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Experimental Section Materials. All of the primary anilines were commercial samples except for 3-methyl-2,4,6-trinitroaniline, which was kindly provided by the late Dr. M. Jorgenson. These substances were recrystallized from ethanol or ethanol-water mixtures until they had constant melting points which agreed with accepted literature values. 2,4-Dimethoxyazobenzene was prepared by coupling diazotized aniline with m-methoxyphenol and then methylating the free hydroxyl group with dimethyl sulfate. l o This coupling reaction produced two substances, both of which gave 2,4-dimethoxyazobenzene upon methylation; that fact plus their nmr spectra identified them as the positional isomers 2-hydroxy-4-methoxyazobenzene and 4-hydroxy-2-methoxyazobenzene. 2,4-Dimethoxyazobenzene was purified by alternate recrystallization from 95 ethanol and from hexane until its melting point was constant and in agreement with the literature value. This preparation gave a mixture of cis- and rruns-2,4-dimethoxyazobenzene, but isomerization of the cis isomer to the more stable trans form is acid catalyzed and very rapid at the acidities employed for the indicator measurements.20 Thus, these measurements refer only. to trans-2,4-dimethoxyazobenzene Deionized water was purified further by distillation from alkaline permanganate in glass apparatus. All other materials were best available commercial grades and were used without further purification. Density Measurements. Solutions were prepared by pipetting 10-ml quantities of 95% ethanol into 50-ml volumetric flasks and then filling the flasks to the mark with aqueous sulfuric acid of the appropriate concentration. With concentrated acids, considerable heat was evolved during this dilution; the acid was therefore added in small portions and the flask was cooled between additions. In all cases, final volume adjustments were made with the flask and its (19) N . Kaneniwa, J . Pharm. Soc. Jap., 76,261 (1956); V. L. Horner and U.Schwenk, Justus Liebigs Ann. Chem., 579,204 (1953). (20) A . J. Kresge and G. L. Capen, unpublished work.
contents in temperature equilibrium with a bath operating at 25.0 f. 0.05". Densities were determined using Weld pycnometers of 10-ml nominal volume; these were filled in the recommended wayz1while suspended in the 25" constant temperature bath. Weighings were performed on a Mettler type B6 semimicrobalance and were ccrrected for the effect of air buoyancy.21 Each measurement was made in duplicate with each of two pycnometers; the results are therefore averages of four separate determinations. lo Some density measurements of wholly aqueous sulfuric acid were also made; these agreed with published29 values to within 0.001 g/ml. A few density measurements of 20z ethanolic sulfuric acid have been made before,ab but the values reported are consistently lower than the present results by ca. 2 x ; the reason for this difference is not known. Indicator Measurements. Stock solutions of indicators in 95 ethanol were prepared at concentrations (ca. M ) selected to give maximum absorbance readings. Aliquots of these solutions were then diluted with sulfuric acid as described above for density measurements, and spectra were recorded from 500 to 350 nm using spectrometers (Beckman DK-2 or Cary 11) with cell compartments thermostated at 25.0 i 0.1". Absorbances were estimated to 0.001 from the recorded traces at absorption maxima and also at positions 5 nm to either side. The values of A so obtained were transformed into indicator ratios using the relationship I = ( A B - A ) / ( A - A m +), where A B and ABH+ are the absorbances of soluticns containing indicator completely in its basic and acidic forms, respectively. These limiting absorbances were measured at acidities at least 3 H units to either side of the indicator pK.. The spectrum of each solution was usually recorded three times, and most values of I are therefore averages of nine measurements. lo (21) N. Bauer, Tech. Org. Chem., 1,253 (1949). (22) "International Critical Tables," Vol. 111, McGraw-Hill, New York, N. Y., 1926, p 24.
Electrochemical Studies of Heme Proteins. Coulometric, Polarographic, and Combined Spectroelectrochemical Methods for Reduction of the Heme Prosthetic Group in Cytochrome c S t e p h e n R.
Betso, M i c h a e l H. K l a p p e r , and Larry B. Anderson*
Contribution f r o m the Department of Chemistry, The Ohio State University, Columbus, Ohio 43210. Received December 22, 1971 Abstract: T h e detailed electrochemical behavior of native horse-heart cytochrome c is described. This heme protein is shown to reduce a t a variety of electrode materials producing freely diffusing ferrocytochrome c that is fully active in the cytochrome oxidase enzyme system. Adsorption of the protein onto the electrode surface has significant influence o n the observed electrochemistry, but it does not cause electrode fouling or loss of the electrode's ability to transfer electrons. O n the basis of these results, it is not possible t o distinguish between an electron transfer mechanism involving charge conduction through the protein fabric a n d a mechanism wherein electron transfer occurs only a t the exposed heme edge. T h e relaxation techniques developed here appear suitable for electrochemical study of high molecular weight proteins in general.
