280 E. BOERI, A. EHRENBERG, K. G. PAUL, H. THEORELL VOL. 12 (ig53) 



"Type I"^ must have been a more or less denatured product, since the enzymic 

 activity is lost at such acidities, which are low enough to bring about its appearance. 



A comparison of the old and new magnetic data discloses that the former values 

 generally were higher. The preparations used at that time were of low purity (0.7) and 

 they might accordingly have contained some non-cytochrome iron. Our preparation 3 

 gave x^ = 2600-10-^ cgs emu for Cyt as compared to i^ = 2120 for preparation 4. 



A priori one would not expect to find close analogies between the reactions occur- 

 ring with ferricytochrome c and horse radish peroxidase (HRP) or catalase upon acidi- 

 fication. Ferricytochrome c at neutral pH is undoubtedly a hemichrome with nitrogen 

 attached to the iron atom by covalent bonds on both sides of the ferri-porphyrin disc, 

 whereas in HRP and catalase the iron atom is held by essentially ionic bonds. It has 

 been supposed to be linked on one side to the protein and on the other to a water mole- 

 cule or a hydroxyl ion. Moreover, cytochrome c is stable and gives reversible compounds 

 in the acidity range applied, whereas the acid compounds of HRP and catalase studied 

 by Maehly' seem to be irreversibly altered products, with the exception of HRP- 

 chloride, "Compound A". 



Nevertheless certain parallels can be found between the present results on cyto- 

 chrome c and those of Maehly on HRP. Maehly's "Compound A" has its Soret band 

 maximum at 407.5 m/x, ^ = 20-10'' (recalc), while Cyt-2H+-2C1~" has its maximum at 

 402 m/x, j8 = 25.8 • 10''. This difference is the same as the one found by Theorell between 

 the wavelengths of the band maxima of certain protohemin compounds and comparable 

 derivatives of cytochrome c. The displacement of the bands depends upon the fact that 

 in the prosthetic group of cytochrome c the number of conjugated double bonds is re- 

 duced by two because of the thioether structure at the side chains in positions 2 and 4. 

 Maehly has not published any calculation of the number of chloride ions involved in the 

 formation fo Compound A. 



Maehly's "Compound B" and our Cyt-2H+ are rather similar. The wavelengths 

 of the Soret band maxima are 396.5 and 395 m/x respectively, thus at 11 and 7 m/x 

 shorter wavelengths than for the corresponding chloride compounds. The intensities of 

 the bands are, however, rather different: jScomp. b = 30-10" (calc. from £ = 130 cm-^ 

 X mM-i) and iScyt_2H+ = 5o-io^ cm^ x mole-^ Maehly points out that the value 

 £ = 130 is obtained only in the absence of halogen ions. Sulphuric acid developed 

 Compound B, but a further addition of sulphate ions did not cause any additional spec- 

 tral change, contrary to what was the case with cytochrome c. The transformation from 

 HRP to Compound B involved only one proton ; the same value was found for the corre- 

 sponding change in catalase (Fig. 10 and 11 in (7)). Kinetic experiments, however, indi- 

 cated that the rate of acid splitting was proportional to the square of [H+]. In order 

 to explain this, Maehly considered the simultaneous breaking of one iron-protein bond 

 and one bond between a propionic acid residue and the protein. M.aehly's conversion 

 B-^C is perhaps comparable to the strong increase of the dissociation constant (Kq_) 

 between Cl~ and Cyt-2H+ when pH is changed from 2 to i. Denaturation of the HRP 

 protein or reversible change of shape of the cytochrome c upon acidification to pH i 

 could both lead to a diminished affinity for chloride ions. 



In summary a comparison of the reactions of HRP and cytochrome c in acid solu- 

 tions discloses certain similarities; the interpretation of the data is less certain for HRP 

 than for cytochrome c, because HRP in contradistinction to cytochrome c undergoes ir- 

 reversible changes under the experimental conditions. Nevertheless it appears possible 



References p. 282. 



