VOL. 12 (1953) COMPOUNDS OF FERRICYTOCHROME C 277 



perimental data derive from Fig. 3, others from separate experiments not mentioned 

 before. 



At pH = 2.0 the value of ^^^^^ [-q-] = o could be rather accurately determined by 

 extrapolation to 36 X 10', as in Fig. 4. The j8-values for different [C1-] at pH 2.0 were 

 taken graphically from Fig. 3. ^395^ ci- = 00 = 20.5 • lo^. 



36-10^— ^obs 



^obs — 20.5-10^ 

 absorption were too small to allow accurate calculations. 



Evidently the results prove that the acid ferricytochrome c, with two protons added, 

 forms a compound with two chloride ions per molecule, since >ici- = 2 ("Cyt-2H+-2C1~"). 

 Thus the following relations are valid 



[Cyt] [H+]2 



log — ^ is plotted against [C1-] in Fig. 6. At pH = 3 the changes in light 



[Cyt-2H+] 

 [Cyt-2H+] [Cl-]2 



[Cyt-2H+-2C1-J 



= Kn+; (I) 



= Kcr (2) 



We shall now calculate the value of A'q- at pH i, 2 and 3. At pH = i less than 

 0.6% of the total cytochrome is in the form of Cyt, and can be neglected. At log [Cl~] = 

 — 1.07, [Cyt-2H+] = [Cyt-2H+-2C1-J (see Fig. 6). This gives 



Kci-pHx = 10-2-1^ = 7-10-3 M2. 



At pH 2 Cyt, Cyt-2H+ and Cyt-2H+-2C1~ may all be present in considerable 

 amounts. Since [Cyt] ?» [Cyt-2H+] at pH 2.10 and [Cyt] + [Cyt-2H+] = [Cyt-2H+ 

 -2C1~] at [C1-] = 0.035 M (see Fig. 6) we could calculate the mole fractions of the three 

 compounds at pH 2 and [C1-] =0.035 to [Cyt] =0.194; [Cyt-2H+] =0.306; and 

 [Cyt-2H+-2C1-] = 0.500. Inserting these values into eq. (2) gives Kq- pH = 2 = 

 0.7- 10-3 M2. 



A very uncertain value at pH = 3 could be calculated from Fig. 4 and 5, in which 

 the midpoints of the transitions seem to appear near [CI"] =0.1; this gave the value 



Kci-pH3 = 2(± I) -10-3^2 



It was now possible to calculate the true values for ^^^^^ 2H+ and pHjoo/^ for 

 the transition Cyt+2H+ ^f^ Cyt-2H+. We thus found 



i^jgs. Cyt-2H+ = 50-10' cm2 X mole-i, and pHj^o^ = 2.12. 



The difference from the preliminary values at [C1-] = 0.009 ^' 48 •10'', resp. 2.10 

 is so small that obviously practically no errors had been introduced by using the preli- 

 minary values. 



We could now derive the /3-values for the whole Soret-band of the pure Cyt-2H+. 

 Of course one must remember that this compound cannot exist in a pure state, but only 

 in equilibrium with Cyt and some anion compound. The light absorption of pure Cyt was 

 directly measured at pH = 5.5, and that of Cyt-2H+-2Cl-at [C1-] = 2.83 M and at pH 



I-5-3-5- 



The curve for pure Cyt-2H+ could be calculated only in the pH-range 1.7 to 2.2, 

 because outside these values the contributions of Cyt-2H+ in the equilibrium with 

 Cyt-2H+-2C1- and Cyt become too small for obtaining reliable values. From five sets 

 References p. 282. 



