oxidation product have been suggested (10). However, for clarity, the initial oxidation product of MPH4 will be referred
to as quinonoid 6-methyldihydropterin (q-MPH2) in this report. This report describes the application of LCEC to the study of the electrochemistry of 6-methylpterin (MP). The series dual-electrode ampermetric detector is used to investigate the electrode reactions (11). Products generated coulometrically are determined with a parallel configuration dual-electrode detector. The tautomerization of q-MPH2 to 6-methyl-7,8dihydropterin (MPH2) is also studied with the parallel dual-electrode detector. In addition, LCEC is used to show that the product of MPH4 is the same upon electrochemical, chemical and enzymatic oxidation. EXPERIMENTAL SECTION Apparatus. The liquid chromatographic system employed consisted of an Altex 110 pump and a Rheodyne 1070 injection valve with a 2OvL sample loop. A Brownlee MPLC RP-18 5-pm (4.6 mm X 10 cm) column was used. Detection was with a dual-electrode LC-4B amperometric detector (Bioanalytical Systems, Inc., West Lafayette, IN) with glassy carbon working electrodes. Cyclic voltammetry was performed with a BAS-100 Electrochemical Analyzer. The coulometric flow cell has been described previously (12). A Ag/AgCl electrode was used as reference in all experiments and reported potentials are vs. this reference electrode. Reagents. 6-Methyl-7,8-dihydropterin and 6-methyl-5,6,7,8tetrahydropterin were purchased from Calbiochem-Behring (La Jolla, CA). 6-Methylpterin was obtained from Sigma (St. Louis, MO). Octyl sodium sulfate was purchased from Eastman Kodak (Rochester,NY). All other chemicals were reagent grade or better and used without further purification. Procedures. Chromatography. A reverse-phase “ion-pair’’ chromatographic system was used to achieve separation of the various oxidation states of MP. The mobile phase was 3 mM octyl sodium sulfate in 0.1 M sodium phosphate buffer, pH 2.5, with 10% methanol (v/v). The mobile phase were prepared from distilled, deionized water and glass-distilledmethanol. Prior to use, the mobile phase was filtered through a 0.22-wm nylon filter (Rainin Instrument Co., Woburn, MA). In order to remove dissolved oxygen, the mobile phase was continously purged with nitrogen and maintained at a temperature of 40 “C with refluxing. A flow rate of 2.0 mL/min was used in all experiments. Flow Cell. Before use the flow cell was flushed with 0.1 M sodium phosphate buffer, pH 2.5, containing 0.5 mM EDTA to remove metals from the packed-bed electrode. This procedure is necessary because pterins have been reported to react with metal ions (13). The desired potential was applied and the current allowed to decay for several minutes. The sample was then pumped through the column by a peristaltic pump and an aliquot was collected. This sample was subjected to analysis by LCEC. A flow rate of 2.0 mL/min was employed. Chemical Oxidation. Ferricyanide was used to chemically oxidize MPH4. At a mole ratio of 2:l ferricyanide to MPH4, oxidation to MPH2 was complete in a few seconds. If excess ferricyanideis added, M P is gradually formed. Therefore, a slight excess of MPH4 was used to minimized oxidation of MPH4 to MP. Enzymatic Oxidation. For the enzymatic oxidation experiments, phenylalanine hydroxylase was isolated from mouse liver by precipitation between 35 and 45% saturation with ammonium sulfate (14). The ammonium sulfate precipitate was dissolved in 0.01 M sodium phosphate buffer, pH 7.5, and purified on a
G 1984 American Chemical Society 0003-2700/84/0356-0658$01~50/0
ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984 0
O
MP
\
q-MPH2
i-r 40
H
5,S-MPH2
\
H
659
J.
%HLyNlf3 1
I
ia
30
Flgure 3. Cyclic voltammogram of 1.0 mM MPH2 in 0.1 M sodium phosphate buffer: pH 2.5; sweep rate, 50 mV/s.
H2.N
H MPH4
Flgwe 1. Reaction scheme for the electrochemistry of B-methylpterin.