B
e c a u s e c y t o c h r o m e c, a heme p r o t e i n distributed widely in living organisms, has a central r o l e in t h e e l e c t r o n t r a n s f e r reactions of a e r o b i c m e t a b o l i s m , t h e m e c h a n i s m of r e d u c t i o n a n d o x i d a t i o n of t h e protein i r o n is o f great interest. D a t a f r o m h e t e r o g e n e o u s e l e c t r o c h e m i c a l e x p e r i m e n t s may yield i n f o r m a t i o n on r e d o x s t o i c h i o m e t r y , on e q u i l i b r i u m , on t h e transport of e l e c t r o a c t i v e species t o and f r o m the e l e c t r o d e surface, a n d on t h e c h e m i c a l r e a c t i o n s occurring b e t w e e n t h e e l e c t r o a c t i v e species and o t h e r components in t h e s o l u t i o n p h a s e . T h e s e data are o b t a i n e d without a l t e r -
the atomic composition of the solutions under study, since only electrons are added or removed. For t h e s e reasons we have begun what we believe is the first s y s t e m a t i c application of the v a r i o u s m e t h o d s of e l e c t r o c h e m i s t r y t o an e l u c i d a t i o n of t h e b e h a v i o r of native c y t o c h r o m e c. In addition these techniques can serve as clean, synthetic m e t h o d s for the p r o d u c tion of reduced or o x i d i z e d m a t e r i a l without t h e required addition of other redox reagents. E l e c t r o chemical p r o c e d u r e s may a l s o be combined with spectrophotometric methods to increase the amount of a t i o n s in
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information which may be obtained, and to measure phenomena occurring a t very fast times, 1-4 Previous potentiometric studies of cytochrome c5-9 utilizing mediators have provided internally consistent results for the free energy of electron addition to the ferriheme prosthetic group as a function of pH. The equilibrium potential of the cytochrome c system has also been obtained spectrophotometrically using redox indicators i n place of the heterogeneous l1 The two methods have given comparable results. The mechanism of the redox reaction has also been in\. estigated spectrophotometrically. l4 Early polarographic studies of solutions containing cytochrome c were degradative in purpose.15 The electrochemistry described did not pertain to reduction of the prosthetic group. The catalytic wave observed was shown’+-’g to be a “protein double wave” characteristic of cobalt reduction in the presence of proteins containing cysteine groups. Direct polarographic reduction of cytochrome c was reported by Griggio and Pinamonti.9 Three irreversible waves were seen, the first of which ( E I ‘v ~ ~-0.3 V US. sce) was attributed to reduction of the heme prosthetic group. The first successful attempt at macroelectrolysis of ferricytochrome L‘ to its ferro form was reported by Kono and Nak;imura,?O using a platinum electrode. The reduction process was masked by hydrogen evolution, however, and the ferrocytochrome c produced was stated to be only 60% active with cytochrome a?. We describe here the results of a series of experiments designed to test the feasibility of using electrochemical relaxation methods for initiating and monitoring electron transfer reactions in native protein systems. The reduction of ferricytochrome c at both mercury and platinum electrodes was studied, and spectrophotometric examination provided information on the nature and purity of the reduction products.
Experimental Section Materials. Preparations of horse-heart cytochrome c were obtained through the courtesy of Dr. Grant Barlow, Abbott Laboratories, Chicago, Ill. All preparations were stored In a desiccator ( I ) M. Petek, T. E. (1971).
Neal, a n d R. W. Murray, Anal. Chem., 43, 1069
(2) N. Winograd and T. Kuwana, ibid., 43, 252 (1971), and references
therein.
(3) R. N. Adams, “Electrochemistry at Solid Electrodes,” Marcel Dekker, New York, N. Y., 1969, p 255 ff. (4) I . B. Goldberg and A . J. Bard, J . Phys. Chem., 75, 3281 (1971). ( 5 ) R . Wurmser and S. Filitti-Wurmser.J. Chem. Phys., 35, 81 (1938). (6) I
