276 



E. BOERI, A. EHRENBERG, K. G. PAUL, H. THEORELL VOL. 12 (1953) 



3 



6 

 o 



e 



10 



acid solution. This effect arises through an interaction between hydrogen and chloride 

 ions. The cation of the added salt seems to play no role; potassium or calcium chloride 

 gave the same results as sodium chloride. 



Some other anions were also investigated. Br^ 

 and S04~^ gave effects similar to Cl~, but the absorp- 

 tion coefficients were slightly different. 



In view of these new facts it seemed interesting 

 to reinvestigate the magnetic susceptibility of ferricy- 

 tochrome c in acid solution. In Fig. 5A we have plotted 

 the new data on the paramagnetic susceptibility, ^j^, 

 for the cytochrome iron at 20 °C at varied [Cl~] and pH. 

 At low [C1~J %jj^ is strongly dependent on pH, just as 

 the light absorption, but at higher [Cl~] the curves con- 

 verge and reach horizontal asymptotes. Contrary to 

 the spectrophotometric values, the magnetic ones do 

 not approach exactly the same value; the levels of 

 the asymptotes depend to a certain degree on the pH, 

 as indicated in Fig. 5B. Two different concentrations of 

 cytochrome were used for these determinations, 1.54 

 and 0.77 mAf .The lower concentration was used in such 

 determinations where it was important to reach a low 

 pH with a minimum of Cl^ ; in these cases the buffering 



0.2 



0.6 



1.0 



[cn 



Fig. 5A. Magnetic susceptibility 

 of cytochrome c plus NaCI at dif- 

 ferent pH: 1.5 (x), 1.8 (/\), 2.0 

 (O), and 3.0 (#). Single values 

 at [Cl^] = i.o ( + ), pH given in 

 figure. 



Fig. 5B. Variation of Xm of the 

 chloride compound with pH. 



capacity of the cytochrome played a certain role. 



Evaluation of experimental data. Spectrophotometry . From Fig. 3 it is immediately 



seen that the curve for the lowest chloride ion content (0.009 ^) ^^^ the general form of 



a part of a dissociation curve. The asymptotic level 

 at pH 5.5 is experimentally determined ifi-^^^^ pH 5.5 = 

 14.0-10' cm^ X mole-^) but the level at low pH is 

 unknown. 



T) 1 J.J.- 1 roh% PpH 5.5 



By plottmg log 



A 



low pH Pobs 



against pH for the 



experimental points (pH, ^^^^ x 10-') : (2.04, 33.5); 

 (2.24, 25.6);(2.40, 20.8) and (2.54, 17.3) it was found 

 by trial and error that the value of /J^gj^ lo^y pH = 

 48-10' cm^ X mole-^ gives a straight hue with nH+ = 

 2.0 and a pHjoo/^ 2.10. We conclude that an increase 

 in hydrogen ion concentration leads to a new compound 

 by the simultaneous addition of two protons to fer- 

 ricytochrome c. If the neutral cytochrome is denoted 

 Cyt, we may designate the new compound Cyt-2H+. 

 The values of ^395^ Cyt-2H+ and pHjQO/^ obtained in this 

 way must be somewhat too low because of the small 

 amount of chloride ions present. They can, however, be 

 used in the calculations of the influence of chloride ions. 

 At pH 1.0 we had ^395 ^,,^ [ci-] = « = 18.7 - 10', and ji^^^ „,„, [ci-] = 0.009 = 48 • lo' cm^ 



mole. Thus we can plot log — °— versus log TCl-J , as in Fig. 6. Some of the ex- 



° 18. 7-10' 



Log[Ci 



Fig. 6. Graphical determination of 



the number n and [Cl~]5o% for the 



reaction with Cl~ at pH i.o ( + ) 



and 2.0 (o). 



^^ 



obs 



References p. 282. 