A
E, Volts
4
i 2
w
a a
Z B
a a 3 u
E, Volts
I1
lY
I-40 ia 1
Flgure 4. Cyclic voltammogram of 1.0 mM MP in 0.1 M sodlum phosphate buffer: pH 2.5; sweep rate, 50 mV/s.
Flgure 2. Cyclic voltammograms of 1.0 mM MPH4 in 0.1 M sodlum phosphate buffer: pH 2.5; sweep rate (A) 50 mVls, (B)5 mV/s.
DEAE-Sephadex column as described by Shiman et al. (15). The incubation mixture was as described previously (16).The incubation was dowed to proceed for 2 min, after which a 200-pL aliquot of the mixture was diluted to 2.0 mL with 0.1 M sodium phosphate buffer, pH 2.5. This sample was deoxygenated for 2 min and then 20 pL was injected onto the chromatography column. The entire time from the start of an incubation to the injection of the sample was 4 min.
RESULTS AND DISCUSSION Cyclic Voltammetry. The electrochemistryof substituted pterins has previously been investigated by classical electrochemical techniques (17-20). A brief study was done on MP by using cyclic voltammetry to serve as a guide in the investigation by LCEC. A slightly modified version of the electrochemical scheme proposed by Karber and Dryhurst (17) was used for this study (Figure 1). MPH4 exhibits a quasi-reversible couple, &/Ic, at +250 mV (Figure 2). A cathodic wave can be seen at -400 mV. At slow scan rates, a second oxidation wave, 111,, is observed at +750 mV. The process at wave I, is the oxidation of MPH4. I,, therefore, corresponds to the reduction of q-MPH2. If the scan rate is slow enough to permit sufficient tautomerization of q-MPH2 to MPH2, wave 111, can be observed. This wave corresponds to the oxidation of MPH2. The wave III, process is the reduction of MP.
Cyclic voltammetry of MPHB confirms this scheme (Figure 3). MPHB gives rise to two anodic waves, one a t +300 mV (11,) and the other a t +750 mV (111,). On the reverse scan, three cathodic waves are seen. Wave 11, (+200 mV) forms a quasi-reversible couple with 11,. Wave 111, is at -450 mV while wave IV, is at -1150 mV. The process at wave 111, is clearly the oxidation of MPH2, as noted previously for the MPH4 cyclic voltammogram. Wave 111, is then the reduction of MP to MPHB with wave IV, being reduction of MPH2 to MPHI. According to Karber and Dryhurst (17), the II,/II, couple is the reversible oxidation of hydrated MPH2. A typical cyclic voltammogram of MP is shown in Figure 4. On an initial positive scan no waves are observed. However, on the reverse scan two cathodic waves are seen, wave 111, at -450 mV and wave IV, at -1150 mV. After 111, and IV, are scanned, three anodic waves appear, wave IV, at -400 mV, wave I, a t +250 mV, and wave 111, at +750 mV. As assigned for the voltammograms of MPH4 and MPH2, wave 111, is the reduction of MP and wave IV, is the reduction of MPH2. Waves I, and 111, are also as previously assigned, the oxidation of MPH4 and MPHB, respectively. Wave IV, is coupled to 111, and is therefore the oxidation of 5,8-MPH2. That IV, is not seen for MPH4 or MPH2 suggests that the tautomerization of 5,8-MPH2 to MPHB must be rapid. Liquid Chromatography/Electrochemistry. These reactions, both the direct electrode reactions and the subsequent chemical reactions, can be investigated by using LCEC. The series dual-electrode detector can be used to determine the redox potential and chemical reversibility of a reaction. In addition, for chemically irreversible reactions, the nature of the initial product can often be investigated. In the parallel configuration, the dual-electrode detector can be used to study
660
ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984
I
I O 4
I E, Volts I
+0.5
A T
i
I
-0.1
+03
I
-0.3
P 10.6 Figure 5. Hydrodynamic voltammograms for the stable Oxidation states of 6-methylpterin,obtained by liquid chromatography/electrochemlstry in a mobile phase of 0.1 M sodium phosphate, pH 2.5, 3 mM octyl sodium sulfate, 10% methanol (v/v): ( 0 )MP, ( 0 )MPHP, (+) MPH4.
