VOL. 12 (1953) BIOCHIMICA ET BIOPHYSICA ACTA 273 



ON THE COMPOUNDS OF FERRICYTOCHROME C 

 APPEARING IN ACID SOLUTION 



by 



E. BOERI*. A. EHRENBERG, K. G. PAUL and H. THEORELL 



Biochemical Department and the Wallenberg Laboratory of 

 Physiological Chemistry, Medical Nobel Institute, Stockholm {Sweden) 



Twelve years ago Theorell and Akeson^ found that ferricytochrome c shows five 

 different types of absorption spectra at different pH values. These investigations were 

 extended from pH — 0.3 to 13.8, because the absorption data seemed to show that 

 ferricytochrome was so stable that only reversible changes would occur within this re- 

 markably wide range. Keilin and Hartree, in 1940^, had stated that cytochrome c 

 as judged by their enzymic test was entirely stable in 0.3 M HCl at room temperature 

 for 18 hours, whereas a decrease of 50% in activity followed in o.i M KOH at the same 

 temperature and for the same time. Paul^ more recently demonstrated that the enzymic 

 activity of cytochrome c, as determined with succinic oxidase from kidneys and succinic 

 acid is somewhat less stable to extreme pH values. He found the limits for practically 

 indefinite stability to be pH 1.6 and 12.3. Below pH 1.6 down to 0.66 there was a slow 

 decrease of the activity. The inactivation in this region seemed slow enough to permit 

 spectrophotometric and magnetic experiments to be made during the first half or per- 

 haps whole hour after acidification without any serious degree of disturbance from in- 

 activation. 



In the following we shall thus restrict ourselves to experiments at pH values higher 

 than around i. 



The spectrophotometrically determined dissociation curve indicated a pA' of 2.5. 

 Magnetometric measurements* indicated that the essentially covalent bonds in ferri- 

 cytochrome c in neutral solution were shifted to essentially ionic bonds in acid solution. 

 An attempt to correlate the spectrophotometric and magnetic values with one another 

 gave curves that did not agree entirely. At that time we did not know whether the dis- 

 crepancies were due to experimental errors, or to some unknown factor interfering. 

 Since we have now at our disposal an accurate magnetometric apparatus requiring only 

 small amounts of materiaP, it appeared to be of interest to reinvestigate the spectro- 

 photometric and magnetic properties of ferricytochrome c in acid solution. The magnetic 

 apparatus has in the last year been considerably improved, as will be described elsewhere. 



For several years we have had reason to believe that chloride ions influence the 

 light absorption of ferricytochrome c in acid solutions^. The existence of a fluoride com- 

 pound of cytochrome c around pH 3 was reported as early as 1941^. Maehly'^ recently 

 found that acidification with HCl, or some other acids, of a solution of horse radish 

 peroxidase (HRP) was followed by spectral changes in the Soret band region, from which 



* Rockefeller Foundation Fellow. 

 References p. 282. 



