In view of the observations, an adsorption mechanism is proposed as follows. Hydroxide ions are strongly adsorbed a t the bronze surface in a deoxygenated system establishing a large negative potential (-1200 mV or less). When 0 2 molecules are introduced, they are also adsorbed a t the surface, displacing the negatively charged hydroxide and resulting in a positive shift in electrode potential. A similar mechanism would also explain the 0 2 response in KC1. The success of EDTA titration in basic solutions could similarly be explained if the positive ions act in a manner similar to the oxygen molecules in displacing adsorbed OH-. The action of Li+ in LiOH may have a similar explanation.
high degree of sensitivity attainable and the magnitude of the potential change per unit change of oxygen concentration, The relative ease with which potentiometric measurements can be made and the simplicity of the equipment add to the utility of measuring systems using these electrodes. Work in progress includes the development of a portable device for monitoring dissolved oxygen in surface and waste waters.
ACKNOWLEDGMENT
CONCLUSION
The authors wish to acknowledge the assistance of Howard R. Shanks of the Ames Laboratory for providing the tungsten bronze crystals which were used in this investigation and of Patrick R. Montoya for his help in conducting the experiments.
The tungsten bronzes have been shown to be highly useful as indicating electrodes in the potentiometric determination of dissolved oxygen. Obvious applications are foreseen in the environmental field, resulting from the
Received for review September 11, 1972. Accepted December 18, 1972. Paper presented a t the 20th Annual Anachem Conference, October 9-11, 1972, Detroit, Mich.
Indirect Coulometric Titration of Biological Electron Transport Components F r e d M. Hawkridge and Theodore Kuwana Department of Chemistry, Ohio State University, Columbus, Ohio 43210
The approach of utilizing an electrochemically generated titrant to transfer charge to an electron carrier enzyme has been demonstrated in the mediator/spinach ferredoxin-NADP-reductase/NADPH system. Present work describes further developments aimed toward the general application of spectroelectrochemical methods using optically transparent electrodes (OTE’s) to evaluate the stoichiometry, energetics, and kinetics of enzymatic electron transfer sequences. Electrochemical and spectral data indicate that reagents such as potassium ferrocyanide, 1 , l ‘-dimethyl-4,4’-bipyridyl dichloride (methyl viologen), and 1,l ’-ethylene-2,2’-bipyridyl dichloride can also act as electron mediators and that their essential properties are unaffected by the presence of a protein. These reagents undergo electron transfer at the electrode and in turn transfer charge to the enzyme. Anaerobic redox titrations ( 0 2 5 5 X 10-7M) of horse heart cytochrome c, modified horse heart cytochrome c, and sperm whale myoglobin are reported. Results indicate that the n values of these heme proteins can be evaluated within f3% of the expected values. Concurrent potentiometric data have also been obtained for certain titrations. Experimental details of cell design, oxygen .removal by vacuum degassing, procedures in charge injection, and the rapid acquisition of spectral information are discussed.
In the understanding of the mode and sequence of electron transfer in biological systems, the accurate evaluation of stoichiometry and energetics is of considerable importance. The present study has been directed to the development of a spectroelectrochemical approach to such
an evaluation and has been applied to some of the heme proteins involved in the mammalian respiratory system. The approach is to electrochemically generate a t an optically transparent electrode (OTE), a reducing or oxidizing titrant which in turn transfers charge to the heme and also, in many instances, acts to couple the heme protein and/or electron transfer enzyme to a potentiometric electrode for E“’ measurements (E” denotes formal potential). The titrant redox couple in this latter role has been called a “mediator” by bio-types. The mediated sequence is diagramatically shown in Figure 1. The optically transparent electrode functions to transfer electrons to the reagent and the generated titrant in turn transfers charge to the heme protein or any biological electron transport component(s). The potential of this working electrode governs whether a reagent is undergoing reductive or oxidative charge transfer. In the present study, both an oxidative and a reductive reagent are utilized for the indirect coulometric titration of the heme proteins. An oxidative sequence is illustrated by the following reactions
where the homogeneous electron transfer reaction 2 serves to oxidize the heme protein and regenerate the electroactive species. Thus, the conditions are similar to those required for the so-called, catalytic or regenerative, electrochemical process. In the ideal sequence, reactions 1 and 2 are both reversible. Of course, in the “real” biological electron transfer sequence such as is found in the respiratory system, many, many more individual components are involved. The equilibrium position of reaction 2 is determined by the respective E”’ values of the two redox couples involved A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 7 , JUNE 1973