4 is the normalized current response.
Table I. Cyclic Voltammetric Peak Potentials and Hydrodynamic E,,, Potentials reaction MP reduction MPHB reduction MPHB oxidation MPH4 oxidation
HDV E,,, > v -0.37 -1.00 +0.71 +0.25
cv
10.8
Figure 6. Hydrodynamic voltammogram of q-MPH2, chromatographic conditions as in Figure 5: (0)q-MPHP generated by oxidation of MPH4 upstream, (+) MPH4. E , Volts
E,, v -0.40
-1.15 t 0.75
+0.25
chemical reactions following electrochemicalreactions and to investigate the final products of electrochemical reactions. In the following sections these techniques will be illustrated for the electrochemistry of MP. Hydrodynamic Voltammetry. The electrochemical potentials of the reactions observed with cyclic voltammetry can also be obtained with LCEC. Figure 5 shows the hydrodynamic voltammograms (HDV’s) for the oxidation of MPHI and MPHB and for the reduction of MPHB and MP. The potentials found in this manner are fairly consistent with those obtained by cyclic voltammetry (Table I). Due to extremely high background current, the EIlzfor the reduction of MPHS is only an estimate. In the series configuration, the dual-electrode detector can be used to study the electrochemistry at the downstream electrode of products generated at the upstream electrode. In this manner, the reversibility of electrode reactions can be studied. At a flow rate of 2.0 mL/min with a 100-pm gasket, the time interval from production at the upstream electrode to detection at the downstream electrode is approximately 75 ms. Therefore, only fast following reactions have time to occur. The oxidation of MPH4 is an example of a chemically reversible reaction. When the upstream electrode is operated at +500 mV (on the limiting current plateau for the oxidation of MPH4), the q-MPH2 generated can be detected at the downstream electrode. Figure 6 shows the HDV obtained at the downstream electrode when MPH4 is oxidized upstream. The anodic current at potentials more positive than +150 mV is due to oxidation of excess MPH4 as shown in Figure 6 by the overlap of the HDV for the oxidation of MPHI. If the upstream electrode is operated at a potential where no oxidation of MPH4 occurs, no cathodic current can be observed at the downstream electrode. This indicates that the reductive reaction downstream is dependent on the prior oxidation of MPH4 upstream.
J 1.0
Figure 7. Hydrodynamic voltammogram of the reduction product of MPHP, chromatographic conditions as in Figure 5: (0)MPHP reduction product, ( 4 ) MPH4.
The reduction of MPHS illustrates the situation for a chemically irreversible reaction. MPHS can be reduced at -1000 mV (while not on the limiting current plateau, this potential was chosen to eliminate excessive background) at the upstream electrode; however, no anodic current is observed at the downstream electrode until a potential more positive than +lo0 mV is applied. The HDV obtained at the downstream electrode when MPHS is reduced at the upstream electrode is identical with that obtained for the oxidation of MPH4 (Figure 7). When the upstream electrode is operated at a potential where MPHB is not reduced, no oxidation occurs at the downstream electrode. These observations indicate that the reaction sequence consists of the reduction of MPHB to MPH4 at the upstream electrode followed by the oxidation of the MPH4 to q-MPH2 at the downstream electrode. The oxidation of MPH2 is another chemically irreversible reaction. MPHP can be oxidized a t a potential of +900 mV (limiting current plateau) at the upstream electrode. Under
ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984 1.0 -
Table 11. Coulometric n Values and Products from Reaction in the Flow Cell
0.8
sample
potential, V
MPH4
+1.00 1-0.50
06 -
04
0c
661
MPHS
1 i
MP
-0.50 +1.00 0.00 -1.00 t 1.00 -0.50 -1.00
n
products
2.0 2.0
q-MPH2,a MPHS q-MPH2,a MPH2' NR MP NR MPH4 NR MPHB MPH4, MPHS
1.9
2.1 2.1 3.8
a Decreases with time. Increases with time. reaction. Minor component.
