Aut 8 rnat ed Electrodic PoteratiQ met ry of Potassium Ferricyanide and Respiratory Components Richard MI. Hendler Section on .Membrane Enzymology, Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 200 14
TRls paper demonstrates the appllcablllty of a new system of electrodic potentiometry for manlpulatlng the electric potentlal of aqueous solutions. I n thls system a mlcrocomputer-controlled current source causes the oxldatlon or reduction of solution components at a working electrode surface. I n this manner, repetitlve potentlometrlc titrations are performed or a serles of optical absorption spectra are taken at dlfferent solution potentials. The present paper shows the potentlometric titrations of potasslum ferrlcyanide, soluble cytochrome c , and insoluble cytochrome b, In E . collmembrane fragments. The role of soluble redox mediators In these tltratlons is studied. A serles of optical spectra at different potentlals Is shown for the ferrlcyanlde and E . collmembrane systems. T h r d h e n d o n a l plottlng technlquoa are introduced to display ?he relationship between absorption spectra and solution potential.
T h e accompanying paper ( 1 ) describes a technique for performing electrcdic potentiometry which, when coupled with spectrometry, is capable of determining the redox potentials of substances that absorb light. The same system can collect a series of spectral scans a t different so!ution potentials. This paper presents iesults obtained with this system in determining the redox potentials of a dissolved inorganic substance (ferricyanide), a dissolved protein (cytochrome c), and an insoluble respiratory pigment in a suspension of membrane fragments (cytochrome bl in E. coli). The need for and influence of solub!e redox mediators in this system are also examilzed. Series of spectral scans obtained a t different voltages are shown for ferricyanide and for various respiratory pigments in E. coli membranes. The use of 3-dimensional plotting techniques displays the results in the form of surfaces. This ability reveals hitherto unappreciated complexities of the response of E . coli respiratory membranes to changes in oxidizing potential. These complexities are being studied and wili be the subject of a future paper. EXPERIMENTAL All electrode potentials expressed in this work are relative to the standard hydrogen electrode. Materials. Mediators. In most experiments, six mediators were used. They were potassium ferricyanide (“FCN”, E = 435 mV, Merck and Co., Rahway, N.J.); quinhydrone (“QH”,E’m = 280 mV, Fisher Scientific Co., Fair Lawn, N.J.); 1,Lnaphthoquinone (“1,2-NQ”,E’m= 143 mV) and pyocyanine perchlorate (“PYC”, E’,,, = -34 mV) (K and K Laboratories, Plainview, N.J.); phenazine methcsulfate (“PMS”,E’m= 80 mV, Calbiochem, La (“2-OH-NQ”,E ‘,,, JoUa, Calif.; and 2-hydroxy-l,4-naphthoquinone = -145 MV, Eastman Organic Chemicals, Rochester, N.Y.). The mediator solutions were freshly made for each experiment in stock solutions of 12 mM for FCN and 6 mM for each of the other five. In some experiments, a mixed stock solution was used, where all of the dry mediators were first ground together in a mortar. ‘The mediators are light-sensitive and therefore are always kept in the dark. Desired experimental mediator concentrations could be achieved with 10 to 50 pL aliquots per 3 mL. In addition to the 1814
* ANALYTICAL CHEMISTRY,
VOL. 49, NO. 13, NOVEMBER 1977
mediators mentioned above, dimethyl dipyridyl chloride (dimethyl viologen, E’, = -440 mV, ICN Pharmaceuticals, Inc., Plainview, N.J.) was used at 0.5 mM concentration in the experiment shown in Figure 4. Other. Cytochrome c was salt-free, A grade purchased from Calbiochem, Los Angeles, Calif. E. coli membranes (T-fraction) were prepared as previously described ( 2 ) . Procedure. The substance to be analyzed was placed in 3 mL of an aqueous solution containing 0.2 mL each of 2 M KC1 and 1 M K3P04buffer (pH 7.0), plus mediators as desired. The pH of the solution was 7.0 k 0.05. Final concentrations of the samples, unless otherwise noted were 0.5 mM for K,Fe(CN),, 0.67 mg/mL for cytochrome c, and 6.7 mg protein/mL for E. coli membranes. The cuvette, containing the so!ution, was closed with the electrode-containing cap assembly ( 1 ) and argon passed over the mechanically stirred solution at a rate of about 65 mL/min. All channels of A/D and D/A conversion were checked and adjustments made if necessary, using a calibrations program ( 1 ) . After about 20 min of gassing, either a potentiometric titration or a programmed series of spectral scans was started by activating the appropriate program ( I ) . The voltages of the reference and auxiliary electrodes were checked before placing the cap assembly on the cuvette and again at the end of each experiment. This was done by reading the voltage of each against both a standard calomel and a standard Ag/AgCl electrode. The voltage of the reference electrode did not usually change by more than 3 mV during experiments sometimes lasting about 8 h. For potentiometric titration experiments, dual wavelength spectrophotometry was used to determine the extent of oxidation or reduction. The Wavelength pairs used were 420 nm and 460 nm for ferricyanide, 550 nm and 540 nm for cytochrome c, and 560 nm and 550 nm for cytochrome bl. For spectral scans experiments, split beam spectrophotometry was used. The reference cuvette contained water in the ferricyanide experiments and a neutral density Delrin plastic (to balance the light scattering) in the E. coli membrane experiments. At the end of all of the experiments, the communications program was activated and all data were transmitted to the PDP DECsystem-IO computer (I). Analysis of Data. Data from potentiometric titrations were analyzed by MLAB, a mathematiml modeling system programmed for the DECsystem-10 as previously described (3). The RD[E] function was used. This procedure fits the data to a system of one or more components obeying the Nernst relationship,