1021
-+ e hv,A
DISC
w--
,
Figure 2. Disassembled OTE cell (see text for description)
A ELECTRODE
EO’
(OTEI
Figure 1. Diagram depicting the indirect coulometric titration scheme using electrogenerated titrants Reducing mediator A which has a E”’ more negative than E”HP of the heme protein is coulometrically generated to transfer electrons to the heme protein: similarly. the potential of the OTE when stepped more positive can generate the oxidizing Mediator B
and when it lies sufficiently to the right, it is convenient to perform exhaustive coulometric titration of the heme protein with the electrogenerated titrant. For the heme proteins of sperm whale myoglobin, horse heart cytochrome c, and modified horse heart cytochrome c discussed in this paper, the heterogeneous electron transfer rate to the protein is sufficiently slow, in the potential ranges employed, that the faradaic charge can be considered to be quantitatively transferred to the titrant. For reductions, methyl viologen was reduced to the radical cation. For oxidations, ferricyanide was generated from ferrocyanide. When increments of charge are introduced and then the optical absorption spectra are taken to follow the extent of the reduction or oxidation of the heme protein, the use of an OTE cell is convenient. Such a cell has also proved useful for spectroelectrochemical determination of kinetic parameters. A previous work has already described a kinetic rate study for a mediated transfer in the reduction of NADP to NADPH in the presence of spinach ferredoxin-NADP-reductase ( 1 ) . For the chemical redox titrations of electron carrier enzymes, potassium ferricyanide is commonly used as an oxidant while NADH (2-4), ascorbate (3, 5 ) , sodium dithionite (6-g), phthiocol ( I O ) , and chromous acetate (10) have been used as reductants. In these titrations, aliquots of a standard solution of the titrant are added with the titration vessel either under positive pressure of an inert O gas or in uacuo. A gravimetric procedure in U ~ C U employing solid sodium dithionite in potassium chloride as a diluent has been used extensively (6, 7). (1) M. Ito and T. Kuwana, J. Eiectroanai. Chem., 32, 415 (1971). (2) K. Minnaert, Biochem. Biophys. Acta. 110, 42 (1968). (3) P. L. Dutton, D. F. Wilson, and C. Lee, Biochemistry, 9, 5077 (1970), (4) D. F. Wilson and P. L. Dutton, Biochem. Biophys. Res. Comrnun.. 39,59 (1970). (5) D. F. Wilson and P. L. Dutton, Arch. Biochem. Biophys., 136, 583 (1970). (6) W. H . Orme-Johnson and H. Beinert, Anai. Biochem., 32, 425 (1969). (7) W. H. Orme-Johnson and H. Beinert, J. Bioi. Chem., 244, 6143 (1969) (8) F. L. Rodkey and E. G . Ball, Proc. Nat. Acad. Sci. U. S., 38, 396 (1952). (9) P. L. Dutton, Biochem. Biophys. Acta, 226, 63 (1971). (10) F. L. Rodkey and E. G .Ball, J. Bioi. Chem., 182, 17 (1950).
1022
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 7, J U N E 1973
I AUX.
=RASS
BAR
CONTACT
‘HOLDER
Figure 3. Back view of OTE cell (see text for description) The present approach, which is essentially an indirect coulometric titration, has several advantages over the chemical titration procedures. These are: solution conditions remain essentially invarient during the titration; small increments of the titrant (charge) can be added accurately and conveniently a t any repetitive rate; oxygen can be rigorously removed before (less than 5 X lO-7M) and excluded during the titration; small volume cells (less than 2 ml) can be fabricated using OTE; and the heme proteins can be repeatedly cycled through various oxidative or reductive levels. To fully appreciate these advantages, the limitations as to the available quantities and therefore “costs” of highly pure heme proteins or other electron carrier enzymes should be recognized. It is common to have available only milligram quantities of the high molecular weight materials and, therefore, titrations are performed on a total of 10-30 nequiv. Also, the chemical properties impose several experimental problems which are minimized by the above advantages. The OTE cell allows rapid, repetitive charge injection and acquisition of spectral information which are important for materials undergoing time dependent charge. In most potentiometric titrations for the purpose of determining E“’ values, one or more mediators are often added, such as organic dye molecules, which “couple” the proteins to the indicator electrode (platinum) (2-5, 8-13). It is assumed that these mediators do not interact with the protein in any manner as to affect the E“’ value and that the E”’ reflects an equilibrium value. One of the long-range objectives of our work is to test the validity of (11) F. L. Rodkey and J. A . Donovan, Jr., J . Bioi. Chem., 234, 677 (1959). (12) A . H . Caswell, J . Bioi. Chem., 243, 5827 (1968). (13) A . H . Caswell and B. C. Pressman, Arch. Biochem. Biophys.. 125, 318 (1968).
these assumptions. In the present approach, E"' v a l u e s can be determined independent of direct potentiometric measurements from either the i n i t i a l or final portions of the charge-absorbance plots, as for reaction 2 , if the E"' values of the titrant couples are known.