I
A
v
-03
-04
-05
1
-06
E, Volts
Figure 8. Hydrodynamic voitammogram of the oxidation product of MPHP, chromatographic Conditions as in Figure 5: (0)MPHP oxidation product, (+) MP. E, Volts
I
i
1
Flgure 9. Hydrodynamic voltammogram of the reductio0 product of MP, chromatographic conditions as in Figure 5: (0)MP reduction product, (+) MPHP.
these conditions a potential more negative than -350 mV is required to reduce the product at the downstream electrode. Comparison of the HDV obtained for the product of MPHZ oxidation with the HDV of authentic MP shows them to be the same (Figure 8). This supports the proposal that MPHZ is oxidized to MP but that MP is reduced to 5,8-MPH2. The reduction of MP has been reported to be a chemically reversible process based on polarographic and cyclic voltammetric investigations (17, 19). However, with the series dual-electrodedetector, this was not the case on the time scale of these experiments (i.e., 75 ms). When MP was reduced at the upstream electrode, the reverse reaction was not observed at the downstream electrode. Instead, a species with the same voltammetric characteristics as MPHZ was detected downstream. This can be seen from the HDV obtained when the upstream electrode was operated on the limiting current plateau for the reduction of MP, -600 mV, and the HDV for the oxidation of MPHB (Figure 9). LCEC does provide evidence for an intermediate in the reduction of MP to MPHZ. The HDV of MP (Figure 5) exhibits only a single wave. If
'
No
the reduction of MP was directly to MPHZ, then a second wave for the reduction of MPHZ to MPH4 should be observed. These results indicate that the direct reduction product of MP is not MPHZ but does rapidly react to form MPHZ. Coulometric Flow Cell. In the parallel configurationthe dual-electrode detector can be used in conjunction with a coulometric flow cell to study the final products of electrochemical reactions. This combination has previously been used to study the oxidation of acetaminophen (21) and benzidine (22). In addition to determining the products of electrochemical reactions, the number of electrons transferred in each step can be determined. Table I1 lists the products of coulometric oxidation or reduction of MP, MPHZ, and MPH4 at various potentials as determined by LCEC and the number of electrons transferred for each step. All processes are two-electron steps except for the reduction of MP at -1000 mV. The product at this POtential was found to be MPH4. This indicates that MP is first reduced to MPHZ and this is then reduced to MPH4. Again, this shows that the tautomerization of 5,8-MPHZ,the initial reduction product, to MPHZ must be fast. The tautomerization must occur while the 5,8-MPHZ is still in the flow cell for the further reduction to MPH4 to be possible. Oxidation of MPH4 at +500 mV produced a mixture of q-MPHZ and MPHZ. Directly after being oxidized in the flow cell, q-MPHZ was found to be the predominant species. However, the ratio of MPH2 to q-MPHZ increased with time until no q-MPHZ could be detected. This shows that the initial product of oxidation of MPH4 is q-MPH2 which subsequently tautomerizes to MPHZ. If the oxidation of MPH4 is carried out at a potential of +lo00 mV, where complete oxidation to MP could occur, a mixture of q-MPHZ and MPHZ is again found by LCEC. Therefore, the tautomerization of q-MPHZ to MPHZ must be slow relative to the residence time in the flow cell. This also indicates that the MPHZ detected by LCEC is being produced after leaving the flow cell. Tautomerization of q-MPH2. By use of series dualelectrode detection, it has been shown that the oxidation of MPH4 is reversible. In addition, by use of a coulometric flow cell and the parallel dual-electrodedetector, it has been shown that the initial oxidation product, q-MPHZ, undergoes a following reaction to yield MPH2. However, neither of these techniques is well suited to investigating the rate of this tautomerization. LCEC can be used to accomplished this goal using a prior chemical oxidation of MPH4 and the dualelectrode detector used in a parallel configuration. The tautomerization of q-MPHZ has previously been studied by spectroscopictechniques (17,19). These methods are not completely satisfactory because the spectra of the species involved are overlapped. In addition, studies at various pH's are difficult because the absorption maxima shift
062
ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984
w2T MPH4
q-MPH2
WI
1
Lk I , ,
0
1
I
w:;I((I
;;3 i Y
C
T
1
2 4 minutes
IOnA
1
Flgure 10. Separatlon of the oxidation states of 6-methylpterin by dualsiectrode LCEC, chromatographic conditions as in Figure 5:
electrode potentials W1 = -600 mV, W2 = +900 mV.
