THE SPECTROPHOTOMETRIC DETERMINATION OF THE EQUILIBRIUM IN OXIDATION-REDUCTION SYS-

TEMS; THE POTENTIAL OF CYTOCHROME C

BY ELMER STOTZ, A. E. SIDWELL, JR., AND T. R. HOGNESS

(From the George Herbert Jones Chemical Laboratory of the University of Chicago, Chicago)

(Received for publication, February 19, 1938)

Equilibrium constants, free energy differences, or potentials have often been determined by calorimetric analysis, but this method is very limited in its application if confined to the visible region of the spectrum and to one colored constituent. Conse- quently it has been overshadowed by other methods of analysis, in particular by the electrometric titration method which has been so widely used where organic and biological oxidation-reduction systems are involved. One of the notable exceptions to the use of the electrometric titration method for organic and biological systems, which in principle is related to the work reported in this paper, has been the work on the succinate-fumarate system by Quastel and Whetham (1) and by Thunberg (2) who, independ- ently, used methylene blue and its leuco derivative as reference substances. From the ratio of the reduced and oxidized amounts of methylene blue the latter investigator was able to determine the potential of the succinate-fumarate couple.

From our experience with the precision photoelectric spectro- photometer, developed by Hogness, Zscheile, and Sidwell (3), there is every reason to believe that the calorimetric method, which has many advantages, will become more generally appli- cable. With this instrument the determination of the amounts of the oxidized and reduced forms of two substances in equilibrium, even though they absorb only in the ultraviolet region, can be carried out and the potential of the system can be calculated from these data.

Considering the biological importance of cytochrome C and the 11

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12 Potential of Cytochrome C

large discrepancy among the reported values of the potential of this substance, we were led to restudy this problem by this method. The results of this study, which were made possible only by the previous careful studies on oxidation-reduction in- dicators by Clark (4), together with a general description of the method employed, are reported in this paper.

The first recorded potential for cytochrome C was that of Cool- idge in 1932 (5). Coolidge’s preparation of cytochrome C gave very unsatisfactory potentials with the electrode, and the E’o values varied with pH. His procedure, with Keilin’s (6) yeast cytochrome preparation, involved the addition of quinhydrone (to stabilize the potential) and the subsequent addition of small amounts of oxidant or reductant to a point where the cytochrome spectrum (hand spectroscope) changed. At this point the poten- tial was recorded. He obtained an average value for E’o of +0.260 volt at pH 7.0. The only other values recorded were +0.207 volt at pH 5.0 and +0.235 volt at pH 4.5.

Green (7) believes that the method of preparation used by Cool- idge did not yield cytochrome C and that his concentrations of iron were below the level which could give a stable potential with the electrode. Green, therefore, repeated the determination and reported a lower value (E’o = +0.127 volt between pH 4.59 and 7.14, shown in Fig. 3). Using for the first time a pure cytochrome C preparation from beef heart muscle, we have obtained a value which agrees closely with one of the somewhat uncertain values given by Coolidge.

Method

After examination of the spectra of the oxidized and reduced forms of the two substances under investigation, two suitable wave-lengths were chosen for analysis. In general, the choice was such that there existed a widely different absorption of light at X1 for the oxidized and reduced forms of substance A, and a like difference at XZ for substance B. At each of the chosen wave- lengths it was necessary to make preliminary measurements of four quantities; namely, the absorption for oxidized A, for re- duced A, for oxidized B, and for reduced B. Two simultaneous equations may then be formulated involving two unknowns, namely the fractions of A and B oxidized, and these equations are

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Stotz, Sidwell, and Hogness 13

then applied in the analysis of the equilibrium solution. This method of analysis is independent of spectral changes which may occur with change of pH, since all these measurements are made separately for each experiment.

For the general equation,

where K’,,. is the equilibrium constant for the equation as written, n is the valence change of the equation, and a and b, etc., are integral numbers.

Since in the equilibrium mixture the potential (Eh) must be the same for both substances, we have

El,,, - E’o, = y In K’,,.

The validity of the method was tested with two indicators, the potentials of which have been accurately measured by Clark (4); namely, naphtholsulfonate indophenol and its 2,6-dichloro sub- stitution product. Fig. 1 shows the absorption spectra of the oxidized forms, the reduced forms absorbing no light in this wave- length region. The wave-lengths used were 5000 A. (maximum absorpiion of naphtholsulfonate indophenol, the “red” dye) and 6100 A. (maximum absorption of naphtholsulfonate indo-2,6- dichlorophenol, the “blue” dye).