EXPERIMENTAL Apparatus. The O T E cell is shown in Figures 2 and 3. The cell was made of lucite (3.81 cm in diameter, 1.27 cm deep) and the cell cavity was bored out to 0.953 cm. The cell was polished with Wrights Silver Polish so that it was transparent. One face of the cell was machined to accommodate a n O-ring (0.953-cm inside diameter, 0.159-cm wall, Viton). The bottom of the cell cavity was milled to provide a flat surface on which a small stirring bar operated. This stirring bar and the one to be described later were glass-jacketed. Holes were drilled in the cell to accommodate valves 2, 3, and 4 (Hamilton Company, No. 1MM1) and a length of 22-gauge platinum wire as shown in the figures. Valves 2, 3, and 4 and the platinum wire were epoxied in position ( c f . Figures 2 and 3). About 1 cm of the platinum wire protruded into the cell cavity. A quartz disk was epoxied to the back face of the cell as seen in Figure 2. Tin oxide OTE (Corning Glass Company) were cut into 1.9-cm squares. Brass shim stock was cut in the shape shown in Figure 2 to provide external electrical contact with the OTE. Four holes were drilled in the cell and tapped to accommodate four screws. The cell was assembled by placing the O-ring in the O-ring groove, positioning the brass contact around the 0ring, and clamping the parts to the cell face with the aluminum holder with the four screws. The auxiliary electrode, shown in Figure 3 , was made from an outer joint (Ace Glass Company, joint No. 7602-35) and a funnel fitted with a 10-mm, C porosity frit (Ace Glass Company, No. 7186-06). A silver wire inserted through a serum cap completed the auxiliary electrode. The reference electrode was assembled from an outer joint (Ace Glass Company No. 7605-35), a n inner T 5/20 joint and a reference probe (Leeds and Northrup No. 117147). A silver-silver chloride reference electrode was made by epoxying a length of silver wire into an outer T 5/20 joint. Silver chloride was deposited on the silver wires (reference and auxiliary) by anodizing in a HC1 solution. This reference electrode (silver/silver chloride, 1.OM KCI) was measured with respect to three different saturated calomel reference electrodes and gave a mean value of potential of +0.230 f 0.001 V us. NHE. The vacuum degassing bulb, shown in Figure 3 , was made from a n outer joint (Ace Glass Company, No. 760235), a T 2 stopcock, and an inner T 7/25 joint. A stirring bar was included in the bulb to stir solution during vacuum degassing. Vacuum, about 0.1 Torr, or nitrogen pressure, about 3 psi, could be applied conveniently to the cell cavity through the degassing bulb when joint A was connected to the vacuum/nitrogen train. Two drying tubes containing Drierite and a liquid nitrogen trap were placed in the line between the cell and the vacuum pump. T o remove oxygen, the nitrogen gas was passed through two tubes filled with hot copper turnings (two tubes in furnaces, SargentWelch, No. S-36518 and No. 36517) (6). All connecting tubes in the nitrogen train were either glass or copper. The electrochemical instrumentation was of conventional design. Charge was measured by integrating the current flowing through a 100-ohm load resistor placed between the working electrode (OTE) and ground. The integrator was built employing Teledyne-Philbrick 1009 Operational Amplifier and constructed so that 0.1 V corresponded t o 0.005 C of charge ( R = 500 KQ, C = 1.0 pF). The integrator drift error was less than 0.3%. The rapid scanning spectrophotometer (RSS) is an improved version of an earlier model (14). Modifications include replacement of lens (L2, Figure 1 of reference 14) with a 12-cm focal length concave mirror and the use of a flat front surfaced, vibrating mirror. This latter change necessitated placement of a short focal length lens between slit SI and the vibrating mirror. The dimensions of the vibrating mirror were increased for improvement of resolution and greater throughput of light intensity, at the sacrifice of scan speed. This RSS could scan a t a rate from less than 0.1 Hz to a maximum rate of 500 Hz with a resolution of cn. 5 A a t the optimum wavelength. For the work described here, the incident angle of the grating (600 lines/mm, blazed a t 8"38' and Xmax 5000 A) was adjusted so that a wavelength range of 400-650 nm was scanned. The electronic circuitry remained es(14) J. W. Strojek, G . A. (1969).