markedly with pH. Recently, Haavik and Flatmark (23) described a method involving liquid chromatographywith UV detection for the study of the tautomerization of quinonoid dihydrobiopterin. This method is an improvement over bulk spectroscopy because the quinoniod dihydro and 7,gdihydro tautomers could be resolved chromatographically. Unfortunately, the quinoniod dihydrobiopterin could not be resolved from the tetrahydrobiopterin. The dual-electrode detector in the parallel configuration can readily resolve all of the species involved in the oxidation of MPH4. Although, as for tetrahydrobiopterin, q-MPH2 and MPH4 could not be resolved chromatographically, these species can be resolved by the dual-electrode detector. Since MPH4 is easily oxidized, it can be detected at a potential where q-MPH2 is not electroactive. Likewise, since q-MPH2 is easily reduced, it can be detected at a potential where MPH4 is not electroactive. Through a judicious choice of operating potentials, MP, MPH2, q-MPH2, and MPH4 can be resolved and detected with dual-electrode LCEC. Figure 10 shows a chromatogram illustrating this separation. Figure 11 shows typical chromatograms obtained after MPHI is oxidized by ferricyanide. Initially, a large amount of q-MPH2 is detected with a small amount of MPH2. Subsequent analyses show that the amount of q-MPH2 decreases while the amount of MPH2 increases. The poor peak shape for MPHB in the early chromatograms indicates that some tautomerization is occurring on the chromatographic column. The kinetics of the tautomerization of q-MPH2 to MPHB was studied by monitoring the decrease in peak height for q-MPH2 with time. MPHB was found to be unsatisfactory for studying the kinetics because of the poor peak shape caused by on-column tautomerization. Between pH 2 and 8 the kinetics of the tautomerizationwere found to be f i s t order. Figure 12 shows how the observed rate constant varied with pH. In agreement with Karber and Dryhurst (13,the reaction rate reaches a maximum at pH 5 to 6. Above pH 8 the pterins were found to be extremely unstable and rapidly oxidized to MP so that no useful measurements could be made. Enzymatic Oxidation of MPHI. MPH4 was oxidized enzymaticallyto show that the oxidation product is the same as with chemical or electrochemicaloxidation. Figure 13shows typical chromatograms obtained after oxidation of MPH4 by phenylalanine hydroxylase in the presence of phenylalanine. It can be seen that the same oxidation products occur in the enzymatic oxidation as occur in the chemical (ferricyanide) and electrochemical (flow cell) oxidations. The formation of
WI 0u 2 1
minutes
Flgure 11. Chromatograms of MPH4 oxidation products following
chemical oxidation by ferricyanide, chromatographic conditions as in Figure 5: electrode potentials W1 = -600 mV, W2 = +900 mV; peak identities (A) q-MPHP, (B) MPH4, (C) MPHP.
0.24
I
I 2
4
6
0
PH Figure 12. Varlatlon of observed rate constant for the tautomerization Of q-MPH2 (k2) Wlth pH.
tyrosine indicates that the oxidation of MPH4 in the enzyme incubation is indeed a result of the action of phenylalanine hydroxylase. These results demonstrate that investigating the electrochemistry of MPHI is a valid method for probing the enzymatic reaction. Electrochemical Investigations with LCEC. LCEC can offer several advantages in electrochemical investigations, especially of complicated systems. The chromatographicstep provides a single component sample to the electrochemical detector. This is the case even with very labile compounds such as the reduced pterins where it is virtually impossible to maintain a pure solution. Also when reactions in complicated matrices are investigated, the chromatographic step can be invaluable. Coupled reactions can readily be determined with the series dual-electrode detector, even when the two reactions are widely separated in potential and part of a complicated electrochemical scheme. Only for fast reactions ( t l l z< 10 ms) will the initial product not be detected downstream for following chemical reactions. Finally, the amount
ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984
663
No electrochemical investigation can rest solely on a single method of analysis. Several techniques must be employed to expand and verify the available information. Registry No. MP, 708-75-8; MPHB, 17377-13-8;q-MPHZ, 70786-93-5;MPH4, 942-41-6.