A special anaerobic cell was used in this work (Fig. 2). The two buffered indicators, of strength such that they gave a

log lo/I of approximately 0.5 at their respective maxima (1 cm. cell), were reduced with hydrogen and palladiumized asbestos. Without special precautions to prevent the autoxidation of the leuco forms, they were filtered and 2.0 cc. of each were introduced into the anaerobic cell. The mixture which was already partially oxidized was shaken in a constant stream of oxygen-free nitrogen for at least 30 minutes, closed to the air, and transferred to the cell box equipped with a thermostat (30”) for measurement of the

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14 Potential of Cytochrome C

light absorption at 5000 A. and 6100 8. While the nitrogen was passing through, 0.1 cc. of dilute ferricyanide solution was added to bring about oxidation of the indicators and a new point of

2-

l-

o-

9-

.6 - x

$

.I - i3

2

.6 -

.5 -

.4 -

.3 -

.2 -

.I -

‘WI

REDUCED CYTOCHROME C

II "BLUE"

I I INDICATOR

"RED'

FIG. 1. Relative absorption of cytochrome and indicators (pH 7.0)

equilibrium. Again the light absorption at the two wave-lengths mentioned above was observed. Since both indicators were found to conform with Beer’s law, these values of log 10/l were mul-

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Stotz, Sidwell, and Hogness 15

tiplied by the appropriate dilution factor for purposes of calcula- tion. The operation was repeated several times, the number of repetitions depending on the strength of the ferricyanide used. The exact strength of the ferricyanide does not enter into the cal- culation, but the exact volume added must be known.

The two separate indicators were either allowed to autoxidize completely or were oxidized by addition of a small crystal of ferri-

E A D

FIG. 2. Anaerobic absorption cell. A, represents the gas inlet; B, the ground glass cock for introduction of the materials; C, the gas outlet; D, the chamber for equilibration of the liquid with gas; E, the chamber (approximately 4 cc.) with clear windows (strain-free microscope slide sections) for spectrophotometric determinations.

TABLE I

Sample Data in Equilibria between Naphtholsulfonate Indophenol (Red Dye) and Naphtholsulfonate Indo-2,6-Dichlorophenol (Blue Dye)

pH 6.59 (0.1 M POn buffer); T = 30’.

Log 1,/I, 5000 ip .................. “ lo/I, 6100 “. .................

Fraction of each (calculated) in equilibrium mixture ............

Red dye

Oxi- Re- dized duoed

0.410 0 0.064 0

0.596 0.404

T

Blue dye

Oxi- Re- dized duced

--

0.114 0 0.332 0

Equili- brium

mixture

0.302 0.201

cyanide (small amounts of ferri- or ferrocyanide do not absorb significantly at either wave-length). 2 cc. of each of the indicators thus oxidized were separately diluted to 4 cc. and their absorptions measured at the two wave-lengths. The data and calculations for one experiment are given for the purpose of illustration (Table I).

The data in the last line of Table I were obtained from the si- multaneous equations,0.302 = 0.4102 f0.114~ and 0.201 = 0.064s

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16 Potential of Cytochrome C

+ 0.332y, where x is the fraction of red dye oxidized, and y the fraction of blue dye oxidized.

If we write for the equation oxidized blue dye + reduced red dye = oxidized red dye + reduced blue dye, then

Since at pH 6.59 the El0 of naphtholsulfonate indo-2,6-dichloro- phenol is +0.151 volt (Clark), we must subtract 0.06 log,, 1.24. The E’. for naphtholsulfonate indophenol is then +0.146 volt. Clark found 0.1475 volt. Table II records other values found, in good agreement with those of Clark.

TABLE II

Et0 of Naphtholsulfonate Inclophenol at pH 6.59 with Naphtholsulfonate Indo-2,&Dichlorophenol As Reference Indicator

0.1 M POa buffer; T = 30’. The values are given in volts.

PH I Clark’s valulue

I Found

6.37 0.1605

6.59 0.1475 7.44 0.0960

0.1595 0.1593 0.1598 0.1460 0.0956 0.0959

Cytochrome C

Pure cytochrome C was isolated from beef heart muscle by the excellent method of Keilin and Hartree (8). It contained 0.342 per cent Fe determined by the method of Lintzel (9) modified to the extent of measuring the pink Fe ++-bipyridine complex spectro- photometrically at X = 5200 A. Calculation of the concentration of one of our solutions of cytochrome by this method and by the spectrophotometric method (equation given by Keilin and Hartree (8)) agreed perfectly. For the oxidized form TheoreJl (10) records a P(sq. cm./mole) X 10e7 of 2.75 at X = 5300 A. We found 2.69 for our cytochrome. The ratios of maxima and minima in the spectra agree with those given by Theorell. We therefore have every reason to believe our preparation pure.