sentially identical with the earlier model with the except,ion of the use of operational amplifiers having improved specifications. A Hewlett-Packard (Moseley Division) 7035B X - Y recorder or a Houston Instruments Model 2000 X - Y recorder was used for signal monitoring purposes. The magnetic stirrer motor for the cell was mounted so t h a t it could be withdrawn from the proximity of the photomultiplier tubes. A reference OTE cell similar to the sample cell was placed in the reference beam. All computations were done on a Data General Corporation Nova 800 Minicomputer. Reagents. Phosphate buffer, p H 7.00 f 0.02 (Buffer Titrisol) and sodium chloride, Suprapure, were from E. Merck Company. Potassium chloride (analytical grade) was from Matheson, Coleman and Bell. The nitrogen gas was prepurified grade from J. T. Baker Chemical Company. The l,l'-dimethyl-4,4'-bipyridyl dichloride (methyl viologen) was from K and K Laboratories. The l,l'-ethylene-2,2'-bipyridyl dichloride was prepared in this laboratory by E. Steckhan ( 1 5 ) . The horse heart cytochrome c, modified horse heart cytochrome c, and sperm whale myoglobin were provided by C. Hartzell. Doubly distilled water (all glass still) was used throughout. Procedure. The heme protein samples were stored frozen a t 0 "C and were thawed slowly for solution preparation. All samples were provided in a concentration of 3.5 to 5.0mM. Stock solutions were prepared by a 1:25 dilution of thawed samples in supporting electrolyte solution (0.1M phosphate buffer, 0.1N NaCl a t pH 7.0). Test solutions were prepared by purging the supporting electrolyte with nitrogen and filling the volumetric flask to about 75% full. The electrochemical mediators (in solid form) and an aliquot of the stock solution were then added. The volumetric flask was filled to the mark with supporting electrolyte. The stock and test solutions were stored a t 0 "C. OTE's were washed in isopropanol and doubly distilled water prior to assembly in the cell. Valve 1 and joints A and B (cf. Figure 3) were lightly greased with Apiezon N. The cell was dried between runs by connecting joint A to the vacuum/nitrogen train and applying vacuum with all valves open and was then stored with 3 psi of nitrogen pressure in the cell (valves closed). The reference electrode inner compartment was filled with degassed 1.OM KC1 solution using a syringe and the cap replaced. With valves 1 and 3 open, joint A was connected to the vacuum/nitrogen train and vacuum was applied. Nitrogen pressure was then applied and valve 4 was opened. The vacuum/nitrogen cycle was repeated three times (5 sec for each cycle). This procedure served to remove any oxygen which may have entered the cell when the reference electrode was filled with KC1. Care was taken to prevent the KC1 solution from boiling while vacuum was applied. With positive pressure of nitrogen applied, valves 1, 3, and 4 were closed. Next, the cell was disconnected from the vacuum/nitrogen train at joint A. Valve 1 was opened, 2.5 ml of test sollition was injected with a syringe into the degassing bulb, and then valve 1 was closed. After reconnecting the cell to the train, the cell was rotated so t h a t a magnetic stirrer could be positioned under the degassing bulb. While stirring the solution, three cycles of vacuum/nitrogcn were applied (about 20 sec of vacuum and 5 sec of nitrogen pressure). With positive nitrogen pressure in the bulb, the magnetic stirrer was removed, the cell rotated into the upright position, and valve 3 was opened to fill the cell cavity with solution. The vacuum/nitrogen pressures were repeatedly applied to fill the cavity and remove all gas bubbles. The cell was disconnected while under nitrogen pressure. With valve 4 open, the cap on the reference compartment was opened sufficiently to allow test solution into the reference compartment to a level just in contact with the reference probe. Valve 4 was then closed. Pressure release from the auxiliary electrode compartment was done simply by inserting a syringe needle through the serum cap and solution flowed up to the level of the frit (valve 2 open). Valve 2 was closed and degassed 1.OM KC1 was injected with a syringe through the serum cap of the auxiliary electrode making contact between the auxiliary electrode and the frit. Valves 2 and 4 were opened and valve 3 was closed. All experiments were run at 23 f 1 "C. Potentiometric measurements were made between the platinum wire and the reference electrode using a Corning Model 12 p H meter. All potentials are reported us. NHE. Between runs, the auxiliary and reference electrode glassware and the degassing bulb were removed and cleaned in an ultrasonic cleaner. Doubly distilled water was flushed through the cell cavity. The cell was then assembled and dried as previously described.
Gruver, and T. Kuwana, Ana/. Chern., 41, 481 (15) E.
Steckhan, unpublished results, this laboratory.
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 7, J U N E 1973
1023
r
la
MYOGLOBIN
w
I
1
0
20
I
40
1
I
60
80
100
I
I
120
140
1
160
CHARGE (NEP/MLI
Figure 4. Plot of absorbance vs. charge (corrected) during gen-
eration and removal of methyl viologen radical cation Monitoring wavelength 605 nm; reductive charge correction 15%; oxidative charge correction 1 0 % Solution conditions: methyl viologen dication 0.5mM, ferrocyanide 1.OmM. sodium chloride O.lM, phosphate buffer pH 7.0 CHARGE (Noq/rnl)
Figure 5. Plot of absorbance vs. charge
RESULTS AND DISCUSSION Coulometric Generation. Quantitatiue Aspects and Effect of 0 2 . The quantitative aspects of the OTE cell were tested by following the absorption band a t A,, 605 nm for the generation of MV.+ from MV2+ a t a fixed potential of -0.85 V, and then the oxidation of MV.+ by generation of Fe(CN)63- from F ~ ( C N ) Ga~t -a potential of +1.0 V. The electrochemistry of MVZ+/MV.+ has been extensively examined at the tin oxide OTE ( I ) and gives a reversible one-electron transfer with an E"' of -0.442 f 0.005 V. Ferricyanide/ferrocyanide, on the other hand, gives an irreversible appearing L - E wave a t this electrode and necessitates the potential being stepped to + 1.0 V for an electron transfer rate approaching diffusional control. In Figure 4, the linear change in absorbance at 605 nm is plotted as a function of the corrected coulometric charge, Qc. In the initial titration, the 605-nm band of MV.+ does not appear until the 0 2 is scavenged from the cell. The stoichiometry for this reaction is 4MV.'