LITERATURE CITED
T
B
"iA
ITA
/-
2
4
0
2
4
minutes
Figure 13. Chromatograms of MPH4 oxidation products following enzymatic (I), electrochemical (II), and chemical (111) oxidation. Chromatographic conditions as in Figure 5: electrode potentials W 1 = -300 mV, W2 = +I000 mV; peak identities (A) q-MPHP, (B)MPH4, (C) MPH2, (D) tyrosine.
of sample required is much less than for classical electrochemical techniques. An LCEC investigation requires only a few microliters of a micromolar solution as opposed to several milliliters of a millimolar solution for an experiment such as cyclic voltammetry. Perhaps the greatest utility of LCEC in electrochemical investigations will be in conjunction with classical techniques.
(1) Shoup, R. E., Ed. "Bibliography of Recent Reports on Electrochemical Detection"; BAS Press: West Lafayette, IN, 1982. (2) Kaufman, S. Eiochim. Eiophys. Acta 1958, 2 7 , 420. (3) Kaufman, S.; Fisher, D. B. I n "Molecular Mechanisms of Oxygen Activation"; Hayaishi, O., Ed.; Academic Press: New York, 1974. (4) Kaufman, S. Proc. Natl. Acad. Sci. U . S . A . 1963, 5 0 , 1085. (5) Kaufman, S. Adv. Enzymol. Relat. Areas Mol. Eiol. 1971, 3 5 , 245. (6) Kaufman, S.; Fisher, D. B. J. Bo/. Chem. 1970, 245, 4745. (7) Kaufman, S. J. Eiol. Chem. 1964, 239, 332. (8) Lazarus, R. A.; DeBrosse, C. W.; Benkovic, S. J. J. Am. Chem. Soc. 1982, 104, 6871. (9) Archer, M. C.; Vonderschmitt, D. J.; Scrimgeour, K. G. Can. J. Eiochem. 1972, 50, 1174. (10) Bobst, A. Proc. Net/. Acad. Sci. U . S . A . 1971, 6 8 , 541. (11) Roston, D. A.; Shoup, R. E.; Kissinger, P. T. Anal. Chem. 1982, 5 4 , 1417A. (12) Mlner, D. J.; Kissinger, P. T. Eiochem. Pharmacol. 1979, 2 6 , 3285. (13) Vonderschmitt, D. J.; Scrimgeour, K. G. Eiochem. Eiophys, Res. Commun. 1987. 2 6 . 302. (14) Kaufman, S. 1n'"Methods in Enzymology, Voi. 5"; Academic Press: New York. 1962: D 809. (15) Shiman, R.; Gray, D. W.; Pater, A. J. Eiol. Chem. 1979, 254, 11300. (16) Lunte, C. E.; Kissinger, P. T., submitted for publication in Anal. Eiochem . (17) Karber, L. G.; Dryhurst, G. J. Electroanal. Chem. 1982, 736, 271. (18) Kwee, S.; Lund, H. Eiochim. Eiophys. Acta 1973, 297, 285. (19) Archer, M. C.; Scrimgeour, K. G. Can. J. Eiochem. 1970, 4 8 , 526. (20) PradBE, J.; Pradlcovl, J.; Homoika, D.; Koryta, J.; Weber, J.; Siaik, K Cihar, R. J. Nectroanal. Chem. 1976, 7 4 , 205. (21) Miner, D. J.; Rice, J. R.; Kissinger, P. T. Anal. Chem. 1981, 5 2 , 2258. (22) Rice, J. R.; Klssinger, P. T. Eiochem. Eiophys Res. Commun. 1982, 104, 1312. (23) Haavik, J.; Flatmark, T. J. Chromafogr. 1963, 257, 361.
.
RECEIVED for review October 6,1983. Accepted December 27, 1983.