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Stotz, Sidwell, and Hogness 17

An anaerobic titration of reduced cytochrome (spectrophoto- metrically) with standard ferricyanide proved a 1 electron shift in the oxidation of cytochrome. We have thus confirmed the work of Hill and Keilin (11) on the oxidation-reduction equivalent of cytochrome C.

.35 I I I

I I I

1 NAPHTHOL SJLFONATE INDO-2.6.M.CHLOROPHENOL X II ORTHO-CRESOL INDO-2,6-DtCHtOROPHENOL # \\- ‘REEN*S DATA

III 26DtCHLOROPtlENOL INCOPHENOL 0 I ORTMXHW0PHENOL INDOPHENX . P 6ENZOQUIM)NE a- PI NAPHTHOL SULFONATE INDOPHEND

T

L m

T - 3oT. \\

I I I I I I\=>

6 PH 7 8 .05

5

FIG. 3. E’o-pH relations of cytochrome C and indicators (30”)

In the determination of the potential of cytochrome C prelimi- nary experiments were necessary to choose indicators (at various pH values) which had potentials reasonably close to that of cyto- chrome. This was quickly accomplished by observing the per- centage reduction of the indicator and cytochrome at equilibrium. Fig. 3 illustrates the E’o-pH relations of cytochroome and the indicators finally used. The choice of X1 = 5200 A. and .XZ =

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18 Potential of Cytochrome C

5550 A. depended upon the facts that (1) at X1 a marked difference in light absorption of oxidized and reduced cytochrome occurred, and (2) at X2 the light absorption of the two forms of cytochrome is identical, so that the course of the reaction is readily observed during measurements. At the latter wave-length there occurs a great light absorption by the oxidized indicator. The qualitati!e relationships may be seen in Fig. 1. This choice of XZ = 5550 A. allows the simple calculation of the fraction of oxidized dye (y) by the equation, log IO/I,,. = C + yD, where log IO/I,,. is the ob- served log of the equilibrium mixture, C = log IO/l for oxidized (or reduced) cytochrome alone, and D = log lo/l for completely oxidized indicator.

The fraction of re$uced cytochrome (x) was calculated from data obtained at 5200 A. according to the following equation, log lo/l,,. = E + ~$8’ - E) + yG, where E = log I,/1 for completely oxidized cytochrome, F = log IO/I for completely reduced cyto- chrome, and G = log IO/I for completely oxidized indicator.

We have therefore the four quantities necessary for the calcula- tion of the equilibrium constant for the equation, + reduced dye + oxidized cytochrome = 3 oxidized dye + reduced cytochrome, which is

Ke,. = oxidized dye * X

reduced cytochrome

reduced dye oxidized cytochrome

The data and calculation of the equilibrium constants and E’o of cytochrome of a typical experiment are shown in Table III. In- spection of the constants obtained illustrates the validity of the method. This particular experiment, which was chosen because it involved more check determinations than any of the others, represents one of our best. However, there was never more than 2 millivolts deviation in the E’o found for any given experiment.

The determination of E’o at various pH values requires the use of several indicators.

Since there were no indicators of Clark’s indophenol series available with potentials sufficiently high to form satisfactory equilibria with cytochrome C at pH values above 7.5, we resorted to benzoquinone. In this case quinhydrone was utilized in a concentration 100 times as great as that of the cytochrome, whereas in the indicator experiments, the concentration of cyto-

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Stotz, Sidwell, and Hogness

chrome and indicator was approximately equimolar. Since the reaction, under these stoichiometric conditions, should not change the ratio of hydroquinone to quinone appreciably, the Eh of the equilibrium mixture with cytochrome was considereod to have the Efo of the benzoquinone system. With X = 5475 A. the propor- tion of oxidized and reduced cytochrome was measured directly. The relatively small absorption of the quinone, as obtained from a blank, was subtracted from the observed log lo/l.