+
4H'
+
O2 = 2 H 2 0
+
4MV2+
(3)
and there is no evidence for any H202 remaining in the solution when MV. + is present in excess. From the A-Q plots, the efficiency of electrogeneration in terms of the n value can be determined if t b is known AAFV
lln
=
aQctb
(4)
where t is the molar absorptivity in l./mol/cm, b is the optical path length of the cell in cm, S Q c is the corrected charge in coulombs, J-4 is the absorbance change corresponding to A Q c , F is the Faraday, and V i s the volume of the cell in cm3. I t is convenient to simplify this equation in terms of
n =-e AQ AA
(5)
where A Q is the charge in nanoequivalents per milliliter and AA' is the change in absorbance in absorbance per centimeter. 1024
A N A L Y T I C A L CHEMISTRY, VOL. 45, NO. 7, JUNE
1973
( a ) 1,lf-ethyIene-2,2'-bipyridyl dichloride 0.5mM, mygiobin 30.6pM, monitoring wavelength 560 nm. ( b ) Methyl viologen dication 0.5mM, modified cytochrome c 5 2 . 7 p M , monitoring wavelength 550 nm. Ferrocyanide l.OmM. sodium chloride O . l M , phosphate buffer pH 7.0
Corrections to Q for background charge, which is due to the changing of the double-layer and to any extraneous faradaic process, was performed by stepping the potential to the same value for the same time increments as in the previous experiment with only supporting electrolyte present in the cell. The order of magnitude of the corrections amounted to 10-20% for reductions and 5-1070 for oxidations. The exact total charge consumed was determined periodically for a given heme protein titration. This background charge did not vary significantly when the experiment was repeated in the presence of heme proteins. The value of e was 12,500 for MV-+ a t a wavelength of 605 nm, and b was determined from calibrations using known standard concentrations of ferricyanide ( t = 1020 a t X 420 nm). The calculated n values in Figure 4 are 0.94, 1.05, 1.00, 1.09, 1.07, and 1.01 for the reductive and oxidative titrations on the same solution and give an average value of 1.03 f 0.04. Thus, it appears that MV2t can be quantitatively reduced to MV.+ and then the MV.+ can be quantitatively oxidized, principally through reaction with electrogenerated ferricyanide, a t the tin oxide OTE. It should be noted that the total amount of MV.+ generated in this experiment is only a small portion of the total quantity of MV2+ present in the solution and amounts roughly to the equivalents (20 to 50 nequiv) of heme proteins which were evaluated. As mentioned earlier, 0 2 is scavenged first during the reductive titration. Using the vacuum/nitrogen degassing procedure described in the experimental section, the level of 0 2 is less than 4 X l0-7M as calculated from the charge consumed prior to the 605-11, band moving (estimated from the charge passed at the foot of the A us. Q plot in Figure 5 before a change in absorbance occurred). The ability to remove and maintain an 0 2 free solution is
Table I . Indirect Coulometric Titration of Sperm Whale Myoglobin and Modified Cytochrome Ce n Valuesb Titration sequencea
%
1
2
3 4 Av value
MyoglobinC 0.97 0.95
1.00 1.02
1.08 1.01 1.00 1.01
Modified cytochromecd
1.00 1.01 1.01 1.03
1.01 f 0.02
1.19
1.04 0.97 0.95
1.04 f 0.08
Titrations 1 and 3 are reductions: 2 and 4 are oxidations. Calcuiated from A - Q slopes using Equation 5 (negative slopes for oxidations). (Myoglobin) = 3 0 . 6 ~ M . ( 1 , l '-ethylene-2,2'-bipyridyi dichloride) = 0.5mM. each column represents a new solution. E'' myoglobin = +0.045 at pH 7 ( 1 6 ) . (Modified cytochrome c ) = 52.7pM, (Mi!'+) = 0.5mM e All solutions contained 0.1N NaCI, 0.1M phosphate buffer, pH 7.0, (Y4Fe(CN)6)= 0.5mM.