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where E represents solution potential and E’,,, midpoint potential. The term “Nernstian” refers to adherence to this relationship. All lines drawn in figures showing potentiometric titration data represent the computer best fit to the experimental data, shown as points. Three-dimensional representations of surfaces showing the influence of voltage on spectra were drawn using Omnigraph, a general purpose interactive graphics system programmed for the DECsystem-10 ( 4 ) . RESULTS AND DISCUSSION Figure 1 shows the results of two experiments, each with five successive titrations alternating between oxidations and reductions. In one experiment, five redox mediators were present and in the other, none. Plates A, B, C, and D show four of the five titrations performed in the presence of mediators. Plate E shows all five. In plate F are shown five
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Flgure 1. Potentiometric titrations of potassium ferricyanide. Plates A through D show sequential tiations on 0.5 mM ferricyanide in 0.133 M KCI and 0.067 M potassium phosphate containing 0.1 mM each of OH, 1,2-NQ, 2-OH-NQ, PYC, and PMS at pH 7.0. Plate E shows all five ttrations of the sample and plate F shows five titrations performed in the absence of mediators. 100% oxidation represents 0.43 absorbance unit
titrations performed in the absence of mediators. In this
figure as well as i n all figures showing potentiometric titrations, t h e points show experimental data and t h e lines show computer best-fit (i.e., to the RD[E]function (3))curves. The E , calculated from the data shown in Figure 1E was 439 f 0.39 mV and from the data shown in Figure lF, it was 435 f 0.33 mV. It is obvious that the system is capable of reproducibly titrating ferricyanide in both directions and that mediators are not required to couple the ferricyanide to the measuring and working electrodes. The mediators, however, do help stabilize the system by virtue of their redox buffering action and as a result greatly facilitate the control of the solution potential. In the case of cytochrome c, two successive titrations could be accomplished with good reproducibility. Additional titrations frequently deviated from the earlier c m e s , most likely because of cumulative protein denaturation. Figure 2 shows oxidative and reductive titrations plotted as one-component and two-component systems. In previous titrations, performed manually with chemical titrants, we obtained an E , of 262 mV for the one-component fit and of 266 mV for the major component and 195 mV for the minor component of a twocomponent fit ( 3 ) . T h e two-component fit was closer than the one-component fit and the relative amounts of the two-components were 88 and 12%. The results of five pairs of successive oxidative and reductive titrations obtained with the newer electrodic system were as follows: 1)For the one-component solution, the E , A SEM was 245 i 1.8 mV. 2) For the two-component solution, the major component comprised 64 h 4.7% and had an E , of 266 i 2.3 mV. The minor component comprised 36 f 4.7% and had an E , of 182 f 13 mV.
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The lower one-component E , (245 mV compared to 262 mV) is consistent with the higher apparent content of the second component of lower E , found in the two-component fit of the samples of cytochrome c used in this work (Le., 36% compared to 12%). The higher potential component ( E , = 266 mV) was the same and was determined with high precision in both the current and previous titrations. The lower potential component was subject to greater uncertainty and its identity is unknown. The five pairs of titrations were performed on different days, all with six mediators present. The concentration of cytochrome c was either at 0.38 mg/mL or 0.67 mg/mL. The concentrations of mediators ranged as follows: FeCN from 0.2 to 0.68 mM; QH, 1,2-NQ, 2-OH-NQ, and P M S from 0.1 to 0.34 mM; PYC from 0.04 to 0.34 mM. Variations in the concentrations of individual mediators or in their relative concentrations did not seem to affect the results. However, in the absence of mediators, a poor distribution of voltages was obtained because of the absence of redox buffering action. The one-component E,,, values obtained in the absence of mediators in successive oxidative and reductive titrations were 283 mV and 266 mV. The total amounts of absorbance change were the same in the presence and absence of mediators. In our earlier studies with cytochrome bl of E . coli membranes, we found three Nernstein species present (3). In the current work, we also found three species. A comparison of the earlier results obtained by a manual chemical titration with the newer results (five titrations in three separate experiments) obtained with the automated electrodic system is shown in Table I. T h e high and middle E,,, values are the same, but the low E , value found in the current work is somewhat lower than found previously. The slightly different quantitative distribution of species could be explained by the fact that ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
1915
Table I. Comparison of Chemical and Electrodic Potentiometric Titrations of E. coli Cytochromes b , Chemical Electrodic titration (3) titration Species Percent of each 39 33 28
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