These determinations were admittedly less certain than the indicator type of experiment (1) because only one equilibrium was obtained, and (2) because of the steady increase in the color of the quinhydrone solution with time even under anaerobic conditions.

TABLE III

Equilibria between Cytochrome C and 2,GDichlorophenol Indophenol

T = 30”; pH 6.47 (0.1 M Pod); El0 of indicator = 0.260 volt (Clark).

Indicator

Reduced Oxidized

per cent per cent

57.4 76.0 80.4 87.2 93.3

42.6 23.0 19.6 12.8

6.7

Cytochrome

Reduced Oxidized

per cent per cent 46.3 53.7 36.2 63.8 32.8 67.2 27.8 72.2 20.7 79.3

KW.

1.00 1.03 0.99 1.01 0.99

E’o (calcu- lated) for

cytochrome

IJolt 0.2600 0.2605 0.2600 0.2600 0.2600

The latter factor becomes of such magnitude between pH 7.6 and 8.0 that the values obtained in this region are certainly not to be relied upon to the same degree as those of the other determina- tions.

In Table IV is recorded a summary of the E’o-pH data obtained with the indicators designated. Each K,,.,. and E’o is an average of from three to five values obtained in the particular experiment. Table IV includes experiments in which either one or both of the systems were reduced. The equilibrium constants obtained by these variations were always the same. The concentrations of indicator and cytochrome were varied around a mean value of 0.5 X 10P4 mM per cc. in the equilibrium mixtures. Variation in concentration also did not affect the value of E’o at a given pH.

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20 Potential of Cytochrome C

It is of interest that the Efo values found are independent of the pH of the solution within this range. This concurs with Barron’s (12) belief that the “influence of the hydrogen ion concentration on the E’o values of the hemochromogens . . . seems to depend on the affinity of the heme for the nitrogenous compound.”

TABLE IV E’,,-pH Data jor Cytochrome C (30”)

PH Buffer (0.1 M) Indicator

5.04 Acetate

5.25 5.51 5.87 6.08 6.29 6.43 6.47 6.47 6.53 6.60 6.85 7.10 7.28 7.28 7.37 7.45 7.50 7.61 7.82 8.01

‘I

PO1-citrate ‘L ‘I

PO4 ‘I I‘ ‘I IL I‘ ‘I ‘I ‘C ‘I “ I‘ ‘I ‘I

Borate “

Naphtholsulfonate indo-2,6-dichloro- phenol

‘I ‘<

o-Cresol indo-2,6-dichlorophenol 2,6-Dichlorophenol indophenol o-Cresol indo-2,6-dichlorophenol 2,6-Dichlorophenol indophenol

“ I‘ “ “

o-Chlorophenol indophenol ‘I I(

2,6-Dichlorophenol indophenol IL I‘ ‘I ‘I

Benzoquinone I‘ I‘ I‘ (I I‘ (I ‘I

E’o of cyto-

chrome C

volt

-0.2625

0.2620 0.2635 0.2615 0.2630 0.2620 0.2620 0.2600 0.2610 0.2610 0.2630 0.2630 0.2635 0.2640 0.2650 0.2620 0.2615 0.2630 0.2600 0.2580 0.2550

A few electrometric titrations of cytochrome C were carried out, which were not altogether successful. The best results were ob- tained by titrating completely reduced cytochrome with the com- pletely oxidized material. Stable potentials were obtained against the standard calomel cell, yielding an E’o of cytochrome of +0.262 to +0.266 volt. This was considered satisfactory additional proof for the validity of our method, but was far too uneconomical of cytochrome to pursue further.

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Stotz, Sidwell, and Hogness 21

DISCUSSION

The application of the method described in this paper has cer- tain obvious advantages and disadvantages. We depend upon electrometric potential measurements of the reference substances, although it is conceivable that if such data were not available, primary standards could be measured by the spectrophotometric method. Careful spectrographic consideration must be given to the systems under investigation. It is apparent that extraneous material (turbidity, inert colored substances) which could influ- ence the light absorption would necessarily but not unduly com- plicate the procedure. Whereas, in electrometric titrations, the E’o of the substance under investigation does not necessarily have to be approximated before experiment, in this method is must be.

Barring substances which interfere calorimetrically, this method is independent of impurities, since it is only necessary that the two substances in question come into equilibrium. The spectrophoto- metric method is much more economical of the usual small amounts of biological materials available, and does not necessitate choosing the proper electrodes, titrating agents, etc., as does the electro- metric method.