'
extremely important, particularly with certain heme proteins, as we shall find with cytochrome c. Indirect Coulometric Titration. Myoglobin and M o d i fied Horse H e a r t C y t o c h r o m e c. In Figures 5a and 5b, the linear A-Q plots for sperm whale myoglobin and modified horse heart cytochrome c (methylated methionine) are shown. The absorbances a t wavelengths of 560 ( l e = 8,700) and 550 nm (le = 11,500) were measured for various increments of charge for myoglobin and cytochrome e, respectively. The reductions by electrogenerated MV. + and subsequent oxidations by electrogenerated ferricyanide proceeded smoothly in both cases and linearity in the A-Q throughout the entire titration indicates that the E"' values for both these hemes are sufficiently different from both the MVzf/MV- + and ferricyanide/ferrocyanide couples that the titrations are essentially quantitative up to each end point. Both of these hemes are expected to undergo only a one-electron titration and the experimentally determined n values calculated using Equation 5 are within =t2% of the expected value. The data and titration conditions are summarized in Table I ( 1 6 ) . In the case of myoglobin (Figure Sa), the reagent, 1,l'ethylene-2,2'-bipyridyl dichloride, was employed. In the reduced form, the titrant does not absorb at the wavelength of 560 nm and, therefore, the absorbance does not change any further a t the end point of the titration. This titrant, although a , weaker reducing agent than methyl viologen (EO' for l,lf-ethylene-2,2'-bipyridyldichloride is -0.362 compared with -0.446 for methyl viologen), reacts quantitatively. In Figure 5b, the absorbance continues to increase a t the end point due to the absorbance by the excess of reducing titrant which absorbs at the same wavelength as modified cytochrome e. Although myoglobin functions in the mammalian respiratory system as an oxygen storer, it can undergo a homogeneous electron transfer reaction involving one electron with the titrant used. It has served as one of the hemes for a test of the present approach for n value determination since its behavior has been well characterized chemically. Modified cytochrome c also served as a known system and its properties will be discussed further after results for the horse heart cytochrome c are presented. Titration of Cytochrome e. Spectra obtained during the reductive titration of horse heart cytochrome c by electrogenerated methyl viologen radical cation are shown in Figure 6. The bands a t 520 and 550 nm both increase during reduction. The molar absorptivity of the 550-nm band was evaluated as 21,000 l./mol/cm which is in good (16) W. M. Clark, "Oxidation-Reduction Potentials of Organic Systems," Williams and Williams Co.. Baltimore, 1960, p 451
Figure 6. S p e c t r a taken after equal incremental additions of r e ducing c h a r g e for the reductive titration of c y t o c h r o m e c Solution conditions similar to those in Figure 4 with the addition of 14.6 pM cytochrome c
-
I O0.4 -T-'
U, 0.3 v,
m
4 W
0 2
m U
v,
s
0.1
0 .o
0
20
40
60
80
100
120
140
C H A R G E (nEq/rnl)
Figure 7>PIot of a b s o r b a n c e vs. c h a r g e ( c o r r e c t e d ) ( a ) Solution conditions same as in Figure 4 except 21.4 pM cytochrome c added. ( b ) Same as above except O2 in solution
agreement with literature values ( 17, 18). The solution used for obtaining these spectra also contained ferrocyanide which is oxidized electrochemically during the oxidative titration of reduced cytochrome c. A consequence of the presence of ferrocyanide is reflected in the nonlinear change of absorbance as a function of charge. This is clearly evident in the A-Q plots for reduction and subsequent oxidation (and recycling) for a 18.1pM solution of cytochrome c as seen in Figure 7 . The curvature in the (17) V. Massey, Biochim. Biophys. Acta, 34, 255 (1959). (16) B. F. Van Gelder and E. C. Siater, Biochim. Biophys. Acta, 58, 593 (1962). A N A L Y T I C A L C H E M I S T R Y , VOL. 45, N O . 7, J U N E 1 9 7 3
1025
Table II. n Values Calculated for Various Reductive and Subsequent Oxidative Titrationsa of Cytochrome c Run No.
(Cytochrome c),pM
(MVZf), mM
(K4Fe(CN)6). mM
24.5 16.7 16.7 16.7 26.7 24.5
0.5 0.5
0.5
2.0 1 .o 1 .o
0.5 0.5
1 .o
0.5
0.5
1 2 3 4
5 6
1st ox
2nd Red
2nd Ox
1.10 1.25 1.23 3.36b 1.02 1.08
1.01 0.94 0.92 1.24 1 .oo 1.09
1.09 0.87 0.93 1.09 0.84 1.28
1.07 0.94 0.96 1.10 0.99 1.17
1.03 f 0.09
1 . 0 2 f 0.14
1 .o
Av a
1 st Red
1.14
*
0.08
1.04 iz 0.08
All solutions contain 0.1M NaCI. 0.1N phosphate buffer, pH 7.0. Not included in the average.
A-Q plots is due to the small value of Keq of reaction 2. Ordinarily in such situations, the linear portions near the end of the titration are utilized for obtaining slopes and calculating n values. However, the concentration of cytochrome c was sufficiently low in most titrations, that the entire A-Q curve was affected. Since the total concentration and E"' of the titrant couple are known and the concentration of oxidized to reduced forms of cytochrome c can be evaluated from the absorbance a t 550 nm, the A-Q curves can be computer simulated for various values of n and E"' for cytochrome c, assuming the validity of the Nernst expression.
AE"'
=
Eo'Fe(CN), - E"'Cytoc
0.059 -lode, n
(6)
The solid line in Figure 7, trace a, is a "best fit" with the experimental points and is computed for a AE"' of-0.166 V, assuming an n value of unity. Using the experimentally determined E"' equal to 0.424 V us. NHE for ferricyanide/ ferrocyanide, the E"' of cytochrome c is calculated to be 0.258 f 0.005. This value is in excellent agreement with the average of 19 literature values (0.257 f 0.017 V us. NHE) (3, io, 13,19-28). Assuming the validity of the AE"' value of 0.166 V, all the experimental A-Q curves can be corrected for the presence of ferricyanide. The n values were then determined from the slopes in the usual manner. Data from several runs with varying solution conditions are summarized in Table 11. There is one additional solution variable which becomes evident during the initial reductive titration. The amount of residual 0 2 in the solution affects the slope. That is amply demonstrated in the titration curve of Figure 7 , trace b, where a partially degassed solution is titrated by MV.+. The absorbance in this trace increases fairly linearly with charge but a t a much lower rate. What is happening is that native cytochrome c reduces molecular oxygen a t a very slow rate a t pH 7 (29). A nonequilibrium condition exists and cytochrome c and oxygen are reduced by MV.+ in a parallel reaction. Thus, the absorbance corresponding to formation of reduced cytochrome c in(19) €. G.Ball, Eiochem Z., 295,262 (1938). (20) H. E. Davenport and R. Hill. Proc. Roy. S O C , Ser. E , 139, 329 (1952). (21) R. W. Henderson and W. A . Rawlinson, Eiochem. J., 62, 21 (1956). (22) k. W.' Henderson and W. A. Rawlinson, Nature (London), 177, 1180 11956). (23) K. G. Paul,Arch. Eiochem., 12,441 (1947). (24) E. Stotz, A. E. Sidwell. Jr.. and T. R. Hogness, J. Bioi. Chem., 124, 1 1 (1938). (25) R. Wurmser and S. Filitti-Wurmser. J , Chim. Phys., 35,81 .(1938). (26) D. E. Green, J . Jarnefelt, and H. D. Tisdale. Biochem. Eiophys. Acta, 31,34 (1959). (27) T. 6. Coolidge, J. Bioi. Chem., 98,755 (1932). (28) F. M. Stone and C. B. Coulter. J. Gen. Physiol., 15,629 (1932). (29) A. L. Lehninger, "Biochemistry," Worth Publishers, New York. N. Y., 1970,p 377. 1026
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creases a t a rate dependent on the amount of 0 2 present. The effect of 0 2 is in contrast to denatured cytochrome c or to the previous cases of modified cytochrome c or myoglobin where those hemes in their reduced forms reacted a t a rate sufficiently rapidly with 0 2 so that the absorbance did not change until all of the 0 2 was scavenged from the solution. The error due to the presence of residual 02 in the cell can be easily taken into account by relying on n values determined only after the first reductive titration. For example, the n value is high in the first reductive titration of run No. 4 in Table I1 due to 0 2 ; however, the following oxidative and reductive titrations give n values close to the expected value of unity. Potentiometric Results. Potentials (Pt) were measured during several of the reductive and oxidative titrations of cytochrome c. These titrations were taken to a predetermined end point measured in terms of absorbance, since any significant excess of MV.+ resulted in a large hysteresis of the potential. When the data from these measurements were plotted as E us. log (AAT - AA)/AA and a least-square analysis was made, the slope was 0.039 and E"' was equal to 0.262 V a t the half-titration point. This potential is close to the value determined from the A-Q plots. The n value calculated from the slope gives a value equal to 1.55 which is not in agreement with the expected value of unity. This discrepancy is probably due to the fact that the ferricyanide/ferrocyanide ratio, being very low during most of the titration, does not adequately establish a well-poised system with the electrode and, consequently, the measured potentials do not reflect the true redox level of the solution. Further studies are in progress to test this point.
CONCLUSION The accurate evaluation of the stoichiometry ( n values) and energetics (E"' value) of biological components is important in the understanding of biological electron transport mechanisms. The indirect coulometrh titration technique described seems to offer several advantages over conventional potentiometric titration techniques. In particular, the ability to repetitively cycle the redox level of the solution reasonably rapidly and reproducibly is significant in the examination of time and redox state dependent effects. It is believed that these advantages will be extremely useful in the evaluation of other heme proteins, mixtures of heme proteins, and of enzymes which possess multiple component, transfer sites.
ACKNOWLEDGMENT The authors appreciate the many helpful discussions with Wm. R. Heineman and C. R. Hartzell. The heme proteins were generously provided by C. R. H. One
of us (T. K.) gratefully acknowledges the kind hospitality and assistance of H. Beinert, Institute of Enzyme Research, Madison, Wis., where preliminary experiments were performed. T. K. also wishes to thank the Department of Chemistry a t the University of Wisconsin and their machine shop for assistance in fabrication of the rapid scanning spectrophotometer during sabbatical leave 1970-71.
Received for review October 20, 1972. Accepted December 20, 1972. The authors gratefully acknowledge the financial support provided by P H S Research Grant GM 19191-01 and the National Science Foundation Grant G P 31236. This investigation was initiated during tenure of T. K. (NIH Special Research Fellowship 1F03 GM 48486) at the Institute for Enzyme Research, University of Wisconsin, Madison.