Just as the construction of a typical sigmoid curve is indicative of a successful electrometric titration, the agreement of calculated equilibrium constants indicates the success of the spectrophoto- metric experiment.

It should be noted that while our cytochrome was prepared from heart muscle, that studied by Green was obtained from yeast. Although there are no obvious differences in the spectra of cyto- chrome from the two sources, there is a possibility that the oxida- tion-reduction potentials of the two may be different. The differ- ence between the values of E’o obtained by Green and by us (AZ’, = 0.135 volt) is very large and is perhaps traceable to the purity of the two preparations.

The high oxidation-reduction potential of cytochrome C found in this work is of interesting biological significance. Its relation to the indophenol dyes is shown in Fig. 3. Such a high potential does not imply that cytochrome C could not function as a respira- tory catalyst, but rather adds greater interest to the properties of cytochrome (indophenol) oxidase. When we consider that at pH 7.4 cytochrome C has a potential even greater (by 74 millivolts)

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22 Potential of Cytochrome C

than 2,6-dichlorophenol indophenol, we can readily understand why it requires an oxidase for its oxidation. Keilin (13) attributes the lack of autoxidizability of cytochrome C to some unique prop- erty of this hemochromogen. Although it is probable from the work of Zeile (14) that cytochrome hemin is not typical proto- hemin, such an explanation for its lack of autoxidizability may not be necessary in the light of the high oxidation-reduction potential found for cytochrome C. Actually we have found that cyto- chrome C, such as we have prepared, has a measurable rate of autoxidation which was only partially blocked by cyanide. This would indicate that the autoxidation was not due solely to traces of heavy metals. The high potential of cytochrome C makes this respiratory catalyst available to reducing agents with a wide range of oxidation-reduction potentials.

SUMMARY

1. A spectrophotometric method for the analysis of the reduced and oxidized forms of two colored substances in equilibrium has been described.

2. The method has been tested in the case of naphtholsulfonate indophenol and its 2,6-dichloro substitution product. Calcula- tion of the potential of one of these from that of the other has given results in agreement with those found by Clark.

3. The oxidation-reduction potential of pure cytochrome C has been measured by this method. E’, was found to be +0.262 volt and was independent of the pH between 5.0 and 8.0. The oxida- tion of reduced cytochrome involves an electron change of 1 per molecule.

BIBLIOGRAPHY

1. Quastel, J. H., and Whetham, M. D., Biochem. J., 18, 519 (1924). 2. Thunberg, T., Skund. Arch. Physiol., 46, 339 (1925). 3. Hogness, T. R., Zscheile, F. P., Jr., and Sidwell, A. E., Jr., J. Physic.

Chem., 41, 379 (1937). 4. Clark, W. M., Bull. Hyg. Lab., U. S. P. H. S., No. 151 (1928). 5. Coolidge, T. B., J. Biol. Chem., 98, 755 (1932). 6. Keilin, D., Proc. Roy. Sot. London, Series B, 106, 418 (1930). 7. Green, D. E., Proc. Roy. Sot. London, Series B, 114, 423 (1934). 8. Keilin, D., and Hartree, E. F., Proc. Roy. Sot. London, Series B, 122,

298 (1937).

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Stotz, Sidwell, and Hogness 23

9. Lintzel, W., Z. ges. exp. Med., 86, 269 (1933). 10. Theorell, H., Biochem. Z., 286, 207 (1936). 11. Hill, R., and Keilin, D., Proc. Roy. Sot. London, series B, 114, 104

(1933). 12. Barron, E. S. G., J. Biol. Chem., 121, 285 (1937). 13. Keilin, D., in Nord, F. F., and Weidenhagen, R., Ergebnisse der

Enzymforschung, Leipsic, 2, 239 (1933). 14. Zeile, K., Z. physiol. Chem., 207, 35 (1932). Zeile, K., and Piutti, P.,

Z. physiol. Chem., 218, 52 (1933). Zeile, K., and Reuter, F., Z. physiol. Chem., 221, 101 (1933).

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HognessElmer Stotz, A. E. Sidwell, Jr. and T. R.

THE POTENTIAL OF CYTOCHROME COXIDATION-REDUCTION SYSTEMS;

EQUILIBRIUM INDETERMINATION OF THE

THE SPECTROPHOTOMETRIC

1938, 124:11-23.J. Biol. Chem. 

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