Determination of Iron, Copper, and Lead in High Purity Synthetic Silver Chloride Crystals by Spark Source Mass Spectrometry A. W. Fitchett and R. P. Buck' William Rand Kenan, Jr., Laboratory of Chemistry, University of North Carolina, Chapel Hill, N.C. 27574
This investigation describes the spark source mass spectrometric determination of iron, copper, and lead impurities through dissolution of the non-conducting silver chloride crystals in high purity ammonium hydroxide followed by reprecipitation on high purity graphite. Relative sensitivity factors are calculated using solution doped synthetic standards. A matrix effect for lead and poor lead detectability in silver chloride crystals are observed. Analysis of synthetic doped crystal sections shows accuracy of this method to be fl0-20%.
Preparation of high purity silver chloride and the growth and use of pure and heavy metal doped silver chloride crystals have been a continuing program a t the University of North Carolina Materials Research Center. Mixed conductivity and dislocations confer unusual properties to the doped crystals which have made them useful as detectors of nuclear particles (1-4). In addition to conductivity and dislocation measurements, quantitative determinations of polyvalent cations, particularly Cu(II), Fe(II), Fe(III), and Pb(II), are essential steps in the characterization of these materials. Qualitative detection and analysis of other trace impurities are frequently required as well. Previously, trace metal analyses in silver chloride have been accomplished by emission spectroscopy (5), extraction followed by colorimetry (61, and extraction followed by atomic absorption spectroscopy ( 7 ) . Spark source mass spectrometry (SSMS), with its capabilities of low detection limits for trace metallic impurities and total qualitative sample analysis, appears suited to the analysis of silver chloride crystals. The intent of this study is to explore techniques and applicability of SSMS as an alternate method of analysis. Achieve1To whom correspondence should b e addressed C. Childsand L. Siifkin, Bull. Amer. Phys. Soc., 6 , 52 (1961). C. Childsand L. Slifkin, Rev. Sci. Instrum.. 34, 101 (1963). C. Childs and L. Slifkin, Brit. J . Appi. Phys.. 16, 771 (1965). C. Childs and T. Parnell, Proceedings of the National Symposium on Natural and Man-made Radiation in Space, Las Vegas, Nevada, 1971. (5) K . Tempelhoff, Z. Ana/. C h e m . , 244, 172 (1969). (6) V. Concialini, P. Lanza, and M . T. Lippolis, Anal. Chim. Acta. 52, 529 (1970). (7) J. W. Edwards, G. D. Lomlnac. and R. P. Buck, Anai. Chim. Acta. 57, 257 (1971). (1) (2) (3) (4)
ment of highest sensitivity by SSMS is limited to metallic samples or other substances exhibiting high electronic conductivity. High purity silver chloride crystals behave in SSMS as non-conductors and special techniques must be developed for successful analyses. Spark source mass spectrometry of whole non-conductor pieces has been the subject of a few papers (8-10), and only this third paper, by Dietze and Ludke, dealt with the problems of analysis of synthetic crystals. The methods suggested by these authors rely on the use of conducting counter-electrodes, usually gold opposed to an electrode of sample material. The major drawback to these counter-electrode methods is that the ratio of ionized sample to ionized counter-electrode material in the plasma is not constant and can not be closely controlled. Integrated beam current is then no longer a valid measure of consumed sample. The method of pressed or compacted electrodes containing sample and intimately mixed metal powders is feasible, provided the impurity content of metal powders is low for those elements to be determined. Our experience with available silver powders was discouraging because of high impurity contents. Errock (11) used the method successfully to study non-conductor samples, by grinding and mixing samples with high purity graphite which were then pressed into electrodes. The major remaining problem associated with mixed powder electrodes is lack of homogeneity, but this has recently been overcome by Morrison and Rothenberg (12). A review of these methods for high resistivity samples has been presented by Socha and Baker (13). Each of these techniques and the direct, forced sparking of crystal pieces were attempted for the analysis of the silver chloride crystals. The results were disappointing. Direct sparking of two crystal electrodes under strenuous conditions (60 kV; 30,000 pulses per second) produced uncontrolled surface breakdown which sputtered silver chlo(8) A. J. Ahearn.J. Appi. Phys., 32, 1195 (1961). (9) J. A. James and J. L. Williams in, "Advances in Mass Spectrometry," Vol. 1, J. D.Waldron. Ed., Pergamon Press, Oxford, 1959. (10) H . J. Dietze and W . Ludke, f x p . Tech. Phys., 17, 289 (1969). (11) G. A. Errock, Tenth International Conference on Spectroscopy, College Park, Md., 1962. (12) G. H. Morrison and A. M . Rothenberg, Anai. Chem.. 44, 515 (1972). (13) A. J . Socha and C. W . Baker, Twentieth Annual Conference on Mass Spectrometry and Allied Topics, Dallas, Texas, 1972. A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 7 , J U N E 1 9 7 3
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